NOTE TO USER
Page(s) not included in the original manuscript are unavailable from the author or university. The manuscript was microfilmed as received.
xiv-xv
This is reproduction is the best copy available
STUDIES on the ROLE of r-ATP CMELSin the PRECONDITIONING of CULTURED HI%U4N VENTRICULAR CARDIOIMYOCYTES
Gideon Cohen, MD, BSc, MSc, PhD
Institute for Medical Sciences, University of Toronto. Division of Cardiovascular Surgery, The Toronto Hospital. Toronto, Ontario, Canada.
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy. Graduate Department of the Institute of Medical Sciences, University of Toronto
Copyright O 2001 National Library Bibliotheque nationale 1*1 of Canada du Canada Acquisitions and Acquisitions et Bibliographic Services services bibliographiques 395 Wellington Street 395, rue Wellington Ottawa ON KIA ON4 Ottawa ON KIA ON4 Canada Canada
The author has granted a non- L'auteur a accorde une licence non exclusive licence allowing the exclusive pennettant a la National Library of Canada to Bibliotheque nationale du Canada de reproduce, low distribute or sell reproduke, preter, distribuer ou copies of this thesis in microform, vendre des copies de cette these sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format Bectronique.
The author retains ownership of the L'autsur conserve la propriete du copyright ~JI this thesis. Neither the droit d'auteur qui protege cette these. thesis nor substantial extracts fiom it Ni la these ni des extraits substaatiels may be printed or otherwise de celle-ci ne doivent etre imprimes reproduced without the author's ou autrement reproduits sans son permission. autorisation. i ABSTRACT
OBJECTIVES: Due to the increasing numbers of high risk patients presenting for CABG, improved
methods of myocardial protection are necessary. Ischemic preconditioning (PC)is the most powetful
endogenously mediated form of myocardial protection known. Udortunately, this phenomenon is
quite ditticult to apply clinically. The following studies were designed to determine the sequence of
events inherent in PC, with an attempt to harness its beneficial effects in the form of a simple
pharmacologic additive. ME'IXODS: We have developed a mode1 of ischemia and repefision in
human ventricular myocytes (HVh4s) obtained from the right ventricular outflow tract of patients undergoing corrective surgery for Tetralogy of Fallot. In this model, ischemia was reproduced by
exposing the cells to low volume anoxic phosphate buffered saline (PBS) for a period of 90 minutes.
Both stabilization and repefision were accomplished by exposing the cells to high volume normoxic
PBS for a period of 30 minutes. Endogenous preconditioning was achieved by exposing the cells to a brief 20 minute episode of ischemia, followed by 20 rinutes of reperfhion. Exogenous preconditioning was achieved by exposing the cells to 50 uM Adenosine with or without SPT, 10 nM
PMA with our without Calphostin-C, and 50 uM Pinacidil or 20 uM Diazoxide with or without
Glybenclamide. Viability and metabolic assessments were undertaken in the cell model, and results were confirmed in both large animal and clinical studies. RESULTS: Ischemic preconditioning conferred significant protection to HVMs. This protective effect was reproduced with adenosine and
PMA, and was abolished with SPT and calphostin-C. The second messenger system included PKC activation and translocation. The final effector in this process was K'-ATP channel opening, as glybenclamide abolished all preconditioning stimuli. Although preconditioning was reproduced with the non-specific R-ATP channel opener pinacidil, since HVMs are quiescent in nature, the final effector was believed to be mitochondria1 specific K--ATP channel opening. Indeed, mitochondrial- specific K'-ATP channel opening with diazoxide conferred protection, preserved cellular ATP .. 11 concentrations, and facilitated oxidative phosphorylation by increasing mitochondria1 matrix volume and upregulating respiratory chain enzymes. PKC was localized to HVM mitochondria1membranes using both Western blotting techniques and imrnunogold localization. Adenosine was found to preserve myocardial ATP concentrations throughout the crossclamp period in patients undergoing
CABG, and diazoxide was found to preserve ATP and improve hnctional recovery of cardiac allografts in a porcine model of cardiac transplantation. CONCLUSIONS: In humans, preconditioning is mediated via adenosine release, PKC translocation and activation. The final effector in this process is the rnitochondrial KLATP channel, which when opened facilitates oxidative phosphoryIation and ATP preservation. iii
THIS WORK IS DEDICATED TO MY PARENTS, MEfR AND M4Lh2 COHEN- NEHE~,AS ~LLAS TO msrsm, EFFRATAND DANYA.
WlTHOUT =EM, 1 WOULD NOT BE MERE IAM TODAY. ACKNOWLEDGEMENTS
First and foremost, I wish to thank my supervisor and mentor, Dr. Richard D. Weisel for his support and
guidance, without which this work would have not been possible. His tireless efforts in pursuit of academic
excellence and his dedication and commitment to his students are unsurpassed, and I am eternally gratell to have
been granted the opportunity to work under his supervision
I wish to thank the members of my thesis committee, Dr. Donald A. G. MickIe and Dr. Stephen E.
Fremes, for their valuable input and guidance. Their expertise in their respective fields provided an invduable
resource throughout my academic training and I am indebted to them for their time and effort on my behalf. As
well, I wish to thank Drs. John Coles and Michael Johnston for agreeing to serve as thesis examiners.
AIthough all experiments and assessments were undertaken by the author, this work would not have been
possible without the guidance of the many laboratory technicians and post-doctoral fellows within the Centre for
Cardiovascular Research at the Toronto Hospital. I would especially like to thank Frank Merank: PhD, Laura C.
Turniati, MSc, Molly K. Mohabeer, BSc, and Ren-Ke Li, MD, PhD, I would also like to thank Cameron
Ackerley, PhD, for his assistance in performing the eIectron microscopic assessments, and Charlene Weisel, RN,
for her multi-media support.
The continued commitment of the Division of Cardiovascular Surgery at the University of Toronto
towards the training of surgical scientists remains a valuable asset and has provided an ideal backdrop for
residents such as myself who are interested in pursuing an academic surgical career. I am indebted to the
members of the division for their continued support and I hope to achieve a standard that is worthy of such
cornmitment in the coming years.
My family and friends have always provided the unending support necessary to achieve my persona1 and
career goals. I am forever gratell to them for their love and commitment.
Finally, I would like to thank the Heart and Stroke Foundation of Canada for their fellowship grants in support of this work. Portions of this work have been published in the follow in^ iournals:
Cohen, et al. Circulation 1998; 9811-164-I96
Cohen, et al. J NY Acad Sci 1999; 874:30&319
Cohen, et al: Ann Thor Surg 1999; 68:1995-2OOI vi
STUDIES on the ROLE ofK'-ATP CHANNELS in the PRECONDITIONING of CULTURED WUWKENTRICUZAR CARDIOIMYOCYTES TABLE OF CONTENTS
Abstract ... I ... Dedication ...111 Acknowledgements ... iv List of Publications ...v
Table of Contents .. .VI
List of Abbreviations ...xii Figure Legends ...mi
Introduction ... 1
Chapter One: KNOWLEDGE TO DATE ... 5
Myocardial Preconditioning
1.1.1 Historical Overview
Adenosine
1-2.1 Historical Overview
Adenosine Metabolism
1-3.1 Endogenous Adenosine Production 1-3 -2 Adenosine Transport 1-3 -3 Adenosine Catabolism 1-3 -4 Regulation of Interstitid Adenosine Concentrations 1-3.5 Adenosine Receptors
Cardioprotective Properties of Adenosine 1.5 Signal Transduction
I,5 - 1 Cyclic AMP System 1 -5-2 G proteins
16 Adenosine Effector Mechanisms
1-7 Protein Kinase C
1-7.1 Involvement of Alternate Protein Kinases
1-8 The Human Connection
1-8.1 Ischemic Preconditioning in Humans 1-8.2 Human Cardiomyocyte Studies of Endogenous Preconditioning 1-8.3 Clinical Studies of Ischemic Preconditioning 1.8 -4 Reproducing Ischemic Preconditioning
1.9 Exogenous Adenosine in Humans
1-9.1 Physiologic Effects 1-9 -2 ElectrophysioIogic Effects 1-9.3 Regulation of Coronary BIood Flow 1-9.4 Haernodynamic and Respiratory Effects
1-10 Adenosine Preconditioning in Humans
1.10.1 Human Cardiomyocyte Studies of Adenosine Preconditioning 1.10.2 Human Cardiomyocyte Studies of Protein Kinase C 1.10-3 Clinical Studies of Adenosine Preconditioning
l.iO.3.1 Adenosine Pretreatment 1.20.3.2 Cardioplegic Adenosine Treatment 1.10-3-3 Adenosine post-treahnerzt 1.10.3.4 Contirtuozis Adenosine Treatment
1.1 1 Underlying Mechanisms of Preconditioning
2-12 Cardiac Potassium Channels
1.12.1 Knowledge to Date
1.13 ATP-Dependant Potassium Channels viii
Overview The Search for a K-ATP Clone Physiologic Properties Distribution and Density Biophysical Properties Voltage Dependence ATP Modulation of K-ATP K-ATP Kinetics Other Intracellular Modulators of K-ATP Pharmacological Modulation of K-ATP
1.14 Therapeutic Implications in the Heart .,.55
1.14.1 Myocardial Infarction .,.57 1.14.2 Ischemia-Reperfhion Injury ...57 1- 14.3 Effects on Coronary Vascular Tone ...58 1.14.4 Myocardial Preconditioning ...58 1.14.5 Role of Sarcolemrnal KlAm Channels in Preconditioning ...60 1.14.6 Role of Mitochondria1 K+.xp Channels in Preconditioning ...61
1-15 Summary of Study Rationale, Hypotheses, and Objectives ... 64
Chapter Two: ATP-MEDIATED POTASSIUM CHANNEL (gATdOPENER STUDIES: &econdilioning is medinred via channel opening in hum ventricular my ocytes ... 66
2.1 Summary .. -67
2.2 Introduction ..A9
2.3 Materials and Methods ..A9
2.3 1 Isolation and Culture of Human Ventricular Myocytes ...69 2.3 2 Experimental Design ..A9 2.3 3 Experimental Protocols: ... 70 I. Optid Dose md Timing of Pinacidil ...70 11. Role of KArP channel opening in human ... 71 preconditioning 2.34 Assessment of Cellular Injury ... 72 2.35 Biochemical Measurements ... 72 2.36 Statistical Analysis ... 73 2.4 Results
2.41 Optimal Dose and Timing of Pinacidil 2.42 Role of channel opening in human preconditioning
2.5 Conclusions ... 75
Chapter Three: MITOCHONDRIALSPECIECC CATPCHANNEL OPENER STUDIES :&econditioning is mediated via mitochondrial rA7p channel opening in human ventricular rnyocytes ... 77
Summary
Materials and Methods
3.3 1 Isolation and Culture of Human Ventricular Mycoytes 3 -32 Experimental Design 3 -33 Experimental Protocols 3 -34 Assessment of Cellular Injury 3 -35 Biochemical Measurements 3 -36 Statistical Analysis
Results
Conclusions
Chapter Four: PROTEIN KINASE C STUDIES: Protein-kinase C mediates preconditioning via mitochondn'al fiTPchannel opening in human ventricular myocytes ... 86
4.1 Summary ...87
4.2 Introduction ...89
4.3 Materials and Methods ...89
4.3 1 Isolation and Culture of Human Ventricular Mycoytes 4.32 Experimental Design 4.33 Experimental ProtocoIs: ...91
1. Studies of Protein Kinase C mediated preconditioning 11. Studies of Protein Kinase C activation/ translocation
4.34 Isolation of Mitochondria1 Membranes ...92
4.35 Immunogold Localization of PKC 4.3 6 Assessment of Cellular Injury 4.37 Biochemical Measurements 4.38 Statistical Analysis
4.4 Results ...94
4.41 Studies of Protein Kinase C mediated preconditioning 4.42 Studies of Protein Kinase C activation/translocation
4.5 Conciusions ...96
Chapter Five: MITOCHONDRIAL STUDIES: Precondirioning affords protection by facilitating mitochondrial metabolic potential in human ventricular myocytes ...98
5.1 Summary
5 -2 Introduction ... 101
5-31 Isolation and Culture of Human Ventricular Mycoytes 5.32 Experimental Design 5-33 ExperimentaI Protocols 5.34 Studies of mitochondrial respiratory chain enzyme activity 5.3 5 Studies of mitochondrial matrix volume 5.35 StatisticaIAnalysis
5.4 Results
5 - 5 Conclusions ... 104 Chapter Sk DISCUSSION
Human Cardiomyocyte Cell Culture Model
The Adenosine Hypothesis
Mechanism of Preconditioning's Protective Effects
KrApChannel Opening: The Final Effector
Study Limitations
Future Studies
Alternate Effector Mechanisms of Ischemic Preconditioning
Summary of Original Contributions
Conclusions
APPENDIX 1 Adenosine cardiopZegia it2 contemporary corotrary bypass surgery: A pros-eciive randomized trial ...133
APPENDIX 2 Dimoxide-enhanced donor blood perf21sionfor prolonged storage of cardiac allografts ... 146
APPENDIX 3 Isolation and cultwe of hzrrnan venmicular myocytes
APPENDIX 4 Ischemiu and reperjhsion model
APPENDIX 5 Biochemical measurements
APPENDIX 6 Mi'tocho~tdrliZstudies xii
Lf ST of ABBREVIATIONS
ADO Adenosine ADP Adenosine diphosphate ANOVA Analysis of variance ATP Adenosine triphosphate ATPase Adenosine trip hosp hat ase BSA Bovine Serum Albumin CABG Coronary Artery Bypass Grafting ~a 2" Calcium CaClt Calcium Chloride CAtC Calphostin C CK Creatine Kinase CK-MB MB &action of creatine kinase co2 Carbon dioxide CP Creatine phosphate OC Degrees Centigrade DAG 1,2-Diacylglycerol DCA Dichloroacetate DMSO Dimethylsulphoxide DNA Deoxyribonucleic acid DZX Diazoxide et al "and others" g gms GLY glybenclamide G protein Guanosine triphosphate binding protein a+ Hydrogen ion HCI Hydrogen chloride EIEPES N-[2-hydroxyethyl]piperazine-N-[2-eth~ acid ELPLC High performance liquid chromatography H2so4 Sulfirric acid HXN Hypoxanthine Immunoglobulin G IN0 Inosine p3 Inositol 1,4,5-triphosphate K+ Potassium K'AP ATP-dependent potassium channel KC1 Potassium chloride KH2EtPO4 Potassium phosphate (monobasic) K2mo4 Potassium phosphate (dibasic) kDa Kilodalton LAD Left anterior descending coronary artery m- Milli- (1 O -M Moles per Iitre MARCKS Myristolated, alanine-rich, C-kinase substrate ~g=+ Magnesium MgCIz Magnesium chloride min minutes moI Mole (6.023 x loz3 particles) mOsm Miliiosmoles mRNA Messenger RNA n- Nano- (10-3 N2 Nitrogen Na+ Sodium NmC03 Sodium bicarbonate NaHS03 Sodium bisulphite NazCO3 Sodium carbonate NaCl Sodium chloride NaOH Sodium hydroxide NazaPOj Disodium phosphate N~IPOJ Sodium phosphate NAD Dihydronicotinamide adenine dinucleotide (oxidized) NADH Dihydronicotinamide adenine dinucleotide (reduced) om 2'-0-methyladenosine 02 oxygen Yo Percent PBS Phosphate buffered saline PIA R(-)N~-(~henyl-2~-iso~ro~~l)-adenosine PIN pinacidil PIP= Phosphatidyl4,5-biphosphate Negative logarithm of hydrogen ion concentration PKC Protein kinase C PMlA Phorbol 1 Zmyristate 13-acetate RNA Ribonucleic acid SAS Statistical Analysis Systems SEM Standard error of the mean SPT 8-p-sulphophenyl theophyiline Micro- (10") NOTE TO USER
Page(s) not included in the original manuscript are unavailable from the author or university. The manuscript was microfilmed as received.
xiv-xv
This is reproduction is the best copy available xvi
FIGURE LEGENDS
Figure 1: A: Schematic structure of adenosine combining a purine base and a ribose moiety. B: Schematic structure of adenosine triphosphate combining adenosine and three phosphate groups.
Figure 2: Adenosiue Metabolism. The cardiac adenosine system is comprised of three components; (1) formation; (2) receptor complex effects; and (3) degradation. 1- Adenosine (ADO) can be formed intracellularly via the adenosie triphosphate (ATP) or S-adenosylhomocyst eine (SAH) pathway, or extracellularly via breakdown of adenine nucleotides- 2 - The adenosine receptor (ADO-R) is coupled to ion channels via the guanine binding regulatory proteins (Gi). Theop hylline (THEO) derivatives act as competitive antagonists for the adenosine receptors. 3-ADO can be transported into the cell and then degraded via deamination to inosine or phosphorylated to adenosine monophosphate (AMP). Dipyridamole can block the cellular uptake of ADO, thus prolonging its effect. ADP=adenosine diphosphate; cAMP=cyclic AMP; GTP=guanosine triphosphate.
Figure 3: Purine Metabolism. Ado=adenosine; Hx=hypoxanthine; Ino-inosine; UA-+c acid. a=ATP consuming reactions; b=oxidative phosphorylation; c=myokinase; d=S- nudeotidase; e=AMP dearninase; +adenylosuccinate synthase and lyase; radenosine kinase; h=adenosine dearninase; Iyurine nucleoside phosphorylase; j=xanthine dehydrogenase; kguanine phosphoribosyl transferase; l=adenine phosphoribosyl transferase.
Figure 4: Summary of the adenosine-protein kinase C mechanism of ischemic preconditioning. Brief ischemia results in the degradation of adenosine triphosphate (ATP) through adenosine diphosphate (ADP) and adenosine monophosphate (AMP) to adenosine. Adenosine freely di&ses across the cell membrane to interact with surface adenosine receptors.(Al). Adenosine receptors are believed to be coupled to inhibitory guanosine triphosphate binding proteins (Gi proteins) consisting of a, b, and g subunits. The activated a subunit stimulates membrane bound phospholipase C (PLC) to convert membrane phosphatidylinositol biphosphate (PP2) to inositol triphosphate (P3) and diacylglycerol (DAG). D?3 induces internal mobilization of calcium stores from sites such as the sarcoplasmic reticulum (SR). As the intracellular calcium concentration rises, inactive cytosolic protein kinase C (PKCinact) translocates to cell membranes and is activated by DAG (PKCact). Activated PKC may now mediate the cardioprotective response through modulation of final effectoh such as ion channels, intermediary metabolic pathways, and gene expression, Figure 5: Simplified summary of the adenosine-protein kinase C mechanism of ischemic preconditioning. Brief ischemia results in the degradation of adenosine triphosphate (ATP) to form adenosine diphosphate (ADP), adenosine monophosphate (AMP) and adenosine. Adenosine dfises across the cell membrane to interact with extraceUuIar adenosine receptors (Al). Through a series of intermediary steps including G protein activation and hydrolysis of membrane phospholipids, protein kinase C (PKC) is activated. Activated PKC goes on to phosphorylate intra- or extracellular final eEectors thereby confemng protection.
Figure 6: Anoxic preconditioning (PCD) reduced cellular injury to a greater extent than did hypoxic preconditioning (PC 16) (+p<0.05). Both forms of preconditioning reduced cellular injury compared to ischemic controls (IC) (*p<0.05 vs. IC) (NIC: Non- ischemic controls)
Figure 7: @per Panel: Extracellular lactate levels were significantly elevated at 50 minutes in the anoxic preconditioning group (PCO), however not significantly. Extracellular lactate concentrations following both "ischemia" and "repefision" did not differ between groups. Lowerpanel: Intracellular ATP levels decreased significantly in the anoxic preconditioning group (PCO) in comparison to ischernic controls (IC; p Figure 8: Lower Panel: Preconditioning with the supernatant ofanoxically preconditioned cells (SUPO) reduced cellular injury to a greater extent than did preconditioning with thesupematant of hypoxically preconditioned cells (SUP 16)(p Figure 9: The protective effects of anoxically preconditioned supernatant (SUPO) were abolished when the non-preconditioned cells and the supernatant were first incubated with either SPT or adenosine deaminase (ADA) (NIC: Non-ischemic controls; IC: Ischemic controls) (*p<0.05 vs. SPT, ADA, and IC; px0.05 vs. SUPO, SPT, AD4 K)- Figure 10: Adenosine dose response curve: Adenosine displayed a 'U' shaped dose-response curve. Adenosine was most protective at a dose of 50 pmol. Figure 11: Upper Panel: Exogenous adenosine was most protective when administered at a dose of 50 umol prior to ischemia @RE). Application of adenosine during ischemia (ISCK) was protective to a significantly lesser degree. The two protective effects were not found to be additive when adenosine was administered continuously (CONTJX). Adenosine administered during reperfusion (REP) was not protective. All groups were compared to both ischemic controls (IC) and non-ischemic controls (MC). All protective effects were abolished when SPT was applied to adenosine treated cells, regardless of timing. Adenosine and SPT had no effect on non-ischemic controls (NIC). Lower Panel: Both PRE and CONTIN groups revealed a preservation of ATP following "ischemia" and "reperfhion" in comparison to ischemic controls (IC). The ISCH group revealed preservation of ATP to a lesser degree. Simultaneous administration of SPT abolished the ATP-preservative effects of adenosine. Adenosine applied during reperfusion did not afford ATP-preservative properties. Figure 12: Extracellular lactate concentrations following "ischemia" and "reperfbsion" (FINAL) were elevated in cells which received adenosine either continuously (CONTIN) or during repefision (REP)(*p Figure 13: The protective effects of preconditioning with either ischemia (PCO), adenosine @RE), or PMA (PMA) were abolished with the addition of Cal-C (+Cal-C) (*p<0.05 vs. NIC, IC). (Cal-C: Calphostin-C; A: Adenosine; NIC: Non-ischemic controls) Figure 14: Representative slot-blot analysis demonstrating isoforrn-specific translocation of PKC in cells exposed to 50 pmol of adenosine (Pretreatment), 100 pmol of adenosine, 50 polof adenosine with SPT, or 10 rn PMA. Results were compared to those of cells which underwent stabilization in normoxic PBS only WC). Densitometric analyses revealed no changes in PKC-aor PKC-E distributions with stabilization. Similarly, PKC-G distributions did not change with either adenosine or the phorbol ester PMA. However, there was a marked cytosolic to membrane translocation of PKC-ain cells exposed to 50 pmol of adenosine (Pretreatment) or PMA. Cells exposed to 100 pmol of adenosine prior to ischemia revealed a less marked translocation. Exposure of the cells to 50 pmol of adenosine with SPT (non-selective adenosine receptor antagonist) prevented differential translocation. Figure 15: Hypothetical membrane-folding models for potassium channel subunits of voltage- activated channels belonging to the S4 supe~arnily(A), inward rectifier channels (B), and minimal K+ (minK+) channels (C). Figure 16: Structural model for the predicted UOMK-1 channel protein. The proposed pore- forming P segment of ROMK- 1 (P) is located between membrane spanning segments M1 and M2; the H5 region is indicated. The MO segment is assumed not to span the membrane; as such, the N terminus is depicted in a cytoplasmic location. A single putative ATP-binding site identified by the PO Ioop is associated with a group ofbasic amino acids and potential phosphorylation sites and may form a domain directly involved in regulating channel opening. Potential phosphorylation sites for PKC and cyclic AMP dependent PKC (or PKA) are shown (p). Figure 17: Comparison of the amino acid sequences of human and mouse BRAmino acids are indicated in the single letter code. The amino acid residues of mouse BIR (mBIR) different fiom those of human BIR (hBLR) sequences are shown below that of hBIR. Predicted transmembrane (MI and M2) and pore (HS) segments are indicated. Potential cyclic AMP-dependent protein base phosphorylation sites and protein kinase C-dependent phosphorylation sites are indicated by * and #, respectively. Abbreviations for the amino acid residues are as follows: A, Ma; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr- Figure 18: Representative photomicrographs of primary cultures of human pediatric (A) and adult (B) ventricular cardiomyocytes.(200x magnification) Figure 19: Schematic diagram of simulated "ischemia7' and "repehsion" model. Culture dishes of human ventricular cardiomyocytes are placed in an air-tight plexiglass chamber. To ensure anoxic conditions, t 00% nitrogen (N2)gas bubbled through two oxygen traps is utilized to continuously flush the sealed chamber thereby displacing any ambient oxygen. Four culture dishes are placed in the chamber which is equipped with a central sampling dish to enable verification of anoxic conditions and to allow temperature monitoring with each ischemidrepefision experiment. Figure 20: K-ATP Channel Opener Studies: In study I), cells undenvent either anoxic (PCO), adenosine mediated (Pretreat), or pinacidil mediated (PIN) preconditioning for a period of 20 minutes prior to prolonged ischemia and repefision. In study 2), cells treated with various preconditioning stimuli (PCO, Pretreat, PIN) were pretreated, simultaneously treated, and post-treated with the K-ATP channel inhibitor glybenclamide (GLY). All groups were compared to non-ischemic controls (NIC), which underwent 190 minutes of stabilization, and ischemic controls (IC), which underwent 70 minutes of stabilization folIowed by prolonged ischemia (90 minutes) and repefision (30 minutes). To rule out the possibility of a direct injurious effect, glybenclamide was applied at varying doses during ischemia.(A=Adenosine) Figure 21: Light micrograph of cardiomyocytes stained with Trypan Blue. Lefl Panel: cardiomyocytes stabilized in phosphate-buffered saline for 30 rninutes show little evidence of cellular injury. Middle Panel: cardiomyocytes preconditioned with 20 minutes of "ischemia" followed by 20 minutes of ccrepefisionyyreveal relatively few injured cells (denoted by arrows) following prolonged "ischemia7' and "reperfUsion"Right Panel: non-preconditioned cardiomyocytes reveal large numbers of injured cells (denoted by arrows) following prolonged "ischemiay7 and "repefision". (200x magnification; scale bal-LOpm) Figure 22: A) Trypan blue dose response experiments revealed that 50 polwas the dose which provided maximal preconditioning when applied to human ventricular myocytes prior to prolonged ischemia and repefision. B) When applied at this dose, pinacidil afforded protection which was similar in magnitude to that observed with ischemic preconditioning (PCO),and greater in magnitude to that observed with adenosine pre- treatment (Pretreat). (NIC-Non-ischemic controls; CC=Ischernic controls) Figure 23: A) Comprison between groups revealed that cells which were treated with pinacidil (PDQ prior to prolonged ischemia and reperfhion experienced significant ATP presewative effects in comparison to ischemic controls (IC). This ATP preservative effect was similar in magnitude to that observed with adenosine pre-treatment (Pretreat). B) Unlike the case with anoxic preconditioning (PCO), ATP concentrations immediately following pinacidil treatment did not fall significantly in comparison to controk. (NIC=Non-ischemic controls; IC=Ischemic controls) Figure 24: A) Trypan Blue dose response experiments of glybenclamide (GLY) as a pinacidil (PIN) antagonist revealed that 20 nmol was the lowest dose with which significant anti-preconditioning effects were demonstrated. B) Glybenclamide applied at 20 nmol effectively abolished the protective effects of pinacidil, as well as those of anoxic preconditioning (PCO) and adenosine pre-treatment (Pretreat). (NIC=Non-ischemic controls; IC=Ischernic controls) Figure 25: Glybenclarnide (GLY) applied prior to, during and following preconditioning effectively abolished the ATP preservative effects of adenosine pre-treatment (Pretreat) and pinacidil (PIN). (Stab=Stabilization; Precond=Preconditioning; K=Ischemic controls) Figure 26: Diazoxide Studies: A) To determine the optimd dose of diazoxide (DZX), cells stabilized for a period of 30 minutes were treated with varying doses of diazoxide for a period of 20 minutes, followed by 20 minutes of pre-ischemic repefision, and prolonged ischemia and repefision. Certain plates were simultaneously treated with the K-ATP channel antagonist glybenclamide (GLY). All groups were compared to non-ischemic controls @TIC) which underwent 190 minutes of stabilization, and ischemic controls (IC) which underwent 70 minutes of stabilization followed by prolonged ischemia (90 minutes) and repefision (30 minutes). B) Trypan Blue dose- response experiments of diazoxide-mediated preconditioning revealed that 20 pmol was the dose which provided maxima1 preconditioning when applied to human ventricular myocytes prior to prolonged ischemia and reperfirsion. Figure 27: A) Comparison between groups revealed that cells which were treated with diazoxide @ZX) prior to prolonged ischemia and repefision experienced significant ATP preservative effects in comparison to ischemic controls (K). Glybenclamide (GLY) effectively abolished such ATP-preservative effects. B) Trypan Blue experiments revealed that glybenclamide applied at a dose of 20 mol effectively abolished the protective effects of diazoxide. fNIC=Non-ischemic controls) Figure 28: A) To determine the sequence of PKC in the preconditioning cascade, cells were treated with the PKC agonist PMA, with or without the K-ATP channel inhibitor glybenclamide (GLY). To confirm the role of K-ATP channel opening down-stream to PKC in the preconditioning cascade, cells preconditioned with anoxia (PCO), adenosine (Pretreat), PMA, pinacidil (PIN), or diazoxide (DZX) were simuItaneously treated with the PKC antagonist Calphostin-C (Cal-C). Comparison was made with ischemic and non-ischemic contro!~(IC, NIC). (see Figure 29); B) PMA partially reproduced the protective effects of anoxic preconditioning, adenosine pretreatment, pinacidiI and diazoxide. These protective effects were abolished with the addition of glybenclarnide (GEY). Figure 29: Inhibition of PKC activation/translocation with Cdphostin-C (Cal-C) abolished the protective effects of anoxic preconditioning (PCO), adenosine pre-treatment (Pretreat) and PMA, but not the protective effects of pinacidil (PIN) or diazoxide (DZX), suggesting a K-ATP channel opener effect which exists downstream to PKC activation/translocation in the preconditioning cascade. (NIC: Non-ischemic controls; IC: Ischemic controls) Figure 30: Representative slot-blot analysis demonstrating an isoform-specific translocation of PKC to mitochondrid membranes of human ventricular rnyocyt es preconditioned with either anoxia (PCO),adenosine (Pretreat), or PMA. Digitalized densitametric values are also shown. (NIC: Non-ischemic controls) Figure 3 1: Electron microscopy of human ventricular myocyte preconditioned with adenosine pre-treatment. IrnmunogoId localization revealed the intracellular localization of PKC-a to the surface of intracellular organelle resembling mitochondria. Figure 32: Analysis of respiratory chain enzymes (COX, Complexes I+m, Complexes IItIII) revealed an increase in enzymatic activity with anoxic preconditioning (PCO), adenosine pre-treatment (Pretreat) and diazoxide @ZX) in comparison to controls. Figure 33: Electron micrographs of human ventricular myocytes following: A) stabilization in phosphate buffered saline (non-ischemic controls), and B) treatment with 20 pnol of diazoxide. Mitochondria (denoted by arrows) of cells treated with diazoxide appear swollen in comparison to non-ischemic controls. (N=Nucleus) Figure 34: Schematic representation of mitochondrial erectron transport chain exhibiting the mitochondrid inner membrane, the mitochondria1 K-ATP channel, and the system of electron carriers (Complexes I, 11, 111, and IV). K-ATP channel opening leads to potassium (Kt) influx which facilitates forward flux of the electron transport chain secondary to either mitochondria1 matrix volume swelling or ionic equilibration. INTRODUCTION Despite tremendous advances in the treatment and prevention of cardiovascuIar disease, coronary artery disease remains the single leading cause of death in Canada. According to current estimates, more than 6 million Canadians suffer &om coronary artery disease. In Ontario done, coronq artery disease accounted for 13% of all hospital admissions and 18% of all inpatient resource utilization between 1996 and 1997.' Not surprisingly, coronary artery bypass graft surgery (CABG) has become the most commonly performed surgical procedure in North America, with current estimates projecting a doubling in the number of CABG procedures by the year 2018. In 199 1, the average rate of CABG in the province of Ontario was 75 per 100,000. By 1998, the rate increased to 99 per 100,000. A gradually aging population along with the continued success of surgical intervention has created a growing demand for coronary bypass surgery which will likely extend into the current millennium. The Problem The beneficial effects of contemporary coronary artery bypass surgery (CABG) are well documented. I-' In patients with left main coronary arterial obstruction, double vessel disease or triple vessel disease, coronary bypass surgery has been shown to significantly reduce mortality when compared to medical management alone?* In addition, surgical revascularization has been shown to be an effective means of relieving symptoms in cases where more conservative measures have been unsuc~essful.~Recently changing trends in the population at risk, however, have introduced new challenges for cardiac surgeons in their attempt to minimize perioperative morbidity and mortality. Longer average life spans and significant technological advances have made cardiac surgery accessible to individuals who were previously deemed inoperable, and have enabled previous CABG patients to return for second and third time revascularization procedures. This trend is likely to continue owing to a gradually aging population, with a progressively increasing average life span Moreover, patients who would have previously succumbed to a myocardial infarction are now surviving due to the success and growing availability of thrombolytic therapy. Since progression of disease is rarely halted, such surviving patients are likely to return for surgical management at higher risk due to more advanced and complex disease. Numerous studies have confirmed the growing numbers of such high-risk patients presenting for CABG. A review by Christakis and colleagues revealed a higher incidence of patients greater than 65 years of age, patients undergoing urgent surgery for unstable angina, and patients with preoperative ejection fractions of less than 40%~' Although operative mortality did not change significantly, the risk of non-fatal morbidity rose steadily, contributing to longer hospital stays and increased resource utilization. In a similar, more recent review by Maharajh and colleagues, elderly patients 075 years of age) undergoing CABG. experienced operative mortalities as high as 14%.1° in both studies, the most common factor contributing to death or increased duration of hospital stay was low output syndrome (LOS - the requirement in the intensive care unit for inotropic and/or mechanical support for greater than 30 minutes to maintain systolic blood pressure above 90 mmHg and a cardiac index greater than 2.1 ~/min/m~),which occurred in 494 patients (6.7%). Later studies revealed that in some high risk subgroups the risk of LOS approached 70%, and that patients who developed LOS had an operative mortality of 17%, whereas those who did not, had a mortality of 0.9%. 11*12 In the absence of intraoperative complications, postoperative LOS is the direct result of inadequate intraoperative myocardial protection. Not surprisingly, recent advances in cardiac surgery have centred upon improved methods of intraoperative cardioprotection in the hope of preventing postoperative ventricular dysfunction and improving overall outcome. To date, strategies aimed at minimizing the risks associated with coronary bypass surgery have almost exclusively involved manipulation of ischemia and repetfusion conditions. Parameters such as cardioplegic compositior?, temperature, and flow rate have been extensively evaluated in the hope of optimizing intraoperative myocardial protection. In the mid 1980's, a major innovation involved the conversion fiom unoxygenated crystalloid cardioplegic solutions to oxygenated blood cardiopiegia. ClinicaI studies revealed that blood cardioplegia enhanced aerobic metaboLsm, improved postoperative ventricular function, and reduced anaerobic lactate production.13 Further studies demonstrated the benefits of a terminal infusion of warm blood cardiopIegia in repleting myocardid ATP levels and increasing postoperative diastolic compliance.14 Later, tepid (29'~)cardioplegia was shown to prevent the potential hazards associated with normothermic or hypotherrnic cardioplegia by reducing lactate and acid production during cardioplegic arrest and improving postoperative ventricular Studies of combination antegrade and retrograde cardioplegia demonstrated a reduction in lactate production, a preservation of ATP stores and improved perfhion of the heart during cros~clam~.~~Recent studies involving variable flows revealed that a cardioplegic flow rate of 200 mL/rnin improved the washout of detrimental metabolites resulting in improved ventricular function. " Despite such advances, mechanical and metabolic dysfunction of the myocardium following coronary bypass surgery remains a frequently encountered complication. Such a reality has prompted clinicians and researchers alike to search for yet additional, less traditional methods of protecting the heart against the effects of ischemia and repefision. The Solution: Myocardial l+econdilioning Perhaps most intriguing in the realm of myocardial protection is the advent of myocardial preconditioning. Unlike previous approaches, the aim of preconditioning at its inception was to "condition" the heart in the hope of affording an increased tolerance to the effects of subsequent ischemia. Various endogenous and pharmacoIogic promoters of preconditioning have been identified, including nitric oxide," ~-ar~inine,"insuIiqlg monophosphoryl 1i~id-4~'opioids, 21.22 cat echo la mine^,^^^ angiotensin II,= and inhalational anaesthetics such as de~flurane.~~Physical stimuli have also been shown to facilitate preconditioning including thermal energy," systemic pressure, and hyperdynamic circuf ati~n.~' However, though numerous preconditioning stimuli have been proposed, ironically, none have been as successful or profound as that of ischemia itself. CHAPTER ONE= Knowledge to Date MY0cX.ItLlU.LPECONDITIONlNG: Historical fieniew The effects of ischemia on the heart have been studied for centuries. As early as 1698, a report by Chirac documented the depressant effects of coronary artery ligation on myocardial function in a canine In 1912, Herrick concluded that permanent coronary arterial occlusion resulted in myocardial infkr~tion.~~However, despite initial observations, researchers soon came to realize that myocardial ischemia resulted in varying degrees of injury based primarily upon the duration of the ischemic insult. Blumgart and colleagues revealed that coronary ligation of less than 20 minutes duration in anaesthetized dogs resulted in reversible injury which could take hours or days to resolve." Further studies would reveal that this reversible injury was associated with loss of tissue high energy phosphates and contractile dysfhction.32-34Braunwald and Honer would employ the term ccmyocardialstunning7' to describe this period of reversible hnctional abnormality, which was thought to be characterized by fiee radical mediated injury and defects in ionic homeostasis.35-37 Moreover, Reimer and colleagues demonstrated that the injurious effects of repeated exposures to short (10 minutes) episodes of ischemia were non- cumulative, and that ATP levels, although decreasing by 60% after the first episode, did not decrease with subsequent episodes.38 The finding that an ischemic stimulus need not necessarily result in injury introduced a significant chapter in the study of ischemia. However, it was not until the mid 1980's that the possible beneficial effects of ischemia were described. In 1986 Murry, Jennings an Reimer coined the tern "ischemic preconditioning", to describe what remains by far the most powerfbl endogenously mediated form of myocardial protection In their canine model, the degree of myocardial infarction produced by a 40 minute circumflex coronary arterial occlusion was reduced by 75% when the myocardium at risk had first been subjected to four 5 minute coronary occlusions, each separated by 5 minutes of reperhion. Further studies have revealed that this protective attribute may be indirectly af5orded to myocardial regions adjacent to the area at risk, thus suggesting the presence of a humoral mediator. Przyklenk and colleagues were able to demonstrate this feature by revealing a reduction in canine left anterior descending territory infarction following brief circumflex territory ischemia.40 Such findings seemed to suggest the presence of a humoral mediator which conferred the protective effects of ischemic preconditioning and determined its distribution. To date, the most commonly implicated mediator in this process has been adenosine: an endogenous nucleoside produced physioIogically in a variety of organ systems- The role of adenosine in myocardid preconditioning has become most intriguing to both scientists and clinicians alike, owing to its profound protective properties and possible impIications in clinical practice. ADENOSINE: Historical Overview It is now 83 years since the first recorded administration of adenosine in humans.41 Initial interest in the potential therapeutic uses of adenosine arose in 1929 following a landmark report by Drury and Szent-Gyorgyi describing the isolation of crystalline adenosine monophosphate (AMP) fiom extracts of ox heart muscle, as well as observations of the effects of AMP and adenosine on the heart and circulation of several mammalian species.42 The authors determined that the agonist activity of adenosine depended entirely on both the 6-amino group of the purine base as well as on the ribose moiety.(Figure 1) The rate of metabolism determined the duration of action. The electrophysiologicaI effects of adenosine were found to be sinus bradycardia and heart block, and predisposition to one or both of these effects was entirely species dependent. Despite the remarkable insights developed by these investigators, no mention was made of the role of adenosine in cardiovascular physiology. Two years following the initial findings of Dnuy and Szent-Gyorgyi, Lindner and Rigler crystallized adenosine from heart muscle extracts and confirmed its potent coronary vasodilatory properties in a number of species." Based on adenosine's existence in the heart and its vasoactive properties, Lidner and Rigler hypothesized that the main physiologicd role of adenosine was to regulate coronary blood flow in vivo. Unfortunately, this concept gained little support and the advent of adenosine quickly fell fiom the forefront of clinical research. With the exception of a small series of publications, 44-5 1 interest in the cardiovascular effects of adenosine languished for the next three decades. Modem adenosine research was not revived until the mid 19601s,when two landmark studies by Berm and Gerlach 5253 demonstrated the release of adenosine catabolites from ischemic or hypoxic heart muscle. Modifications of an enzymatic spectrophotometric assay made It possible to demonstrate that adenosine exists in normally oxygenated as well as ischemic heart rnu~cle.~' Further work attempted to document a relationship between cardiac oxygen consumption, interstitial adenosine concentrations and coronary blood flo~.'~-~~ - The discovery in 1970 of the adenosine-stimulated accumulation of adenosine 3',5'-cyclic monophosphate (CAMP) in brain sections and the specific antagonism of this effect by theophyuineS8was the first definitive evidence of the existence of specific adenosine receptors. Further studies would soon reveal receptor subtypes mediating either the stimulation or inhibition of adenylate cyclase, the rate limiting enzyme involved in the formation of CAMP.'^ Perhaps most sigdcant, however, was the discovery of adenosine receptors which were coupled to cardiac effectors other than adenylate cyclase, including G-proteins and potassium channels.60161 Adenosine has since reached the forefront of cardiovascular research due largely to increasing evidence suggesting its role in the regulation of normal cellular functions via control of both intra- and extracellular metabolic processes. ADENOSNE METABOLISM Endogenous Adenosine Production Adenosine is produced via the enzymatic hydrolysis of either of two ubiquitous substrates, adenosine monophosphate (AMP) or S-adenyl homocysteine (SAH).(Figure 2) I) Adenosinefiorn hydrolysis of AMP: Hydrolysis of 5'-AMP by 5'-nucleotidase accounts for the vast majority of adenosine production in heart muscle, liver, and blood leukocytes. Five'- nucleotidase is present in two forms: membrane bound (ecto-5'-nucleotidase) and free cytoplasmic (cytosolic-5'-nucleotidase), both of which are thought to contribute synergistically to adenosine production during myocardial i~chernia.~'AMP is derived from a number of intracellular sources, including cytosolic and mitochondria1 stores, as well as from extracellular sources of adenine nucleotides such as platelets and endothe~iurn.~~ Although a variety of factors combine to influence adenosine production by the heart, levels are primarily increased when myocardial oxygen demand exceeds supply, thus influencing cellular energy state.64 The metabolic pathway that generates adenosine from adenosine triphosphate (ATP) has two key linkages to celluiar energy state? First, the consumption of ATP determines the availability of ADP which subsequently undergoes dismutation by myokinase to form AMP, the immediate precursor of adenosine. Second, AMP exists at a branch point in the pathway of ATP degradation.(Figure 3) The cytosolic ATP potentid (the chemical potential that drives ATP-consuming reactions and regulates respiratory rate) mediates the catalytic activities of the two enzymes which degrade AMP to form adenosine, namely, 5'-nucleotidase and AMP deaminase? However, global oxygen deficit is not a necessary precondition for adenosine production. In fact, a significant amount of adenosine is produced during normo~ia.~~*~' Numerous studies have shown that cycIical flow through the microcirculation of the heart often produces spatial and temporal heterogeneity of tissue oxgenation despite nomoxic condition^.^'*^' Such small, local imbalances in oxygen supply and demand could collectively act as the stimulus for adenosine production in an organ which is otherwise well pefised and well oxygenated. Thus, global ischemia may act to enhance overall adenosine production. 11) Adenosinefi.om hydi-olysis of SAH: S-adenosylhomocysteine (SAH) is a byproduct of transmethylations in which S-adenosylmethionine (SAM) is the methyl donor!' SAH is hydrolyzed by SAH-hydrolase to adenosine and hornocysteine. In isolated perfused guinea pig hearts, the overall adenosine production rate has been reported to be very similar to the hydrolysis rate of SAW suggesting that during normoxia, adenosine is mostly produced from sAK.~O During ischemia and hypoxia, adenosine release increases approximately 50-fold, while the transmethylation rate increases only 1-5-fold. Although SAE3 contributes relatively little to overall adenosine production (maximum measurable levels are enough to account for the adenosine found in the cardiac interstitiurn only)," its additional role as an adenosine binding protein accounts for the large intracellular pool of adenosine in most tissues.Q73 In in-situ dog hearts, the intracellular compartment has been shown to account for greater than 90% of the total adenosine pool.74 Unlike the case with AMP, no correlation exists between the catalytic activity of SAH and cellular energy stzte, suggesting that SAH derived adenosine has no role in the metabolic regulation of coronary blood flow.6" m> Adenosine porn hydrolysis of exh.acellular adenine mcleotides. Adenosine Transport Adenosine crosses cell membranes by specific nucleoside transport (facilitated dfision) or via passive difision into cells.75-77 The carrier which mediates facilitated diffiision controls both the uptake and release of adenosine, and may transport other nucleosides which may in turn act as competitive inhibitors of adenosine transport?'* In dog hearts, this carrier has been shown to be particularly sensitive to structural modifications in the ribose moiety. 79 The ribo sides which are the best known inhibitors of adenosine transport include 6-S-(p-nitrobenzy1thio)guanosine (NBTGR) and 6-S-@-nitrobenzy1thio)inosine (M~MPR)." A number of drugs, including theophylline and caffene have also been shown to inhibit adenosine transport both in vitro and in vivO-8 1.82 The sensitivity of adenosine transport to inhibition varies among cell lines," between species," and between different cell types within the same organ (i-e. cardiomyocytes and endothelial cells within the heart).'' Although the intracellular catabolism of ATP generates extracellular adenosine, only a small portion of this intracellular adenosine is exported, the remainder being bound to SMor recycled to AMP via either adenosine kinase or purine salvage pathways.65-86-88 Extracellular adenosine, whether formed by catabolism of extracellular nucleotides or released &om within cells, is eEciendy sequestered by transport into endothelid cells on passage through myocardial capillary beds.8990 This process is saturatable, and is effectively inhibited by dipyridamole (Figure 2). Dipyridamole, commonly utilized for pharrnacologic cardiac stress testing, inhibits the transport of adenosine intracellularly, resulting in a net extraceuular accumulation of adenosine.'' In some species, sequestration is also accomplished by red blood cells.92 Adenosine catabolism Adenosine is cleared fiom tissue via phosphorylation to AMP by adenosine kinase, or via deamination to inosine by adenosine deaminase. Although extracellular adenosine deaminase does exist (ecto-adenosine deaminase), both enzymes are primarily located within the cell, thus necessitating transport intracellularly prior to degradation.63 The purine salvage pathway which can recycle hypoxanthine to W and AMP, ensures that purines are not irretrievably lost with adenosine deamination.(Figure 3) Although in the heart, exogenous adenosine is primarily sequestered by vascular endothelium for eventual incorporation into the cellular adenine nucleotide pool, this process is easily saturatable, allowing for maintenance of measurable extracellular concentrations. 899093 Similarly, although endogenous adenosine is largely categorized intracellularly (as is evident by the steady-state release of adenosine degradation products from the heart) a certain amount is exported or maintained extracellulady prior to degradation.88.94-97 In those organs which have been studied to date, much of the available adenosine deaminase exists within the endothelid cells of the appropriate vascular bed.98*g9 Moreover, studies of both rat and rabbit cardiornyocytes have shown little or no intracellular adenosine deaminase- Regulation of Interstitial Adenosine Concentrations Adenosine present within the interstitium of the heart represents the physiologically active fraction that is available to react with cell surface adenosine receptors. Adenosine enters the interstitium either by release from parenchymal cells or by ecto-phosphatase hydrolysis of extracellular adenine n~cleotides.~~Adenosine is removed from the interstitial compartment by uptake into parenchymal cells, by washout into the venous drainage and lymphatics, or via degradation by cell surface ecto-adenosine deaminase. Studies employing radio-labelled adenosine in isolated rat hearts have shown that no more than 15% of interstitial adenosine comes from endothelid cells, the remainder being released by cardiomyocytes.89,100 Adenosine Receptim Studies demonstrating the inhibitory eEect of theophylline on adenosine-stimulated accumulation of CAMP in brain sections provided the first definitive evidence for the existence of specific adenosine receptors.s8 Burnstock el al subdivided adenosine receptors into two types depending upon the natural ligands that they recognized: PI receptors recognize adenosine (and possibly AMP), and P2 receptors recognize ATP and AD~.'~'(~able1) Van Calker el al. further subdivided PI receptors into A1 and A2 varieties based upon their effects on adenylate cyclase, A1 being inhibitory and A2 being stimulat~r~.~~~(~abie2) Classification of adenosine receptors has since been accomplished based upon both pharmacologic and biochemical criteria. Isolation of the differing receptor subtypes was made possible by the discovery that adipocyte membranes contained only A1 receptors, while platelet membranes expressed only A2 receptors.'03 A~ receptors have been shown to have a very high af£inity for adenosine, requiring agonist concentrations in the nanornoIar range for activation. Although activation of this receptor has no effect on basal adenylate cyclase activity, activation inhibits the receptor mediated stimulation of this enzyme by alternate agonists. Conversely, A2 receptors are low affinity receptors having an affinity for adenosine approximately three orders of magnitude lower than that of A1 receptors.65 In vitro, adenosine has also been found to inhibit adenylate cyclase through a distinctive "P site". Although the P site has been described as a ligand binding peptide , the characteristics of this site make it quite different from a receptor in that inhibition is only seen under conditions where adenylate cyclase is fbnctionally uncoupled from its G protein.(see section on signal transd~ction)~' Whereas Al and A2 receptors are activated by nanomolar and micromolar concentrations of adenosine respectively, inhibition of the P site requires concentrations of adenosine in the micromolar to millimolar range. Selective antagonists to the P site remain unknown, and its physiological role remains undetermined. More recently, a newly identified adenosine receptor, the Aj subtype, was found to be expressed on both animal and human ventricular cardiomyocytes-104-106 In a cultured chicken ventricular myocyte model, the protective effects secondary to adenosine A3 receptor activation were found to exceed (in duration ) those related to Al or Az receptor activati~n.'~~The selective A3 receptor antagonist MRS 1191 caused a biphasic inhibition of the protective effects of anoxic preconditioning. When the Al receptor antagonist DPCPX was applied simuitaneously, the biphasic dose inhibition curve was converted to a monophasic curve. Thus, activation of both A1 and A3 receptors was required to mediate the cardioprotective effects of anoxic preconditioning. In the same model, cardiac atrial cells were found to lack native A3 receptors, possibly accounting for the shorter duration of cardioprotection following preconditioning.. However, when atrial cells were transfected with cDNA encoding the human adenosine A3 receptor, a prolonged duration of cardioprotection was demonstrated.lo5 In rabbits, selective activation of the adenosine A3-receptor reduced infarct size in a LangendodF model of myocardial ischemia.lo6 Furthermore, the degree of A3 dependent cardioprotection was similar to that provided by A1 receptor stimulation or ischemic preconditioning. Finally, in a model of superfused human atrial trabeculae exposed to ischemia and reperfusion, selective stimulation of both Al and A3 adenosine receptors conferred protection similar to that observed with ischemic preconditioning, as assessed via recovery of baseline contractile Thus, the cardiac adenosine A3 receptor is believed to mediate a sustained cardioprotective effect during prolonged ischemia and reperfusion, and may represent a new cardiac therapeutic target. CARDIOPROTECTWE PROPERTES OF ADENOSINE Various mechanisms have been proposed for the cardioprotective effects of adenosine, including: I) Coronary vasodilatation: Adenosine release by cardiomyocytes during episodes of ischemia or hypoxia may facilitate coronary vasodilatation via stimulation of A2 receptors. The resultant increase in myocardial pefision improves metabolic fbnction and thus, contractility; 107.108 2) Antiadrenergic effects: Adenosine may be released to counteract the stirnulatory effects of catechoIarnines on cardiac hction via A1 receptor stimulation and by inhibiting the release of noradrenaline ftom sympathetic nerves. The resultant decrease in myocardial oxygen demand may thereby confer protection; 109. I 10 3) Protection of endothelium: Adenosine inhibits neuuophil adherence to endothelid cells and prevents neutrophil release of oxygen-derived fkee radicals from neutrophils via A2 receptor stimulation;111.112 4) Prevention of rnicrovascuIar obstruction: Adenosine inhibits platelet aggregation and platelet adherence to endothelid cells via A2 receptor activation; 113,114 5) Increased energy stores: Adenosine may facilitate glucose uptake by cardiomyocytes and stimulate glycolysis (indirectly, by increasing glucose-6-phosphate levels), thereby promoting the production of high-energy phosphates. In addition, exogenous adenosine may replete energy stores by acting as a nucleoside substrate for the creation of AMP, ADP and ATP; 115,116 6) Neovascularization: Adenosine is believed to increase the proliferation of endothelid cells and to promote myocardial neovascularization under conditions of prolonged hypoxia. 'I7 However, notwithstanding the above hypotheses, by far the most commonly implicated cardioprotective role for adenosine has centered upon its involvement in the Ischemic Preconditioning cascade. Adenosine, believed to be a mediator of ischemic preconditioning, may reproduce the inherent cardioprotective effects of this remarkable phenomenon via an Al receptor mediated process. "(Figures 4, 5) Unfortunately, despite numerous investigations, the mechanism whereby adenosine achieves such cardioprotective effects remains undetermined. The following attempts to delineate the initial sequence of the adenosine-mediated preconditioning cascade, as will be hypothesized in this thesis. SIGNAL, TRANSDUCTION All living cells must possess the ability to interact with their surrounding environment. Such communication between extracellular and intracellular compartments requires an intricate transmembranous system by which cells can respond to or react to extracellular stimuli. One of the methods by which this can be achieved in eukaryotic organisms is via signal transduction. Through this phenomenon, extracellular signals represented by ligands either penetrate celI membranes or activate external membrane receptors. Stimulated receptors, in association with membrane bound transducers, may then activate intracellular effector mechanisms either directly or indirectly.11g By way of such transduction systems, extracellular ligands may act as cofactors for intracelIuIar enzymes. Adenylate Cyclase System The adenylate cyclase system was the first plasma membrane signaf transduction system to be characterized. 'Ig Its speciticity and simplicity provided an ideal mechanism for the actions of adenosine on its receptors. Not surprisingly, initial adenosine nomenclature was based solely upon the adenylate cyclase system. Burnstock and colleagues subdivided adenosine receptors into two types depending upon the natural ligands that they recognized, PIreceptors recojpkhg adenosine (and possibly AMP), and PZ receptors recognizing ATP and ADP. able 1) Van Caker fbrther subdivided PI receptors into At and Az varieties based upon their effects on adenylate cyclase, Al being inhibitory and A2 being stimulatory. '02(~able2) Adenylat e cyclase facilitates the production of cyclic AMP (CAMP) which acts as a second messenger to stimulate the release of calcium &om L-type channels. Such calcium release can potentiate ischemia- reperfhion injury by promoting the production of oxygen-free radicles. As such, any protective (or preconditioning) mechanism would act to prevent the production of CAMP. Indeed, Lochner and colleagues revealed that ischemic preconditioning prevented CAMP accumulation during sustained ischemia through an attenuation of the beta-adrenergic response of preconditioned hearts. This attenuated adrenergic response was believed to be secondary to increased phosphodiesterase activity.lzO However, in an earlier report by Sandhu and col1eagues involving in-vivo rabbit hearts, such findings were refuted. Although preconditioning was found to lower CAMP concentrations during sustained ischemia, this effect was not necessary for protection to be realized, since raising CAMP levels (using adenylate cyclase agonists) did not abolish protection. 12' Later studies would reveal the coupling of adenosine receptors to cellular effector systems other than CAMP, based upon differing transduction mechanisms. G Roteins Among the most highly characterized transduction mechanisms are the G proteins. These heterotrimeric proteins, so named because they bind guanine nucleotides, consist of a-,P-, and y- subunits and play a pivotal role in coupling cell surface receptors to one or more effector systerns.(Figure 4) This coupling effect can be characteristically blocked by ribosylation of G proteins with pertussis toxin. The a-,P-, and y- subunits differ corn one kind of G protein to another in molecular size and structure as well as in fi~nction.~'It has been suggested that the diversity of subunits may provide the means by which one kind of receptor is coupled to more than one kind of effector mechanism. 122-124 Most hnctional differences between G proteins seem to be dependent on the molecularly heterogeneous and hydrophilic a-subunits. For example, A1 receptors are believed to be coupled to inhibitory G proteins (Gi), thereby inhibiting adenylate cyclase activity, whereas A2 receptors are believed to be coupled to stimdatory G proteins (Gs), thereby stimulating adenylate cyclase activity.lu Conversely, the P- and y- subunits are more hydrophobic and tend to remain associated as a single P,y- complex after the dissociation of the a-subunit during signal transduction. The P,y- subunit also functions to anchor the a-subunit to the cell membrane. Although less diverse than the a-subunits, recent evidence suggests two types of P-, and two types of y- subunits, thus implying four possible kinds of P,y- complexes, each able to influence the selective coupling of a receptor to its effector. Thus, at the Ievel of G proteins there are a variety of means by which to confer selectivity on the transduction of a signal from a receptor to an end effector. Which, if any, of these factors regulate adenosine receptors is unknown. Moreover, animal data linking both ischemic and adenosine preconditioning to G protein stimulation is controversial. Thornton and colleagues reported that pretreatmeat of isolated perfised rabbit hearts with pertussis toxin blocked the protective effects of ischemic pre~onditioning.'~~However, studies using rat models have been more variable. Although Lasley et al reported that pertussis toxin blocked adenosine A1 mediated protection of the ischemic rat heart,'" Liu and colleagues demofistrated that preconditioning against infarction in the rat heart did not involve a pertussis toxin sensitive G protein.'28 Adenosine Effector Mechanisms Despite initial beliefs, little confirmatory data was available with which to support the adenylate cyclase hypothesis of preconditioning, and existing data was controversial. Although Szilvassy and colleagues demonstrated a reduction of adenylate cyclase activity in preconditioned rabbit hearts, both Iwase ef aZ and Fu ef a1 showed no effect on adenylate cyclase activity with preconditioning of rabbit and swine hearts, respectively. 129-131 Indeed, krther research into the mechanisms of signal transduction suggested that adenosine expressed its biological actions through effectors other than adenylate cyclase. The ability of G proteins to couple cell surface receptors to more than one effector mechanism supported such a possibility. Although most adenosine receptors acting via alternate effectors are of the A1 variety, it is not known whether this hnctional diversification reflects the coupling of a single kind of A1 receptor to different transduction mechanisms or whether there is molecular diversity of A1 receptors similar to that of other receptors. 65,132 The first example of an alternate mechanism, A1 receptors coupled to potassium channels, was discovered in cardiac tissue. 60.133 The following is a list of such alternate effectors: I) Al- POTASSIUM RECEPTOR: In the 1970fs, electrophysiological studies involving atrial cardiac tissue revealed that adenosine shortened cardiac action potential duration by facilitating an outward potassium conductance. Further studies employing patch clamping and protein modification techniques revealed that G proteins were involved in the signal transduction mechanism for this end effector.6 1.134 II) Al- GUANYLATE CYCLASE RECEPTOR: Adenosine promotes an accumulation of cGMP in cultures of aortic smooth muscle cells and stimulates a guanylate cyclase in partially purified plasma membranes from aortic media. 13' ID) Al- CALCIUM RECEPTOR: Several studies in nerve cells have shown adenosine receptors to be coupled to calcium channels via specific G proteins. 136,137 To date, however, no such receptor complexes have been identi£ied in the heart. IV) Al- GLUCOSE RECEPTOR: Adenosine has been shown to stimulate glucose transport in adipocytes independent of CAMP.'^' These receptors have also been demonstrated in myocardium, where adenosine facilitates insulin-stimulated glucose uptake, likely by increasing the Vmax of the glucose transporter. 139,140 Such a phenomenon suggests an effect of adenosine which is distal to the insulin receptor. In fact, insulin's effect on glucose transport may be dependent upon activation of the adenosine receptor.'*' V) AI- PHOSPHOLIPASE C+ and -41- PHOSPHOLIPASE C- RECEPTORS:Adenosine has an - indirect effect on the histamine H1 receptor-initiated hydrolysis of membrane inositol phospholipids by phospholipase C, and a direct effect on cellular free fatty acid production by p hospholipase-A2. 65.14 1 The coupling of A1 receptor stimulation to the hydrolysis of membrane phospholipids impIicates yet another possible adenosine-mediated transduction mechanism. In 1983, Streb and colleagues revealed that inositol- 1,4,5-triphosphate (IP,), a product of membrane phosphatidyl 4,s-biphosphate (PIP2) hydrolysis, was released into the cytoplasm, likely in response to extracellular receptor stimulation. The release of P3, in turn, resulted in the mobilization of ca2' from intracellular stores.'" The other product of PIPz hydrolysis, diacylglycerol (DAG), was found to remain within membranes, and to facilitate the activation of a specialized enzyme known as protein kinase C (PKC). '43 PROTERVR7NASE C The protein kinase C (PKC) family of enzymes transduces a number of signals, which promote lipid hydrolysis. The prevalence of PKC in cellular sigmlling is partially attributable to the diverse transduction mechanisms that result in the production of protein kinase C's primary activator, diacylglycerol.(Figure 4)lM Stimulators of G-protein-coupled receptors, tyrosine kinase receptors or non-receptor tyrosine kinases can promote DAG production either rapidly, by activation of phospholipid C, or more slowly, by activation of phospholipase D to yield phosphatidic acid and then diacylglycerol. PKC activity may be krther mediated by phospholipase AZdependent fatty acid generation. Phorbol esters, also known to be activators of PKC, result in prolonged activation due to their long half-life in vivo. Regardless of the method of activation, some PKC isozymes require ionic ca2' and all PKC isozymes require the cytoplasmic Iipid phosphatidylserine for their activation. All PKC isozymes have in common their single polypeptide structure consisting of an N- terminal regulatory region and a C-terminal catalytic region. After initial cloning of selective isozymes in the mid-1980ts, Coussens and colleagues reported the presence of four conserved domains, CI-~4.'"The C1 domain forms the diacylglycerollphorbol ester binding site and is immediately preceded by an auto inhibitory pseudosubstrate sequence.'46 The C2 domain forms the recognition site for acidic lipids, and in some isoqmes, the ca2+ binding site. Calcium increases the afkity of conventional protein kinase C for negatively charged lipids. The C3 and C4 domains represent the ATP and substrate binding sites of the base core.14' The regulatory and cataiytic halves are separated by a hinge that becomes proteolytically labile when the enryme is membrane bound, thus fieeing the kinase domain fiom inhibition by the pseudosubstrate and rendering the enzyme active. 14* To date, 11 PKC isozymes have been identified and are classified into three groups based on their structure and cofactor regulation.149 The a,P (variants I and II), and y isozymes were the first to be characterized and are distinguishable by their regulatoxy ca2' binding site. The next well characterized are the novel PKC isozymes which include 6,~,q,Qand p. These isozymes resemble the conventional PKCs with the exception of their C2 domain, which does not conth a ca2' binding site. The third group is comprised of the atypical isozymes 5 and h, which are the least well characterized. These isozymes differ significantly in structure fiom the more typical isozymes and are insensitive to phorbol esters both in vitro and in vivo. Protein kinase C typically p hosp horylates serine or threonine residues, however, displays far less specificity than other conventional kinases such as protein kinase A."' Moreover, unlike protein kinase 4 PKC has the ability to autophosphorylate in ~itro.~~'In addition to catalyzing phosphorylation reactions, PKC possesses both ATPase and phosphatase activities.1S2 Under resting conditions, PKC is present mainly within the cytoplasm in an inactive form. PKC is rendered catalytically competent by phosphorylations, which correctly align residues for catalysis. These same phosphoryiations localize protein base C to the cytoplasmic compartment.149 Stimulation of PKC is associated with removal of its pseudosubstrate fiom the kinase core rende~gthe enzyme active.''' Accompanying this activation is a rapid ca2' dependent translocation of PKC to membranes, possibly along cytoskeletai structures such as microt~bules."~This translocation has been shown to be stimulated by DAG and phorbol esters. Both DAG and phorbol esters act as hydrophobic anchors to attract protein kinase C to the membrane while increasing the enzyme's membrane a51.ity.'~' Thus, PKC is regulated via two distinct mechanisms: by phosphorylation which regulates the active site and subcellular localization of the enzyme, and by second messengers which promote PKC's membrane association and resulting pseudosubstrate exposure.149 Activation of PKC by A1receptor stimulation may occur not only through phospholipase C, but also through stimulation of phospholipase D (PLD). Phospholipase D forms phosphatidic acid fiom phosphatidyl chohe, and hydrolysis of phosphatidic acid by phosphatidic acid phosphohydrolase produces DAG. 156.157 This PLD pathway has a much higher capacity for DAG production than the PLC pathway because the content of its substrate (including phosphatidyl choline) in the sarcolernma far exceeds the concentrations of phosphatidyl inositol 4,s- biphosphate, the substrate for PLC. Cohen and colleagues demonstrated that both preconditioning and the adenosine receptor agonist PIA increased PLD activity in the rabbit heads8 Moreover, sodium oleate, a PLD activator, afforded significant protection to rabbit cardiomyocytes exposed to prolonged periods of ischemia. Such protective effects were abolished with the addition of high dose propranolol, which effectively antagonizes phosphatidic acid phosphohydro~ase. Such findings suggest that both PLC and PLD contribute to cardioprotection induced by adenosine receptors in preconditioning. How adenosine receptors couple to PLD, however, remains unclear. lS6 Involvement of Alternate Protein Kinases Although numerous investigators have confirmed the hypothesized involvement of PKC in the ischemic preconditioning cascade, more recent studies have questioned the necessity for PKC activation in different animal models. In studies involving swine hearts, Vahlhaus and colleagues fded to demonstrate inhibition of infarct reduction when preconditioned hearts were pre-treated with the PKC inhibitor ~tauroo~orine.'~~Similar findings were also found in canine models of ischemia and repefi~ion.'~~Miura and coworkers attempted to elucidate the reasons for such discrepancies in PKC studies. They demonstrated that the effects of PKC inhibitors differed depending upon the number of preconditioning episodes eli~ited.'~' While staurosporine and poi- B completeiy abolished the protective effects of single cycle preconditioning, they failed to modify repetitive preconditioning. Thus, a protective mechanism other than PKC may be elicited by repetitive cycles of preconditioning. Tyrosine kinase (TK) may be involved in such a phenomenon. To this end, Vahlhaus and colleagues demonstrated that, in contrast to their previous findings,L59the addition of genistein (a tyrosine kinase inhibitor) to staurosporine in preconditioned porcine hearts did indeed abolish ~ardio~rotection.'~~Such findings seemed to suggest that tyrosine kinase may be involved as yer another second messenger in the preconditioning cascade. Indeed, current evidence indicates that after stimulation by adenosine A1 receptor activation, PKC activates a tyrosine kinase which in turn induces cardioprotection through opening of ATP-mediated potassium channels. 16' Unfortunately, more than 1000 different tyrosine kinases have been identified to date, exhibiting different subcellular !ocalizat ions, different substrates, and differing biological fbnctions. Because of the lack of selective inhibitors, it is unknown which specific family of tyrosine kinases is involved in ischemic preconditioning. 164,165 In non-cardiac cells, the Src family of tyrosine kinases has been shown to serve as an end effector for PKC.'~~*~~~Using a the selective inhibitor lavendustin 4 Ping and colleagues were the first to demonstrate that ischemic preconditioning induces the selective activation of two members of the Src family of tyrosine kinases, Src and Lck, in the hearts of conscious rabbits? The activity of five other Src tyrosine kinases expressed in the rabbit heart (Fyn, Fgr, Yes, Lyn, and Blk) was not affected by preconditioning. Furthermore, ischemic preconditioning had no effect on the activity of epidermal growth factor receptor kinases. Various studies in non-cardiac cell types have reported the involvement of tyrosine kinases downstream to,I6' parallel to, or upstream to PKC.'~~Thus, Src tyrosine kinases, while necessary for preconditioning to occur, may act through independent signalling cascades. In an attempt to clarify this issue, Ping and colleagues endeavoured to determine the sequence by which PKC and SrcLck mediate cardioprotection during ischemic preconditioning.168 Their results demonstrated that the activation of PKC was fully manifest immediately following ischemic preconditioning and persisted for 30 minutes thereafter. Conversely, the activation of Src and Lck became hiIy manifest only 30 minutes after the ischemic stimulus of preconditioning. Thus, the activation of PKC preceded the activation of Src and paralleled the activation of Lkc, a pattern which supports the hypothesis that the recruitment of Src and Lck is a downstream event that follows the activation of PKC. Such results were supported by the finding that tyrosine kinase inhibition with lavendustin A did not prevent PKC activation, while the PKC antagonist chelerythrine effectively abolished both SrcLkc activation and preconditioning.'68 Taken together, these findings suggest that tyrosine kinases may provide the link between PKC and its final effector (Le. KAp). ZHE 'UU2MANYCON?VECTION Ischemic Preconditioning in Humans Although various authors have documented some form of preconditioning in humans, the degree of success is variable and results are somewhat controversid- Initial reports involved observations of patients undergoing cardiac stress testing. In 1980 Jaffe demonstrated a reduction in ST segment depression in patients who underwent two consecutive exercise tests separated by 30 minutes of walking and 20 minutes of rest.16' Similarly, Williams and colleagues demonstrated an increased exercise tolerance in patients undergoing the second of two consecutive periods of pacing-induced angina separated by 5 to 15 minutes of reperfi~ion."~Such findings led some clinicians to propose a possible cardioprotective effect of stable angina prior to infarction. Muller and colleagues reviewed 775 patients who received post-infarction reperfusion with either thrombolysis or angioplasty and found that those patients with previous chronic angina demonstrated lower reinfarction rates and lower in hospital mortality rates.17' Similarly, Kloner reported that patients who experienced angina within 48 hours of infarction displayed smaller infarct sizes, fewer complications of infarction, and lower in hospital mortality. - Subsequent studies would attempt to confirm the ability to precondition human myocardial tissue in vitro. To demonstrate preconditioning, Walker and colleagues utilized a model of isolated right atrial trabeculae exposed to a 90-minute episode of rapid atrial pacing in conjunction with hypoxic perfision (controls). Trabecular preparations exposed to a 3-minute episode of rapid pacing and hypoxic perfhion (followed by slow pacing and normoxic pefision) prior to the more prolonged episode (preconditioned group) demonstrated a preservation of developed pressure in comparison to controls.173 Human Cmdiomyofyre Studies of Endogenous eecondihbning Our group was the first to demonstrate the ability to precondition cultured human ventricular myocytes using simulated ischemia and reperfhion. 118,174 In preliminary graded preconditioning studies performed by the author, we revealed that hypoxic preconditioning for 20 minutes with a p02 = 20 m.g(PC20) significantly reduced the cellular injury associated with prolonged ischemia and repefision (Figure 6). Anoxic preconditioning for 20 minutes with a p02 = 0 mmHg (PCO) reduced cellular injury to a greater extent than did hypoxic preconditioning (PC20). ExtracelIuIar lactate concentrations in cells which underwent anoxic preconditioning (PCO) prior to prolonged ccischemiay'and "repefision" were elevated immediately following preconditioning, although no significant dBerences were found in comparison to ischemic controls during the same time periods (Figure 7, upper panel). Intracellular ATP levels decreased significantly in the PC0 group immediately following preconditioning. Although during ischemia, the reduction in ATP was less profound in the preconditioned group, no differences were found in ATP levels following prolonged "ischemiay' or ccrepefision"in comparison to ischemic controls (Figure 7, lower panel). In our supernatant preconditioning studies, preincubation with the supernatant of cells preconditioned using anoxic PBS (SUPO) significantly reduced cellular injury, however to an extent less than that seen with anoxic preconditioning (PCO) (Figure 8; lower panel). Our preliminary studies also involved the measurement of adenosine concentrations in supernatants of cells, which underwent either anoxic (pOz = 0 mmHg) or hypoxic @02= 20 mmHg) preconditioning. HPLC analysis revealed a grezter concentration of endogenous adenosine in the supernatant of anoxicdy preconditioned cells (SUPO) rather than hypoxically preconditioned cells (SUP20) (Figure 8; upper panel). Finally, in our adenosine receptor antagonist studies, cellular injury after prolonged ischemia and reperfhion was significantly reduced following pretreatment with the supernatant of anoxically preconditioned cells (SUPO). The protective effects of the anoxically preconditioned supernatant (SUPO) were abolished when the supernatant and the non-preconditioned cells were fist treated with the adenosine receptor antagonist (SPT) .(Figure 9) These studies were the first of their kind to demonstrate the protective effects of ischemic preconditioning on human ventricular myocytes undergoing ischemia and reperhion, and the role of adenosine in this process. Clinical Studies of Ischemic Preconditioning Although such impressive results have served to re-introduce the benefits of preconditioning into the forefront of modem cardiovascular research, the clinical applicability of ischemic preconditioning remains in question. Accordingly, clinicians have attempted to apply preconditioning to the clinical scenario via studies of intermittent balloon occlusion in angioplasty patients, studies of intermittent cross clamping in patients undergoing conventional CABG, and studies of intermittent coronary snaring in patients undergoing 'off-pump' coronary revascuIarization. Kerenslq et al. examined nine patients who experienced acute ST segment elevation during balloon angioplasty with complete resolution during the procedure. Seven of the nine had far less ST segment elevation with the second balloon inflation, suggesting that preconditioning had occurred.175 Similarly, Deutsch and colleagues demonstrated that in patients undergoing balloon angioplasty, the second of two consecutive 90 second balIoon occlusions was associated with less anginal discomfort, less ST segment depression, and a reduction in coronary sinus iactat e production. 17' Alkhulaifi and colleagues randomized twenty patients undergoing elective CABG to receive either two 3 minute crossclarnp periods (each followed by 2 minutes of reperfbsion) prior to prolonged ischemia (preconditioned group) or prolonged ischemia alone (control group). Intraoperative myocardial biopsies revealed a preservation of tissue ATP levels in the preconditioned group and a sigm6cant lowering of creatine phosphate levels.ln Similarly, in a randomized trial undertaken by Jenkins and colleagues, patients who received a preconditioning stimulus (in the form of intermittent cross clamping) prior to surgical coronary revascularization revealed decreased serum troponin levels in comparison to non-preconditioned patients. 17' Finally, Jacobsohn et al. reported their experiences with ischemic preconditioning in the setting of off-pump coronary bypass surgery.179 The authors employed intennittent brief periods of LAD occlusion to precondition myocardial regions at risk prior to prolonged occlusion which accompanied the 'of-pump' distal anastomosis. The protocol for ischemic preconditioning consisted of 3 minutes of ischemia (occlusion) and 3 minutes of repefision, followed by 5 minutes of ischemia and 5 minutes of repefision. Once preconditioning was completed, the LAD was occluded and the left internal mammary arterial anastomosis was pefiormed under direct vision. Ischemic time was approximately IS minutes while the anastomosis was being performed. Baseline serum, echocardiographic and pressure-volume loop assessments, performed after each episode of ischemic preconditioning as well as after anastornotic completion, revealed no evidence of ischemia with preserved functional in dice^."^ Despite such promising tindings, subsequent studies of preconditioning in humans would yield contradictory results. In a review of 4,447 patients who suffered myocardial infarction, Barbash reported that previous angina was associated with a higher in-hospital Similarly, several angioplasty studies failed to show any advantage to intermittent balloon occlusions for the purposes of preconditioning. 181.182 Moreover, initial favourable results were attributed to the recruitment of coronary arterial collaterds. Menasche et. al. reported that patients preconditioned with 3 minutes of cross clamping prior to institution of cardioplegia revealed increased levels of creatine kinase ME? and lactate release at the end of cardioplegic arrest. In addition, molecular biology data previously shown to be related to the preconditioning process (i-e. expression of m-RNA for both c-fos and heat shock protein 70) did not suggest a protective effect of preconditioning. Finally, in a cIinicaI trial involving patients undergoing off pump coronary bypass surgery, Mallcowski and colleagues found ischemic preconditioning to be detrimental to myocardial fbnction.lS4 A 5 minute LAD occlusion (preconditioning stimulus) was found to precipitate ischemic dysfUnction as evidenced by worsening left ventricular wall motion scores and increased pulmonary arterial pressures. Moreover, ischemic preconditioning did not afford protection against left ventricular systolic dysfhction during subsequent ischemic episodes. Nonetheless, despite such contradictory results, we viewed studies of ischemic preconditioning in humans to be somewhat promising. Taking these conflicting reports into account, we hypothesized that the observed variabiIity in clinicd and experimental outcomes was likely attributable to the variable consequences associated with the preconditioning stimulus itself, which, in this case, was ischemia. Ironically, the beneficial effects of ischemic preconditioning in humans may be masked by the detrimental effects of the initial brief ischemic insult. Thus, a pharmacological mediator that could harness the beneficial effects of preconditioning without the need for ischemia was essential in this regard. Reproducing Ischemic Preconditioning Various agents have been shown to reproduce, to some extent, the beneficial effects of ischemic preconditioning. Direct preconditioning via stimulation of opioid receptors has been demonstrated in both animal'85 and clinical experiments.lg6 Similarly, preconditioning-mimetic effects have been shown with norepinephrine administration prior to prolonged ischemia and reperfhion in isolated rat hearts'" or supefised rat trabec~lae.'~~Nitric oxide (NO) and the NO donor L-arginine have also been demonstrated to provide protection to isolated perfused rabbit hearts exposed to ischemia and repefi~ion.'~'Inhalational anaesthetics such as isoflurane and sevoflurane have been suggested to enhance the functional recovery of post-ischemic reperfused myocardium, possibly via activation of ATP-sensitive ion channels. 190.19 I Other agents which have been proposed to possess preconditioning effects include insulin,1g and monophosphoryl lipid 4Ig2 in addition to physical phenomenon such as thermal stimulationZ7 and hyperdynamic circu~ation.~~However, none of the aforementioned have been found to be as consistent or as effective in promoting myocardial protection as adenosine administration or upregulation. Adenosine, reIeased in sigdicant amounts during myocardial ischemia, represents an ideal mediator for the protective effects of ischemic preconditioning. Adenosine was first administered to humans in the early 1900's and continues to be utilized clinically as a first line antiarrhythmic agent. Recently, adenosine has reached the forefront of clinical research due largely to its presumed cardioprotective properties. EXOGENOUS ADENOS.ZNHU1)Z4NS PhysioIogic Effects The physiologic effects of adenosine are determined by the particular type of receptor present within the effector tissue.(Table 3) Adenosine Al receptors are present within the cardiomyocytes which mediate the sinus slowing and AV-blocking actions of adenosine. Conversely, Az receptors are found in both endothelial and vascular smooth muscle cells and stimulate coronary vasodilatation when activated.lg3 Although improving coronary blood flow, adenosine depresses myocardial hction acutely by reducing heart rate, slowing AV conduction, and antagonizing the inotropic effects of catecholamines. The resultant increase in oxygen supply and decrease in myocardial oxygen demand has been suggested as a mechanism for some of the cardioprotective effects of adenosine under normal physiologic conditions." EZectrophysiologic Effects Adenosine directly shortens the atrid action potential duration, and suppresses the automaticity of the SA node and other cardiac pacemakers, while slowing conduction through the AV node and prolonging refractorine~s.'~~In the ventricle, adenosine antagonizes the positive chronotropy, dromotropy and inotropy induced by circulating catech~larnines.~~~ Regulation of Coronary Blood Flmv Adenosine is a potent coronary vasodilator. Various studies, both animal and human, have shown that increased levels of adenosine are released during ischemia (likely due to degradation of myocyte high energy phosphates), possibly in an adaptive role to stimulate corresponding vasodilatation.~08.155 Although initial evidence was based largely upon measurements of adenosine breakdown products (due to the extremely short half-life of endogenous adenosine), recent technical advances have allowed for more direct methods of adenosine measurement. Fox and colleagues showed that in 13 of 15 patients undergoing cardiac catheterization, pacing-induced angina stimulated a ten-fold increase in coronary sinus adenosine levels-L96 The same group dso studied coronary sinus adenosine levels in patients undergoing cardiac surgery. Adenosine levels were found to be five times those of control levels during cardioplegic arredg7 Such reports were similar to results from earlier animal studies employing models of intermittent coronary occlusion.64,197 Ironically, adenosine administration as an intravenous bolus has been reported to induce angina-like chest pain. 198~'99 Although this pain has been shown to be cardiac in origin, studies have revealed no relation to coronary blood flow or myocardial ischemia.'" Huetnodynantic and Respiratory Effects Biaggioni and colleagues studied the physiologic effects of exogenous adenosine administered to healthy volunteers. Adenosine infused intravenously at a rate of 140 pg/kg/min increased heart rate and systolic blood pressure, but decreased diastolic blood pressure resulting in no change in the mean arterial pressure.200 Adenosine also induced a tachypnea which was not related to bronchoconstriction, hypoxia or hypotension. This respiratory stimulation resulted in a mild fall in PaCOz and a corresponding rise in pH. Conversely, studies by Verani et a[ using similar doses of adenosine in patients undergoing cardiac catheterization revealed a decrease in both systolic and diastolic blood pressure.201These findings were supported in studies by Smits et al, where adenosine was shown to induce a peripheral vas~dilatation.~~~Subsequent studies by Watt and colleagues revealed that both the cardiac and respiratory effects of adenosine were dose related, and resolved immediately following termination of the infusion.203 ADENOSm PRECOhWITIONI1VG IN HUMS Despite an abundance of research into adenosine and its presumed cardioprotective properties, until recently, much controversy existed with respect to its mechanisms of action and optimal mode of application. Moreover, littIe evidence was available fiom human models with which to confirm or refbte the vast amount of animal data. Human Cardiomyocyte Studies of Adenosine Preconditioning Our group was the first to describe the ability of exogenous adenosine to reproduce the protective effects of preconditioning in human ventricular myocytes 1 I8 174 . In preliminary studies, exogenous adenosine administration afforded significant protection against the injurious effects of ischemia and "repefision". Following a dose-response analysis based upon Trypan Blue assessments of injury (Figure lo), exogenous adenosine was found to be most protective at a dose of SO pmol. Adenosine lost its protective effects at doses equal to or above 100 pmol. At a dose of 50 pol, the greatest degree of protection was afforded when adenosine was applied prior to ischemia (Pretreatment) followed by pre-ischemic reperfusion. Application of adenosine during ischemia (Ischemic treatment) was protective to a lesser degree than was adenosine pretreatment. The two protective eEects were not found to be additive when adenosine was administered continuously (Continuous treatment). Adenosine applied during reperhsion (Reperhion treatment) was not protective (Figure I 1, upper panel). Additionally, we found that adenosine treatment resulted in a significant preservation of ATP following prolonged ischemia and reperfusion (Figure 11, lower panel). Comparison between groups revealed that cells which were pretreated with adenosine or continuously treated with adenosine demonstrated the greatest degree of ATP preservation following prolonged ischemia and repefision in comparison to ischemic controls (IC). Unlike the case with ischemic preconditioning, ATP concentrations immediately following adenosine pretreatment did not fall in comparison to controls. Application of adenosine during ischemia (Ischemic treatment) resulted in only partial preservation of ATP. These preservative effects were non-additive when adenosine was applied continuously (Continuous treatment). Adenosine applied during reperfusion (Reperfusion treatment) did not prevent the degradation of BTP. (Figure 1 1, lower panel) Further investigations revealed that in comparison to ischemic controls (IC), supernatant lactate concentrations following simulated ischemia and reperfhion ("Final" lactate) were elevated in groups treated with adenosine either continuously (Continrrous treatment) or during repefision (Reperkion treatment) (Figure 12). To determine the direct effects of adenosine on lactate production, supernatant lactate concentrations were measured either prior to ischemia, at the end of ischemia, or at the end of reperfusion, with and without adenosine treatment. Under such circumstances, supernatant lactate levels were found to be elevated in all groups immediately following adenosine treatment ("Post-Adenosine" lactate) in comparison to non-treatment controls. (Figure 12) To determine whether the protective effects of adenosine were receptor or substrate mediated, cells treated with adefiosine either prior to (Pretreatment) or during ischemia (Ischemic treatment) were simuitaneously exposed to the non-selective adenosine receptor antagonist SPT. SPT abolished the protective effects of adenosine as assessed by Trypan Blue exclusion and measurements of intracellular ATP concentrations. (Figure 1 1) Similarly, to determine if the lactate elevating effects of adenosine were receptor mediated, we measured extracellular lactate levels in cells which were simultaneously treated with adenosine and SPT prior to simulated ischemia (Pretreatrnent+SPT). Cells which were exposed to both adenosine and SPT revealed a sigdicant reduction in pre-ischemic lactate concentrations in comparison to cells pretreated with adenosine alone (Pretreatment). (Figure 12) Human Ccvciiornyocyte Studies of Protein Kinare C After determining that ischemic and adenosine preconditioning hnctioned via a receptor- mediated process, we endeavoured to iden* the underlying sequence of events which afforded cardioprotection following receptor activation- Extracellular receptors are often linked to intracellular effectors by a second messenger system. As noted above, protein kinase C (FKC) represents such a second messenger system. Various studies have suggested that PKC activation is necessary for preconditioning to take place. Ytrehus et a1 demonstrated that in isolated rabbit hearts, PKC stimulation using a phorbol ester reduced infarct size following 30 minutes of regional myocardial ischemia by 77%. Conversely, treatment of preconditioned hearts with the PKC inhibitors polymixin B or staurosporine abolished all protective effects204 Similar resdts were reported by Armstrong el al in cultured rabbit cardiomyocyte models of ischemia and repefision.205.206 Liu and colleagues reported that prevention of PKC translocation using colchicine in rabbit myocardium prevented preconditioning.207 Similarly, Speechly-Dick and colleagues demonstrated a reduction in infarct size when synthetic diacylglycerol analogues were administered to rats prior to prolonged myocardial ischemia. This protective effect was blocked when rat hearts were treated with the PKC antagonist chelerythrine immediately following the preconditioning stimulus.M8 Additional studies suggest a role for PKC in adenosine mediated preconditioning. Studies involving various tissues including thyroid, cerebral cortex, myometrium and collecting tubule have demonstrated that activation of adenosine Al receptors stimulates phospholipase C."' Kohl and colleagues demonstrated that administration of Al receptor agonists to left atrial and papillary muscle models of ischemia and reperfision increased IP3 concentrations and decreased PIP2 concentrati~ns.~~~Nonetheless, other studies have questioned the role of PKC in preconditioning. Studies by Ikeda and colleagues demonstrated increased cellular damage in comparison to controls when PMA was administered to hypoxic murine cardiac cells.z10 Similarly, in isolated perfbsed rat hearts, Yuan and colleagues showed that administration of PMA led to a dose dependent deterioration in contractile fbnction2'' Our group was the first to demonstrate the crucial role of PKC in human preconditioning using human ventricular rnyocytes. 1 I8174 In studies performed by the author, HVMs preconditioned with either anoxic PBS (PCO), PMA, or adenosine pretreatment (Pretreatment), were simultaneously exposed to the selective PKC antagonist Calphostin-C. Calphostin-C was found to abolish the protective effects of anoxic preconditioning (PCO), adenosine pretreatment, and PMA. (Figure 13) In subsequent experiments, both PKC translocation and activity were assayed. Figure 14 displays a representative slot blot analysis which shows an isoform specific translocation of PKC in cells exposed to 50 pol of adenosine (Pretreatment), 100 pnol of adenosine, 50 polof adenosine with SPT, or 10 nm PMA. Results were compared to those of cells which underwent stabilization in nomoxic PBS only @TIC). Densitometric analyses revealed no changes in PKC-a or PKC-E distributions with stabilization. Similarly, PKC-E distributions did not change with either adenosine or the phorbol ester PMA. However, there was a marked cytosolic to membrane translocation of PKC-ain cells exposed to 50 pmol of adenosine (Pretreatment) or PMA. Cells exposed to 100 pmol of adenosine prior to ischemia revealed a less marked translocation. Exposure of the cells to 50 pol of adenosine with SPT (non-selective adenosine receptor antagonist) prevented differential translocation. Digitized densitometry revealed the following membrane:cytosoIic ratios for each group: PM:4.93; 50 pmol adenosine: 6.4; 100 pzol adenosine: 1-85; 50 poiadenosine +100 pol SPT 2-27;MC: 2 -43. In concomitant studies, we measured the effect of 50 pmol of adenosine, 100 pol of adenosine, 50 pmol of adenosine with SPT, TO nrn PMA, or stabilization on total PKC activity. Although both PMA and 50 polof adenosine stimulated PKC activity, the effect of PMA was far more potent (PMA: 0.65+/-0.07; Pretreatment: 0.39+/-0.05; p In summary, our preliminary studies demonstrated that exogenous adenosine applied prior to ischemia with a pre-ischemic reperfusion period effectively reproduced the beneficial effects of preconditioning without the need for an ischemic stimulus. Unlike the case with ischemic preconditioning, adenosine was found to preserve intracellular ATP Ievels following ischemia and repefision in comparison to controls. In addition, adenosine was found to stimulate Iactate production during its application, both during ischemia and during normoxia. Finally, both ischemic preconditioning and adenosine preconditioning were found to be mediated by isoform- specific PKC translocation and activation. Clinical Studies of Adenosine Preconditioning Adenosine pretreutmenr @re-ischemic treatmenr) Kerenslq and colleagues examined nine patients who experienced acute ST segment elevation during balloon angioplasty with complete resolution during the procedure. Seven of the nine had far less ST segment elevation with the second balloon inflation, suggesting that preconditioning had ~ccurred."~In eleven other patients where adenosine was administered into the coronary arteries prior to the first balloon inflation, the amount of ischemia coted during the first inflation was reduced in only one patient during the second inflation. This finding suggested that preconditioning had occurred prior to the first balloon inflation, likely due to the effects of adenosine. Lee and colleagues administered intravenous adenosine to elective CABG patients immediately prior to the initiation of cardiopulmonary bypass. Adenosine pretreated patients had improved cardiac indices and released less CPK during the fist 24 postoperative hours in comparison to Curdioplegic Adenosine treatment (ischemic treatment) In an open label pilot study conducted by Mentzer and colleagues at the University of Wisconsin, addition of exogenous adenosine to conventional coId hyperkalernic blood cardioplegia in patients undergoing coronary bypass surgery resulted in a significant reduction in the requirement for postoperative vasoactive drugs (personal communication). However, in a similar study performed at our institution, no such benefit could be dernon~trated.~" Two separate double-blind, randomized, placebo-controlled trials were performed in patients undergoing primary, isolated, non-emergent coronary bypass surgery. Patients were randomized to receive adenosine 15 urnol/L versus placebo in the first study (n=200), and adenosine 50 or 100 umol/L versus placebo in the second study (n=128). Adenosine was infbsed with both initial and final doses of warm antegrade blood cardioplegia. The dzerent study groups were comparable with respect to all preoperative clinical characteristics, angiographic findings, and intraoperative variables. In both trials 1 and 2, no differences were found between groups in the incidence of primary morbidity or mortality. Similarly, when both studies were combined there was no statistically significant evidence of any consistent treatment benefit with adenosine administration (Death: relative risk w]=1.02, 95% confidence interval [CI]=0.06-16.6; Myocardial infarction by CK-MB: [RR]=0.84, [CI]=0.54- 1.31; Q-wave myocardial infaction: [Ra=1.30, [CI]=0.41-4.13; Myocardial infarction by troponin-T: [RRl=0.7, [CI]=O.40- 1.2 1; Inotrope requirement: [RR]=0.9, [CI]=O.46- 1-79; Low output syndrome: w]=1.38, [CI]=0.29-6.42; any of the above: [RR]=0.98, [CI]=0.78-1.25; p>0.02) We endeavoured to hrther explore the possible protective effects of adenosine, and to determine the optimal mode of administration of exogenous adenosine, but initiating a phase II prospective evaluation in patients undergoing elective coronary bypass surgery (CABG) .214(~ppendix1) Since adenosine' s protective effects were hypothesized to be both receptor and substrate mediated, and since late benefits could be related to a fiee radical- scavenging pathway, the effects of exogenous adenosine administration were evaluated both prior to and during the ischemic crossclamp period, as well as during reperfusion. In comparison to controls where tissue ATP levels decreased by 15% during crossclamp, tissue ATP levels were preserved in both the low dose and high dose adenosine groups with crossclamping.(Appendk 1) Moreover, as was demonstrated in our cellular studies, patients receiving adenosine tended to produce more lactate during the pre- and early XCL periods in comparison to controls. However, no obvious metabolic or hernodynamic differences were noted between groups following XCL removal, and no significant clinical benefit could be attributed to adenosine administration. Adenosine post-treatment (reperfision Peutrnent) In a study by Houltz and colieagues, the effects of a post-bypass adenosine ifision on central hemodynamics, ST segment changes, and systolic and diastolic hnction, were investigated in 20 CABG patients. Adenosine caused a dose-dependent increase in heart rate, cardiac output and stroke volume with no changes in cardiac filling pressures. The mean ST segments were slightly but sipficantly depressed by adenosine. Analysis of left ventricular wall motion showed no differences in comparison to controls.21s Similarly, Owall and colleagues administered a non- hypotensive dose of adenosine to 16 CABG patients for 4 hours following arrival to the intensive care unit. Although adenosine increased heart rate and cardiac index, and decreased systemic vascular resistance, no differences were noted in ventricular hction when compared to controls.216 Continuous adenosine treatment Acadesine (5-amino-l-[beta-D-ribo~ranosyl]imidazole-4-carboxde)is a purine nucleoside analogue belonging to a new class of agents termed adenosine regulating agents. Acadesine has been shown to increase the availability of adenosine locally to ischemic tissues- En a multicentre prospective randomized trial, acadesine was administered to 633 patients undergoing CABG by intravenous ifision starting 15 minutes before anaesthetic induction and continuing for 7 hours, as well as added to the cardioplegic solution. Although the incidence of myocardial infarction by prespecified criteria was not dierent between groups, a post-hoc subgroup vlalysis using a more specified definition of myocardial infarction revealed a Iower incidence of MI and a lower incidence of adverse cardiovascular outcomes in patients who received the higher of two doses (0.1 mgkghin). Moreover, in patients with Q-wave myocardial infarction, the high-dose acadesine group had a lower peak median CKMB and area under the CKMB curve.217 Finally, in a multi-centre double blind, placebo controlled trial performed by Mentzer and colleagues,218patients receiving high dose adenosine both as an intravenous infusion prior to and following aortic crossclamp and as a cardioplegic infusion during crossclamp, demonstrated a trend towards decreased high dose dopamine requirement and decreased myocardial infarction. A composite outcome analysis demonstrated that patients who received high-dose adenosine were less likely to experience one of five adverse events including high dose dopamine use, epinephrine use, insertion of intra-aortic balloon pump, myocardial infarction and death. Table 2. Atiribrrtes of A, nnd A, adenosine receptors and of the P site Action on adenylate cyclase I Inhibit I Stimulate I Inhibit Location in cell 1 surface I surface I Interior Molecular mass of ligand- binding peptide, kDa Alkylxanthine inhibition Yes Yes No GTP dependence Yes Yes No Transduction protein Gi Gs None Toxin for NAD' ribosylation PTX CTX None of G protein Gi, inhibitory G protein; Gs, stirnulatory G protein; PTX, toxin of BordetelZa pertrissis; CTX, toxin of Vibrio cholerne, Table 3. Cnrniac effects of adenosine A, Receptor Effects Direct Decrease SA node automaticity Decrease AV node conduction Decrease atrial contractility Decrease atria1 action potential duration Suppress norepinephrine release Indirect Attenuate chronotropic, dromotropic, and inotropic effects of catecholamines Suppress catecholamine-induced triggered ventricular afterpotentials A, Receptor Erects Vasodilation Decrease blood pressure ?A, or A, Receptor Effects Increase ventilation Cause chest ~ain/discomfort - Purine Base Y Ribose Moiety Figure 1. A: Schematic structure of Adenosine combining a purine base and a ribose moiety- B: Schematic structure of Adenosine Triphosphate combining adenosine and three phosphate groups. :nine Nucl eo ti des ATP a ADP AMP AMPa } Homocysteine + ADO n channel Inosine lE(+ channel GTP G i Figure 2. Adenosine Metabolism. The cardiac adenosine system is comprised of three components; (1) formation; (2) receptor complex effects; and (3) degradation. 1 - Adenosine (ADO) can be formed intracellularly via the adenosine triphosphate (ATP) or S-adenosylhomocysteine (SAH) pathway, or extracellularly via breakdown of adenine nucleotides. 2 - The adenosine receptor (ADO- R) is coupled to ion channels via the guanine binding regulatory proteins (Gi). Theophylline (THEO) derivatives act as competitive antagonists for the adenosine receptors. 3 - ADO can be transported into the cell and then degraded via deamination to inosine or phosphorylated to adenosine monophosphate (AMP). Dipyridamole can block the cellular uptake of ADO, thus prolonging its effect. ADP=adenosine diphosphate; cAMP=cyclic AMP; GTP=guanosine triphosphate. ATP a ADP synthesis 3 C exogenous I )AMP <=> IMP adenine f 0 b Ado :=> no,=> Hx ,=> UA Figure 3. Purine Metabolism. Ado=adenosine; Hx=hypoxanthine; Ino=inosine; UA=uric acid. a=ATP consuming reactions; b-oxidative phosphorylation; c=myokinase; d=S-nucleotidase; e=AMP deaminase; f=adenylosuccinate synthase and lyase; g=adenosine kinase; h=adenosine dearninase; i=purine nucleoside phosphorylase; j-anthine dehydrogenase; kguanine phosphoribosyl transferase; kadenine phosphoribosyl transferase. Figure 4. Summary of the adenosine-protein kinase C mechanism ofischemic preconditioning. Briefischemia results in the degradation of adenosine triphosphate (ATP) through adenosine diphosphate (ADP) and adenosine monophosphate (AMP) to adenosine. Adenosine freely diffuses across the cell membrane to interact with surface adenosine receptors.(AI). Adenosine receptors are believed to be coupled to inhibitory guanosine triphosphate binding proteins (Gi proteins) consisting of a, P, and y subunits. The activated a subunit stimulates membrane bound phospholipase C (PLC)to convert membrane phosphatidylinositol biphosphate (PIP2) to inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 induces internal mobilization of calcium stores from sites such as the sarcoplasmic reticulum (SR). As the i ntracellular calcium concentration rises, inactive cytosolic protein kinase C (PKCinact) translocates to cell membranes and is activated by DAG (PKCact). Activated PKC may now mediate the cardioprotective response through modulation of final effector/s such as ion channels, intermediary metabolic pathways, and gene expression. Final Effector AMP C ADP 4 Figure 5. Simplified summary of the adenosine-protein base C mechanism of ischemic preconditioning. Brief ischemia results in the degradation of adenosine triphosphate (ATP)to form adenosine diphosphate (ADP), adenosine monophosphate (AMP)and adenosine. Adenosine diffi~sesacross the cell membrane to interact with extracellular adenosine receptors (Al). Through a series of intermediary steps including G protein activation and hydrolysis of membrane phospholipids, protein kinase C (FKC) is activated. Activated PKC goes on to phosphorylate intra- or extracellular final effectors therebye conferring protection. * p4l.05 vs. IC, MC + p<0.05 vs. PC16 Figure 6: Anoxic preconditioning (KO) reduced cellular injury to a greater extent than did hypoxic preconditioning (PC16) (+p<0.05). Both forms of preconditioning reduced cellular injury compared to ischemic controls (IC) (*p<0.05 vs. IC). (NIC: Non-ischemicControls). -k ~~0.05vs. Stabilization vs. IC @J 50 rnin, vs. PC0 and IC @ Rep * Jt: pC0.05 vs. PC0 @ SO rnin, I, R vs- IC @ I, R + p Stabilization Ischemia Reperfusior Figure 7: Upperpanel: Extracellular lactate levels were significantly elevated at 50 minutes in the anoxic preconditioning group (PCO), however not significantly. Extracellular lactate concentrations following both "ischemia" and "reperfision" did not differ between groups. Lower pane[: Intracellular ATP levels decreased significantly in the anoxic preconditioning group (PCO) in comparison to ischemic controls (IC; p<0.05) ('AV debt'). However intracellular ATP levels following both "ischemia" and "reperfusion" did not differ between groups. * p=0.018 vs. SUP16 .k: pcO.05 vs. NIC, IC I SUP0 SUP16 Figure 8: Lower Panel: Preconditioning with the supernatant of anoxically preconditioned cells (SUPO) reduced cellular injury to a greater extent than did preconditioning with the supernatant of hypoxically preconditioned cells (SUP 16)@<0.05). Both forms of supernatant preconditioning significantly reduced cellular injury compared to ischemic controls (IC) @<0.05) (NIC: Non-ischemicControls). Upper Panel: HPLC analysis revealed a greater concentration of endogenous adenosine in the supernatant of anoxically preconditioned cells (SUPO)(p=0.018, SUPO vs. SW16). The supernatant of cells which underwent stabilization only revealed the lowest endogenous adenosine concentrations. - * p<0.05 vs. SPT, ADA, IC + p<0.05 vs. SZIW, SIT, ADA, IC 1 NIC SUP0 SPT ADA 1% Figure 9: The protective effects of anoxically preconditioned supernatant (SUPO) were abolished when the non-preconditioned cells and the supernatant were first incubated with either SPT or adenosine deaminase (ADA) (NIC: Non-ischemic controls; IC: Ischemic controls) (*p<0.05 vs. SPT, ADA, and IC; i-pC0.05 vs. SUPO, SPT, ADA, IC). * pc0.05 vs, 1/10/100/500 umol, NIC,IC I NK 1umol 10 umol 50 umol 100 umoC 500 umol IC Figure 10: Adenosine Dose-Response Curve: Adenosine displayed a 'U' shaped dose-response. Adenosine was most protective at a dose of 50 umol (NIC: Non-ischemic controls; IC: Ischemic controls) (*p<0.05 vs. all remaining doses, IC, NIC). * rsO.05 vs. REP, IC + pe0.05 vs. ISCH # p + p4.05 vs. PRE,ISCH, REP, CONTIN, IC .k p<0.05 vs. ECH, REP, IC # p4.05 vs. REP, IC I & p4.05 vs. SPT * PRE ISCH REP CONTIN IC Figure 11: Upper Panel: Exogenous adenosine was most protective when administered at a dose of 50 umol prior to ischemia (PRE). Application of adenosine during ischemia (ISCH) was protective to a significantly lesser degree. The two protective effects were not found to be additive when adenosine was administered continuously (CONTIN). Adenosine administered during reperfusion (REP) was not protective. All groups were compared to both ischemic controls (IC) and non-ischemic controls (NIC). All protective effects were abolished when SPT was applied to adenosine treated cells, regardless of timing. Adenosine and SPT had no effect on non-ischemic controls (MC). Lower Panel: Both PRE and CONTIN groups revealed a preservation of ATP following "ischemia" and "reperfusion" in comparison to ischemic controls OC). The ISCH group revealed preservation of ATP to a lesser degree. Simultaneous administration of SPT abolished the ATP-preservative effects of adenosine. Adenosine applied during repefision did not afford ATP-preservative properties. I .FINAL [7 POST-ADENOSINE CONTROL B ADENOSINE+SPT I * e.05 vs. hlC, PRE, ISCH, IC + m.05 vs corresponding CONT # ~4.05vs. corresponding POST-ADENOSmE NIC PRE ISCH REP CONTIN IC Figure 12: Extracellular lactate concentrations following "ischemia" and "reperfusion" (FINAL) were elevated in cells which received adenosine either continuously (CONTIN) or during reperfbsion (REP)(*p Figure 13: The protective effects of preconditioning with either ischemia (PCO), adenosine (PRE), or PMA (PMA) were abolished with the addition ofCal-C (+Cal-C) (*p 20 uM Adenosine 0 - - sp-[' PKC - E PKC - U. Figure 14: Representative slot-blot analysis demonstrating isoform-specific translocation of PKC in cells exposed to 50 mmol of adenosine (Pretreatment), 100 mmol of adenosine, 50 mmol of adenosine with SPT, or 10 nrn PMA. Results were compared to those of cells which underwent stabilization in norrnoxic PBS only (NIC).Densitometric analyses revealed no changes in PKC-a or PKC-e distributions with stabilization .Similarly, PKC-e distributions did not change with either adenosine or the phorbol ester PMA. However, there was a marked cytosolic to membrane translocation of PKC-a in cells exposed to 50 mmol of adenosine (Pretreatment) or PMA.Cells exposed to 100 mmol of adenosine prior to ischemia revealed a less marked translocation. Exposure of the cells to 50 mmol of adenosine with SPT (non-selective adenosine receptor antagonist) prevented differential translocation. UNDERLHiVG IMECEL4NISMS OF PREC0N.ITION.G Despite the vast amount of experimental data suggesting the protective effects of adenosine during myocardial ischemia, certain limitations exist, not the least of which are protective effects which fall short of those observed with experimental ischemic preconditioning. Elucidation of a final effector which, when stimulated, affords the protective effects of preconditioning may preclude such limitations. Various final effector mechanisms have been proposed in the past including, heat-shock protein stimulation,219,220 upregulation of anti-oxidant defences,22 1,222 alteration of prostaglandin and inflammatory mediator production,2u~n4promotion of intermediary metabolism 116,225,226 and ion channel modulation.227-229 Unfortunately, none of the aforementioned mechanisms have been conclusively shown to be involved in human preconditioning. In view of the ATP preservative effects of adenosine-mediated preconditioning, we believed that a final effector would more than likely involve a mediator tied to the metabolic status of the cell. The ATF-mediated potassium channel channel) may represent such a find efector. CARDUC POTUSIUM CII;QMVELS Knowledge to Date Ion channels exist as hndarnental units in all excitable and non-excitable cells, partaking in cellular signal transduction and in the maintenance of ionic homeostasis. The ion channels which mediate cardiomyocyte excitation consist of high molecular weight proteins spanning the hydrophobic lipid bi-layer of the cell membrane. Aqueous pores within the channel protein are believed to provide pathways of low resistance through which ions travel passively along electrochemical gradients. Preferential binding sights across the various channel proteins, in addition to pore breadth allow for selectivity of channels for particular ions. Cardiac ion channels are classified according to their inherent gating mechanism. Although not mutually exclusive, three superfdes are known to exist: (1) voltage gated ion channels; (2) ligand gated ion channels; and (3) stretch activated ion channe~s."~ Voltage gated channels represent the largest group among the three families. In these channels, conformational changes of charged regions allow for channel gating in response to changes in membrane potential.u1 Alternately, ligands such as neurotransmitters, intracellular kinases, adenosine triphosphate (ATP), and calcium ions may interact directly with membrane bound receptors to open or close channels. Finally, physical factors such as pressure and stretch are capable of gating specific ion channels through various second messenger pathways.232 Perhaps most diverse among the differing groups of ion channels are the potassium (Kt) selective channels. The regulation of plasma membrane permeability to K+ ions is a ubiquitous phenomenon common to all eukaryotic cells. Such differential permeability is intimately involved in the regulation of numerous cellular knctions and determines various properties including action potential firing and cell v~lume."~ Until recently, knowledge of potassium channels was quite limited . However, modem recombinant DNA techniques have succeeded in unravelling both the functional and structural diversification of these channels. An understanding of the molecular basis for this diversity came in 1977, when Jan et. al. described an interesting finding in hit flies of the species Drosophila melmogarter, which would later prove to be instrumental in the development of a genetic clone for the potassium channel.u4 Despite exposure to ether anaesthesia, "Shaker Mutant" fruit flies were found to persistently shake their hind extremities. Upon isolation of Shaker nerve fibres, electrophysiological analysis demonstrated broadened action potenti Js and multiple firings. This feature would later be attributed to a defect in Shaker potassium channels which inhibited conductance, and thus membrane repolarization.z5 Subsequent voltage clamp and recombinant DNA techniques revealed that the Shaker gene coded for an "A-type" channel component homologous to that of mammalian potassium channel^.^' Purification and isolation of the potassium channel was subsequently achieved by Rehm and Lazdunski with the aid of dendrototoxin, a snake venom which has been found to selectively bind such channels.u6 By applying the classical techniques of protein biochemistry along with the relatively new techniques of molecular biology, the A-type potassium channel was further characterized as a fast activating outward channel responsible fcr early repolarization in the majority of fast repetitively spiking cells. In 1988, Pongs et al utilized amino acid sequence data derived fiom cDNA clones to propose a model for the potassium channel. According to this model, the transmembrane potassium channel protein consisted of a hydrophobic core surrounded by hydrophilic domains at the carboxy and amino termini.u7 The transmembrane pore region was proposed to consist of several peptides with hydrophobic side chains. These peptides were oriented in such a way as to form membrane spanning alpha helices, each being 22 amino acid residues in length. The pore itself was hydrophilic and lined by the polar faces of several of these alpha helices .237 Early classifications of Kt channels were based upon the number of potential membrane spanning segments within the channel protein. As such, three original classes were shown to exist. Most prevalent among the three were the voltage dependent K+ channels, formed by the association of four subunits, each consisting of 500-1000 amino acids. This configuration resulted in six putative membrane spanning segments (S 1-S6). Alternatively, the inward rectifier K+ channels were believed to be composed of subunits containing two transmembrane complexes (Ml,M2), while the minimal K+ (minK+) channels, the simplest among the three, were believed to consist of a single polypeptide domain traversing the membrane once.@igure 15)=Oa3 Additional classes of K+ channels, many of which share various structural features with the above classes, have since been identified within the heart based on differing biophysical, physiological and pharmacological properties. Among these are the outward rectifymg, transient outward, calcium activated, sodium activated, ATP-dependent, acetylcholine activated, and arachidonic acid activated potassium channels.238 ATP-DEPENDENT POTASSIUM (GTp)CtL4NNELS Overview Perhaps most intriguing among the various potassium channels are the adenosine triphosphate (ATP)-dependent potassium channels (KfAn)- The uniqueness of these channels stems from their ability to couple cellular metabolic energy state to membrane potential. The advent of patch voltage-clamping and the abiIity to isolate individual cardiomyocytes in cell culture has done much to enhance our understanding of the ionic events underlying normal and abnormal cardiac function. Using such methods, acute myocardial ischemia has been shown to involve a period of rapid transition Erom normal electrical function to a period of metabolic and ionic instability resulting in a significant decline in contractile function and an increased susceptibility to arrhythmogenic activity.u8 The most notable features during this period include action potential shortening and extracellular potassium ion accurnulati~n.~~ The shortening of the cardiac action potential secondary to myocardial metabolic depression has been well documented for years. Since the early 19501s,this phenomenon has been attributed to activation of a large time independent outward potassium current."' The exact mechanism responsible for this effect remained elusive until 1983, when Noma et al, reported the presence of specialized K+ channels in porcine cardiomyocytes, which were primarily regulated by the intracellular concentration of ATP."~ However, it was not until 1992 that the role of the KtAv channel in myocardial ischemia would be definitively elucidated through the pioneering work of G.J. Gross and J-A ~ucham~ach.~~ These "ATP dependent" potassium channels (K+A~)quickly gained attention due to their unique ability to regulate membrane excitability based on cellular bioenergetic and metabolic states. Subsequent research would identify such channels in pancreatic Beta cells,240neurons, skeletal muscle cells,"' vascular and non-vascular smooth muscle cells,242and pituitary tissue.'" Although CATP channels have long been observed in humansY2"most of the data concerning this channel has been derived from animal models involving a variety of species including ratsYug mice,"5 guinea pigs,245rabbitsYzd6 and dogs."' The Search for a KCATp Clone Following Noma's initial discovery in 1983, isolation of a K+ATPchannel clone remained elusive for some time. Although Ashcroft was able to demonstrate the expression of K+ATP channels in Xenopus oocytes injected with rn-RNA fi-om rodent insulinoma cells, a clone could not be established.248 Unfortunately, an early account by M.L.J. Ashford and colleagues claiming to have isolated such a clone from rat hearts (Kir 3.4) was subsequently retracted when it was determined that Kir 3.4 was in fact an intrinsic component of the channel underlying acetylcholine mediated potassium channeIs, rather than a component of KTATP-' 249,250 In 1993, Ho colleagues reported the deduced amino acid sequence and structural features of a novel K+ channel polypeptide (ROMK-I) isolated and cloned fiom mammalian kidney cells.251 When expressed in Xenops oocytes, this protein gave rise to an inwardly rectifying K+ channei sensitive to intracellular ATP. The hypothesized pore forming segment (P) of ROMK-1 was suggested to be located between the two membrane spanning complexes typical for inward rectifiers (Ml, MZ), and was found to be homologous to the pore forming H5 region of voltage gated K+ channels. The regulatory domain for channel opening involves a single ATP binding site in addition to several potential phosphorylation sites for protein kinase C or protein kinase A.(Figure 16) Several inward rectifier K+ channels, including a cardiac KtAm channel (cKAm-I), have since been identiiied based on corresponding biophysical properties. Expression in human tissues, however, was only recently achieved. In a recent report, Inagaki and colleagues described the isolation of a clone representing a novel member of the inward rectifier K+ channel family (Gn-l) corn a murine pancreatic cDNA library.u2 This channel, composed of 390 amino acid residues, has been shown to close in response to 1 rnM ATP, and to open in response to the KrA~p agonist, diazoxide. Northern blot analysis has shown this channel to be expressed ubiquitously in murine tissues, including pancreatic islets, pituitary, skeletal muscle and heart cells. Subsequent screening of human genomic libraries revealed four clones encoding a distinct protein similar to that of designated BIR.~~~urther studies would reveal 71 percent identity of BIR (82% similarity) with murine uKCATp1, and 4 1 to 44 percent identity with ROMK- 1 and cKtAIP 1, respectively.(Figure 17) Furthermore, northern analysis would show BIR messenger RNA to be expressed in large amounts in murine pancreatic islets and glucose-responsive insulin-secreting cell lines. In view of its close association with KCAIP channels in all tissues, the authors attempted to isolate the sulfonylurea receptor (SUN) as a possible link in the elucidation of a human KATe channel clone. In fact, co-expression of BW with SUN reconstituted an inwardly rectirjlng potassium current which was sensitive to ATP, inhibited by sulfonylureas, and activated by dia~oxide.~"Finally, gene mapping analyses revealed that the genes coding for BIR and SUN were found in close proximity on human chromosome 11 at position 11~15.1.~~~These studies would suggest that human pancreatic and cardiac KCApchannels are a complex consisting of at least two subunits; namely BIR and SUN. Such intensive efforts are presently continuing in the hope of isolating a monoclonal antibody to the human K+AP channel. PhysioIogrgrcal l+opertrrtres Although KfAWcharnels have been shown to exist in a variety of cell types, it is only in the pancreatic Beta cell that their true physiological si@cance is apparent. Small variations in extracelluIar glucose concentrations have been shown to lead to changes in intracellular ATPIADP ratios which, in turn., mediate the closing of KfATPchannels. This closure facilitates action potential firing, allowing calcium entry and thus, insulin secreti~n.~~Conversely, such K+A~channels are activated by polypeptide hormones including somatostatin and galanin, both of which are known to inhibit insulin secretion- Such hormonal regulation is mediated via G-protein activation, in contrast to pharmacological channel opening (ie. diazoxide, pinacidil) and closing (ie. glybenclamide), which is mediated via direct receptor activation.z54255 Similarly, p horbol esters and diacylglycerols can either activate or inhibit KTAn channels via protein kinase C rnediati~n?~ Adenohypophyseal cells possess K+ATPchannels that appear to share similar properties to those of Beta cells, such that experimental regulation of such substances as growth hormone and prolactin can be achieved via KtAvopening or closing.z6 Distribution and Density The ubiquitous nature of the KrAn channel remains intriguing in view of its vastly differing fimctions in various cell types. Early reports, however, made use of channels isolated fiom the studied rodent pancreatic Beta cell to uncover basic channel properties common to all cell types. With the advent of patch voltage clamping, investigators have been able to estimate the density of K+ATPchannels by measuring the increase in Beta ceIl conductance (G,) following complete removal of inhibitory intracellular ATP.*' After applying various channel properties to the equation: N = G, /gumma X P,,, where N is the number of channels per cell, gamma is the single channel conductance, and P, is the open probability of the channel, a value of N corresponding to 5000 - 10,000 channels per cell was obtained. When myocyte cell diameter was accounted for, values corresponding to 0.5-1 channel per urn2 were obtained for cardiac in comparison to 10-20 channels per urn2 in both Beta and skeletal muscle cells.240 Bicrpliysical Properties Single KtAm channel conductance is most precisely measured using excised patches of cells where the membrane potential and surrounding ionic concentrations can be easily measured and manipulated. When such cell patches are bathed in a solution containing concentrations of potassium of approximately 150 mM, current flow is found to be inward with conductance values (gamma) of 80 picosiemens @S) in cardiac cells,z8 and 50-60 pS in Beta ce~ls.*~ This conductance is quite large in comparison to the conductances of other cardiac potassium channels and can be effectively blocked by high internal concentrations of sodium, calcium and magnesium, all of which are able to bind to sites within the K*AIP channel itself.260.261 upon exposure to physiologic potassium gradients ([K'i]=150 rnM, G]=5 mM) and in the absence of internal ~a',Mg", and Ca*, an outward current is recorded with a conductance of 27 pS. Thus, during the physiological range of membrane potentials, the KeA~pchannel behaves as an outward At systolic membrane potentials, however, this conductance diminishes due to the voltage dependent blockade of the channel by intracellular Na' and divalent cations.262 The relative permeability of the KCATPchannel to sodium (PNa'/PK+) is quite low regardless of cell type, ranging fiom 0.007 in the Beta cell, to 0.0 15 in fiog sarcolern~na.~~~In the cardiac myocyte, the single chamel permeability to K' (Ph) approaches 1-4 X 10-" cm3/sec at physiologic concentration^.^^^ Higher PL+levels are recorded with low external concentrations of potassium.263 The cell membrane's preferential permeability to K' ions accounts for a resting membrane potential which approximates the equilibrium potential of potassium (-70 mV). The dependent nature of KtAn channel permeability has been substantiated by various studies. In 1987, Spruce et-al revealed that KfAp saturates with increasing concentrations of external potassium.263 This finding supported the hypothesis that conductance was limited by ion binding to sites with in the channel protein itself. More recent studies have shown that KATPis likely a multi-ion pore with binding sites to several cations including K', Na', Mg", and ~b-230.265 Binding sites with affinity to K' at concentrations of 290 rnM and 85 mM have been proposed in Beta cells.26s The latter, higher affinity binding site is postulated to lie closer to the cytoplasm while the former is situated near the extracellular surface. This finding may represent a protective feature which controls channel permeability to potassium at non physiologic concentrations. Voltage Dependence Whole cell experiments have indicated that ATP-sensitive potassium currents are voltage independent and that channel openings are grouped in bursts.u5 Further analysis of cardiac myocyte membrane patches has revealed that for high extracellular concentrations of potassium g>60 rnM) and at a potential of approximately -70 mV, the mean lifetime of channel closures within bursts ranges from 0.3 to 0.7 msec in all cell types.266 Conversely, mean open times have been shown to range between 1.8 and 2.8 msec. Such studies have also determined that the gating of channels is dictated by electromotive forces (M. vs. [K2i) rather than by absolute membrane potential. Channel opening is maximal at potassium equilibration and decreases again at more positive voltages. The opposite is true for channel clo~ure.~~ ATP Modulation of rATp ATP regulation of KtATP channels involves two separate mechanisms. Although the presence of ATP along the cytoplasmic aspect of myocyte cell membranes markedly decreases the probability of CAPchannel opening, ATP dependent channel phosphorylation is necessary in order to maintain channel activity. Conversely, the inhibitory effect of ATP does not require phosphorylation, as has been seen with non-hydrolysabk ATP analogues which have aIso been shown to inhibit channel activity.267 Although removal of ATP activates (opens) KIAp channels within seconds, patch clamp studies have revealed that this activity gradually decreases with time. This "run-down" effect can be reversed by exposure to exttacellular Magnesium-ATP ("refreshment" effect).268.269 Alternate studies involving non-hydrolysable analogues of ATP and Mg"-free ATP are unable to reverse the run-down effect, thus suggesting a magnesiumlphosphorylation dependent phenomenon.270 This may be accomplished by channel protein phosphorylation, possibly via a protein-kinase C mediated pathway.27'" In fact, studies involving pancreatic Beta cells have hypothesized that channel activation is induced by K+Am phosphorylation secondary to diacylglycerol mediated activation of PKC.~~ Application of ATP to cardiomyocyte membrane patches at concentrations as low as 100 pM permits only 50% of maximal K+- channel opening."* Millimolar concentrations of ATP will cause almost complete channel inhibition. When applied to whole cell experiments, 50% maximal inhibition is achieved at concentrations as high as 0.5 mM. The apparent discrepancy likely exists due to the presence of other mediators (ie. Mg2f/H+/Ca2+/ADP) in the whole cell model which act to inhibit KAV- Thus, other investigators have favoured the use of such preparations including cell permeabilization studies, where channel activity can be recorded without disrupting important links between membrane proteins and the cytoskeleton. In 1986, Dunne erd revealed that changes in the glycolytic cycle result in variations of the ATP/ADP ratio which are of greater magnitude than changes in intracellular ATP concentration^.^^^ Later investigations revealed that the ATP/ADP ratio, rather than ATP alone, was the important final mediator of channel activity.275 In fact, the sensitivity of these channels to ATP has been shown to be markedly reduced during metabolic stress. KCAwchannels are normally inhibited by ATP concentrations less than 500 p.M. However, during periods of metabolic poisoning or myocardial ischemia, intracellular levels of ATP seldom fall to concentrations below the miholar range.276 As such, the stimulus for channel opening under conditions of ischemia is unclear. One hypothesis involves the compartmentalization of ATP in myocytes such that during ischemia, only certain ATP pools which modulate KfAw channels are selectively decreased to sufficient levels during ischemia?* In support of this theory are reports that cardiac myocyte KfATPchannels are controlled preferentially by glycolytic rather than mitochondria1 ATI?.~~~This feature would explain the early phase of potassium loss which occurs during myocardial ischemia.239 An alternate hypothesis suggests the existence of a gradient between the mitochondria and cell wall such that during ischemia, ATP concentration is decreased at the site of the channel. Finally, during ischemic conditions, an increase in the levels of alternate channel effective metabolites (ADP, H+, lactate) may decrease channel sensitivity to ATP at a time when less ATP is available, while an increase in channel mediators (adenosine, activated G proteins, protein kinase AK) may directly facilitate channel opening.u8 It is unclear whether one or more molecules of ATP are required for channel modulation. Initial studies revealed Hill coefficients of 3-4 for channel inhibition as a function of ATP, thus implying multiple binding sites.Y5 However, more recent studies involving channel structure reveal that channel closure is stimulated by the association of one molecule of ATP with a single channel receptor.31,262 Although ATP metabolism is not required for channel inhibition, channel exposure to ATP metabolites has shown effects similar to those of ATP. Kakei et-al. reported a 50% blockade with ADP concentrations in the range of 0.8-2 m~.~~~Other purines such as GTP, ITP, and UTP have also displayed partial inhibitory properties at high concentration^.^^^ FATP Kinetics The "burst" theory of K'Ap kinetics suggests that channel openings are grouped in bursts. This theory proposes that the main effect of ATP is to decrease the number of channel openings per burst, and to increase the duration of the long closed periods (approx. 100 msec) between Short closed periods (approximately 0.3 msec) which correspond to closures within bursts, are shown to be only minimally affected by ATP. Other Intraceliular Modulators of AK+*T.p In contrast to the higher concentrations, lower concentrations of GTP, GDP, and other guanosine nucleotides have been shown to increase K4An channel activity.278 In fact, the guanosine nucleotides may link G-protein activation to KfATPchannel regulation. Similarly, both the reduced and the oxidized forms of the pyridine nucleotides NADP and NAD lead to channel activation at low concentrations (10- 100 CIM) aod channel inhibition at higher concentrations (>SO0 pM).267 Fluctuation in intraceUular pH has been shown to significantly affect KIA- channel activity. Experiments conducted using NH&I to induce a transient intracellular alkalosis have reported significant increases in channel activity.279-28 I Accordingly, removal of mCI(increase in m)results in channel inhibition. Other intraceflular cations including calcium and magnesium have also been shown to decrease the open state probability of K'Arp in the absence of ATP. 239,282 PharmacoIogical Modulution of ffAr KtA~pchannels are activated pharmacologicaIIy by K+AV channel openers (KCOs) (ie. pinacidil, cromakalim, nicorandil, aprikalim) and are, for the most part, inhibited by antidiabetic sulfonyIureas (ie. glybenclamide, tolbutamide).283.284 However, response to the various pharmacological agents remains highly dependent upon channel origin. In pancreatic Beta cells, the most potent KC0 has been shown to be diazoxide (effective at 10 pM; diazoxid~pinacidil>cromakalirn),whereas the most effective blocking agent is glybenclamide.ug Conversely, although cardiac KT~nchannels are similarly blocked by antidiabetic sulfonylureas, the finity for this class of drugs is much lower than in pancreatic Beta cells, and is markedly reduced during periods of metabolic stiess.285.286 Nonetheless, cardiac KCATPchannels have a greater sensitivity to ICCAnchannel openers, and are thus activated at much lower concentrations of these drugs than is the case for Beta cell channels. The most potent KCOs in cardiac cells (in order of potency) are crornakalirn, pinacidil, diazoxide, aprikalim and nicorandil. 239,287,288 Although diazoxide has been shown in some studies to be inhibitory in cardiac cells,2g9more recent reports seem to contradict this finding.290 Such results suggest that endocrine and cardiac - KfAp channels belong to different subtype^.^^ TUERAPEUTIC ZMPLICA~ONSin the HEART Although the bction of the cardiac KfArPchannel under baseline physiologic conditions remains unclear, substantial evidence has accumulated to suggest that opening these channels in the ischemic heart plays an important cardioprotective role, as assessed by fbnctional recovery of ischemic heart preparations and by the demonstration of reduced infarct size in intact in situ heard2' KCAmchannels have long been known to open in response to cell ischemia and metabolic inhibition (pharmacologic or otherwise). This activation catalyzes the outward transfer of K+ ions, partly accounting for the early potassium loss and extraceIIu1a.r potassium accumulation seen in the ischemic myocardium. The addition of this outward K+ current to the myocardial action potential facilitates acceleration of membrane repolarization and action potential shortening.29 1-293 Activation of less than 1% of the total myocyte K+ATPchannel conductance reduces action potential duration by at least one halfD4 Action potential (AP) shortening has served as the main basis for the hypothesized cardioprotective mechanisms inherent in RATpchannel opening. Its possible effects are twofold: 1. AP shortening limits calcium entry (which normally takes place during the plateau phase of the action potential) which serves to decrease the magnitude of twitch contractions, thereby conserving ATP; or 2. AP shortening markedly reduces the time for calcium influx via voltage-sensitive calcium channels and increases the time during which the sodium- calcium exchanger may operate in forward mode to extrude calcium. Inhibition of intracellular calcium accumulation during ischemia prevents cell injury or death secondary to activation of oxygen free radical production pathways, and preserves energy stores which would otherwise have been utilized to maintain normal calcium homeostasis. 228,262,295,296 Several lines of evidence exist to support the role of channel opening in cardioprotection during hypoxia and ischemia. Mcpherson et al showed that pharmacologic activation of KfAm channels during global ischemia in arterially pefised guinea pig right ventricular walls resulted in the preservation of high energy phosphates and the reduction of ischemic injury. This effect was abolished and ischemic injury was exacerbated with KCATP blockade.295 Similarly, Grover et a[ revealed preservation of high energy phosphates and contractile bction in isolated pehsed rat hearts treated with various KCOs prior to ischemia. This effect was abolished upon pretreatment with glybenclamide, suggesting a K+A~mediated More recently, Grover utilized a novel cardiac-selective KC0 (BMS- 180448) to afford protection to globally ischemic rat hearts, independent of coronary vasodilatation and without the hypotensive effects typical of conventional ~~0s.~~ In vivo evidence of KAIPmediated protection is largely based upon studies of myocardial stunning initiated by Gross and colleagues. In two separate studies, bimakalim and aprikalim were both shown to improve postischemic recovery of fbnction in stunned myocardium. As in previous studies, glybenclamide was shown to abolish the beneficid effects of aprikalim and to worsen the recovery of hnction with increasing doses. 228,299 Myocardial Infarctiun Initial reports of myocardial infarction in canine regional ischemia models revealed no benefit with exposure to pinacidil.u8'00 However, later investigators would note that these original studies were accompanied by sigdicant hypotension, reflex tachycardia, and decrease in cdateral bIood flow upon pinacidil administration, such that the lack of benefit was not surprising. More recent studies involving anaesthetized dogs have shown that when cromakalim, aprikaIim or pinacidil are administered centrally via intracoronary injection at specified doses, no hernodynamic instability is encountered, and a marked reduction in the extent of myocardial infarction is observed. Conversely, giybenclamide administration significantly increases the extent of infarction. 22830 1 Ischemia-Reperfusion Injury The opening of potassium channels with agents such as pinacidil and crornakalim has been reported to be protective against ischemia-reperfusion mediated cellular damage.= The KCOs aprikalim and bimakalirn have both been shown to attenuate neutrophil infiltration into non- infarcted border zones of ischemic canine myocardium, while nicorandil and bimakalim have been shown to inhibit superoxide production in in-vitro human and canine cardiomyocytes.228,296,302 Effects on Coronary Vascular Tone Several lines of evidence exist to suggest that KCAmchannel opening may also be involved in the mediation of coronary vasodilatation during hypoxia and ischemia by attenuating action potential firing in smooth muscle cells, thereby inhibiting vascular constri~tion.~~In a model of isoIated guinea pig hearts, Daut and colleagues were able to show a reversal of the usual fall in coronary vascular resistance typically seen upon exposure to hypoxic or ischemic conditions. The vasodilatory effects seen during hypoxia were mimicked by pre-ischemic administration of the KC0 crornakali~n.~~~Similarly, Samaha el al showed that infusion of glybenclamide into the coronary circulation of anaesthetized dogs and isolated pe&sed rabbit hearts led to a two-fold increase and a 67% increase in basal coronary vascular tone and tissue ischemia, respectively.304 Although such studies offer compelling evidence to support the role of smooth muscle KlAW channels in facilitating normal coronary resistance during ischemia and hypoxia, little is known regarding the nature of these channels and their relationship to cardiac K+ATP channels and myocardial protection. Myocardial Preconditioning Although, ischemic preconditioning has been shown to be an effective cardioprotective modality, its clinical applicability remains limited. Thus, investigators have long been searching for a final effector mechanism which would allow implementation of this valuable phenomenon in clinicd practice. The ATP-dependent potassium channel may provide the missing link which would enable this important phenomenon to take place. Adenosine, found to confer myocardial protection against ischemia through an as yet unknown mechanism, has been shown to open lKCAv channels in rat and rabbit heart preparations. 3OZJO6 Similarly, K+*p blockade has been demonstrated to abolish adenosine mediated cardioprotection in various animal models."' he mechanism for this effect is likely similar to that hypothesized for ischemic preconditioning. Adenosine receptor activation (possibly by adenosine released during ischemic preconditioning) leads to activation of the alpha unit of a membrane bound G protein. This unit stimulates membrane bound phospholipase C which converts membrane phosphatidyl inositol bip hosphate (PIP2) to inositol triphosphate (IP3) and diacylglycerol @AG). IP3 induces internal mobilization of calcium stores from sites such as the sarcoplasrnic reticulum (SR). As the intracellular calcium concentration rises, inactive cytosolic protein kinase C (PKC) translocates to cellular membranes and is activated by DAG. Finally, activated PKC mediates the cardioprotective response through modulation of a final effector (possibly ~'~~p).~~~(Fig~re4) This sequence of events is supported by recent studies describing the upregulation of K+*rn channels by G-proteins in guinea pig ventricular myocytes, and by similar accounts implicating adenosine AI receptors and protein kinase C in the regulation of KfATPmediated ischemic preconditioning in both canine and human models of paced right atrial trabeculae.309-3 11 Although such data are promising, animal studies of KIATP mediated preconditioning have been quite conflicting. In canine models, blockage of CAPchannels has been found to abolish the anti-infarct effect, but not the anti-arrhythm~ceffect seen with ischemic preconditioning.247.3 12 In rabbits, JCATPblockade decreased myocardial tolerance to ischemia but did not lead to an increase in infarct size.'* Finally, in a rat heart model, CAWblockade was not shown to prevent the infarct sparing or anti-arrhythrmc effects seen with ischemic preconditioning.313 Although such inconsistencies may not be generalizeable to the human scenario, few human accounts are available with which to prove or disprove the KtAn mediated preconditioning hypothesis. Role of Swcolemrnal KCATP Channels in l+econdinuning Despite the ability to reproduce preconditioning using KtATPchannel agonists, the precise mechanism by which KtAp channel opening affords protection remains elusive. Noma and colleagues originally hypothesized that opening of sarcolemmal KfAIPchannels led to a shortening of myocyte action potential duration (by accelerating repolarization), thereby inhibiting calcium entry via L-type channels.*2 Decreased calcium overload would afford protection by limiting the force and duration of myocyte contraction resulting in ATP preservation, and by preventing calcium mediated ischemia-reperfusion injury. Several reports have supported such a hypothesis in various models. Using patch clamping methods, Cole and colleagues demonstrated a profound shortening of action potential duration associated with improved recovery of ventricular finction when isolated guinea pig right ventricular wall preparations were treated with the KATPchannel opener pinacidil.296 This protective effect was abolished with the addition of glibenclamide which effectively inhibited action potential shortening. Similarly, in a canine model, Yao and colleagues found that the RAm channel opener bimakalim lowered the threshold for ischemic preconditioning while significantly shortening action potential duration during prolonged i~chemia.~l4 More recent studies have employed molecular biological techniques to support earlier reports. Jovanovic and colleagues conducted a number of elegant studies using RAm deficient COS-7 cells.3 15.3 16 When such cells were exposed to 3 minutes of chemical hypoxia using dinitrophenol, marked calcium loading was demonstrated (similar to that observed in non- preconditioned cardiomyocytes) irrespective of pinacidil administration. However, when subunits (SUR2A and Kir6.2) of the cardiac sarcolernmal K+ATPchannel were co-transfected into these cells using viral vectors, calcium loading was attenuated in the presence of pinacidil- Role of Mitochondrial RArpChannels in Preconditioning Despite such promising data, conflicting reports began to surface regarding the role of sarcolernmal KtAIP channels in preconditioning. In a study by Schultz et d., although ischemic preconditioning resulted in enhanced shortening of action potential duration in anaesthetized pigs, such shortening was unimpressive (-lo%), and seemed unlikely to account for the magnitude of cardioprotection observed."' Indeed, the first study to question the role of action potential duration in K*ATP mediated preconditioning was published by Yao and Gross in 1994.318 The authors found that in dogs, cardioprotection could be afforded with low dose bimakalim independent of action potential duration shortening. Subsequent studies by Grover et al. demonstrated no correlation between action potential duration and cardioprotection when dog hearts were treated with cromakalim in the presence of dofetilide (a class III anti-arrhythmic) to prevent action potential duration I.addition, studies similar to ours using isolated non-beating rabbit cardiornyocytes showed a role for K+~~~mediated cardioprotection in the absence of a cellular action potential.320 In Iight of such studies, and due to the metabolic effects of KfATP channel openers, sites of action other than the sarcolernmal membrane were proposed. By far the most commonly implicated site of action would prove to be the mitochondria. Inoue and colleagues were the first to establish the presence of ICCAIP channels in the inner 321 membranes of rat liver mitochondria using patch-clamp technology . Since mitochondria take up and extrude various inorganic and organic ions as well as larger substances such as proteins, the technique of patch cIamping provided real-time information on such transport and on energy transduction in oxidative phosphorylation. Using such technology, houe was able to demonstrate a resting mitochondrial inner membrane potential of -1 50 to -160 Further studies would reveal characteristics similar to those of sarcolemmal K+ATPchannels including reversible inhibition by ATP and glibenclamide. Paucek and colleagues would confirm such findings after isolation and partial purification of a I(ChTP channel from beef heart mitochondria." However, whereas sarcolemmal K+ATP channels were primarily involved in membrane based electrical activity, these rnitochondrial specific channels were found to be intimately involved in matrix volume control. The advent of mitochondrial specific KCATPchannel openers furthered such selective findings. In studies by Okuyama and colleagues, although cells transfected with SURZA and Kir6.2 revealed all the characteristics of native sarcolemmal KCApchanneIs, this channel was only weakly activated by nicorandil and was not activated by dia~oxide.~~Indeed, both agents would eventually be demonstrated to have mitochondrial selective properties.324,325 Evidence for the role of rnitochondrial KrATp channels in cardioprotection was first introduced by Garlid and co-workers in 1997.326,327 To determine whether mitochondrial K+ATP and sarcolemmal K+*- hmthe same cell differed pharmacologically, drug sensitivities of cardiac mitochondrial and sarcolemmal CATPfrom reconstituted bovine heart mitochondria were compared.328 According to Garlid's findings, mitochondrial K+Aphm heart and Liver tissue did not Wer si~cantlyin their drug sensitivities (&a values). Moreover, cardiac mitochondrial K+AWand cardiac sarcolemmal KrAn exhibited similar sensitivities to benzopyran derivatives. However, cardiac mitochondrid KCAwwas found to be approximately 2000 times more sensitive to diazoxide than cardiac sarcolemmal KrATp (i.e. diazoxide was found to open mitochondrial K+Aw channels with a KIDof 0.8 poVL, while only opening sarcolernmal KCAlPchannels at 800 POW-326,327 The low sensitivity of reconstituted cardiac sarcolemmal KCAp to diazoxide was entirely consistent with previously published reports.289 Subsequent studies would demonstrate a protective effect of diazoxide at concentrations (5-20 pmol/L) that would not open sarcoIemmal K+*- channels- Recent studies by Liu et and Sato et al.3ZIhave confirmed the selective nature of diazoxide and have suggested that 5-hydroxydecanoate (5-HD:previously believed to be non- specific) may be a selective mitochondrial K+AIP channel inhibitor. Such studies have also attempted to demonstrate a role for protein kinase C in mitochondrial KCA~- channel modulation. Indeed, Sato and colleagues demonstrated that diazoxide produced a marked increase in flavoprotein oxidation in isolated rabbit ventricular myocytes with no effect on I-K*Ap. The phorbol ester PM4 had no effect on flavoprotein fluorescence, but potentiated the effect of diazoxide on K+Apchannel activation.325,326 Both such effects were abolished with the addition of 5-HD. Finally, Gogelein and colleagues have recently described the pharmacology of a new cardioselective sarcolemmal KCAwchannel antagonist, HMR 1883, which has been shown not to block ischemic preconditioning in rabbit hearts at doses which block the shortening of the cardiac action Unfortunately, the mitochondrial K+ATP channel has not been cloned to date, thus Limiting more specific molecuIar bioIogica1 assessments. Intracellular coo- NH+ B) C) x3 Extracellular Intracellular NH+ COO- COO- Figure 15: Hypothetical membrane-folding models for potassium channel subunits for voltage-activated channels belonging to the S4 superfamily (A), inward rectifier channels (B), and minimal K+ (minK+) channels (C). (KukuZjan, et al. Am JPhysiol, 1995; 268) Figure 16: Structural model for the predicted ROMK 1 channel protein. The proposed pore-forming P segment of ROMK 1 (P) is located between membrane spanning segments M 1 and M2; the H5 region is indicated. The MO segment is assumed not to span the membrane; as such, the N terminus is depicted in a cytoplasmic location. A single putative ATP-binding site identified by the PO4 loop is associated with a group of basic amino acids and potential phosphorylation sites and may form a domain directly involved in regulating channel opening. Potential phosphorylation sites for protein kinase C (PKC) and CAMP-dependent PKC (or PKA) are shown (p). (Ho et al. Nature; 362:I993) # # hBIR MLSRKGIIPEEYVLTRLAEDPAEPRYRARQRRARFVSKKGNCWAHKNIREQGRFLQDVFTTLVDLKWPH mBIR T E hBIR mBIR hBIR mBIR hBIR mBIR # # hBIR LEIIVILEGWETTGITTQARTSYLADEILWGQRFVPIVAEEDGRYSVDYSKFGNTIKVPTPLCTARQLD mBIR # * hBIR EDHSLLEALTLASARGPLRKRSVPMAKAKPKFSISPDSLS 390 mBIR R D S AV . 390 Figure 17: Comparison of the amino acid sequences of human and mouse BIR. Amino acids are indicated in the single letter code. The amino acid residues of mouse BIR (mBIR) different from those of human BIR (hBIR) sequences are shown below that of hBIR. Predicted transmembrane (M 1 and M2) and pore (H5)segments are indicated. Potential cyclic AMP-dependent protein kinase phosphorylation sites and protein kinase C-dependent phoshporylation sites are indicated by * and #, respectively. Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.(253} SUMlMARY OF STUDY RATIONALE, HYPOTHESES AND OBJECTIVES Aortic crossclamping during coronary bypass surgery results in global myocardial ischemia.330 Although the detrimental effects of ischemia are lessened with cardioplegia, adenine nucleotides (ATP, ADP, and AMP) are degraded while being used to maintain myocyte integrity. The resulting nucleosides (including adenosine) wash out upon repefision, limiting nucleotide resynthesis and resulting in poor postischemic myocardial function. Accordingly, any efficient method of conserving ATP stores, repleting ATP stores, or facilitating purine synthesis would be both energetically and hnctionally advantageous. Ischemic preconditioning is by far the most powetful endogenously mediated form of myocardial protection known. Unfortunately, this phenomenon is both energetically costly and diEcult to apply clinically. Adenosine, although an intermediary in the ischemic preconditioning pathway, fails to completely harness the beneficial effects of ischemic preconditioning, and yields unwanted side effects which limit its clinical appIicability. Identification of a final effector in the human preconditioning pathway and elucidation of the sequence of events which Lead to its activation may enable successful reproduction of the preconditioning phenomenon within the clinical scenario. Our model of cultured human ventricular myocytes provides an optimal method of delineating both the sequence of events and the final effectods responsible for human preconditioning. We propose a series of experiments designed to test the folIowing hypotheses: 1. Endogenous ischemic preconditioning in human ventricular myocytes is mediated by a second messenger pathway, the final effector of which involves K+ATP channel opening. 2. The protective effects of ischemic preconditioning in human ventricular myocytes can be reproduced by the administration of exogenous IXCATp channel openers and blocked by the administration of K+Aw channel inhibitors. 3. Preconditioning mediated KCAv channel opening is dependent upon adenosine receptor activation and PKC translocation. 4. PKC translocates to the mitochondria with endogenous and exogenous preconditioning. 5. Mitochondrial KCATPchannel opening results in maintenance of mitochondria1 electron transport chain activity under conditions of ischemia. The above hypotheses have never before been demonstrated in a human model. To our knowledge, hypotheses 4 and 5 are being proposed for the first time using such a model. By confirming or refuting the above hypotheses, we hope to develop a model for the clinical application of exogenous preconditioning in patients undergoing coronary bypass surgery. CHAPTER TWO: ATP-MEDIATED POTASSIUM CHANNEL OPENER STUDIES Preconditioning is mediated via LTpchannel opening in human venfriculur myocyt~ SUMMARY OBJECTIVES: To determine the role of ATP-mediated potassium channel (K~A~)opening in human preconditioning (PC). METHODS: Isolated cultures of human ventricular myocfles (n=10 platedgroup) were stabilized in normoxic phosphate buffered saline for 70 minutes (S) followed by exposure to 90 minutes of prolonged simulated ischemia (I)and 30 minutes of reperfirsion (R) (Ischemic Controls; IC). Prior to prolonged I, plates were either exposed to 50 pol of the K+AT~agonist pinacidil (PIN), 50 pol of adenosine (PRE), or to anoxic (POz=O m.)preconditioning for a period of 20 minutes. Certain plates were exposed to 20 pmol of the KIATp antagonist Glybenclarnide (GLY) throughout the pre-ischemic period. AU treatments were followed by 20 minutes of reperfhion in normoxic PBS. Comparisons were made with plates exposed only to buffered saline won-ischemic Controls; MC). Cellular viability was assessed via % Trypan Blue exclusion and by measurement of cellular lactate release and intracellular ATP concentrations. RESULTS: PC0 and PIN provided the greatest degree of protection from the injurious effects of ischemia as expressed by a decrease in Trypan Blue uptake @TIC: I O+/-3, PCO:20+/-4, PIN:22+/-3, Pretreatment:25+/-4,IC:38+/-5% Trypan Blue uptake; ANOVA: p<0.001; differences between PCOIPIN, Pretreatment and IC p Multiple Range Test). GLY abolished the protective effects of PCO, Pretreatment and PIN, and exacerbated the injurious effects of ischemia (PCO+GLY:43+/-5, Pretreatment+GLY:37+/-4, PMA+GLY:3 6+/-3, PIN+GLY:3 8+/-4, IC:3 8+/-5; NS). Although adenosine Pretreatment stimulated lactate production under both normoxic and ischemic conditions, PIN had no such effect. However, similar to adenosine pretreatment, PIN did prevent ATP degradation following prolonged ischemia and reperfusion (NIC: l.95+/-0.3, PW: 1.73+/-0.3, IC:0.75+/-0.4 mmol/gm - DNA; ANOVA p<0.00 1; differences between groups p<0.05 by Duncan's multiple range test). This ATP preservative effect was abolished with the addition of GLY (+GLY:0.46+/-0.4 mmoVgm DNA; p<0.01 vs. NIC/PIN). CONCLUSIONS: KtAn opening effectively reproduced the protective effects of anoxic preconditioning by decreasing cellular injury and prese~ngintracellular ATP following simulated ischemia. This protective effect was greater than that observed with adenosine pretreatment. GLY abolished the protective effects of anoxic preconditioning, adenosine pretreatment and pinacidil. K+ATPopening is a key intermediary and may represent the find rate-limiting step in the human myocardial preconditioning cascade- INTRODUCTION This chapter describes experiments designed to assess the ability of a K+ATPchannel opener to reproduce the protective effects of preconditioning. Although the beneficial effects of exogenous adenosine have been widely reported, the protection afforded by ischemic preconditioning has consistently been shown to surpass that provided by adenosine treatment alone. Thus, a more crucial determinant, or final effector of preconditioning has yet to be realized. The ATP-dependent potassium channel (ICATP)may represent such a hal effector. The following studies attempt to determine: 1) whether K+*~channel opening affords protection to human ventricular myocytes exposed to simulated ischemia and reperfusion; and 2) whether K+Am channel opening is a crucial intermediary or final effector in the preconditioning cascade. lMATERIALS and METHODS Isolation and Culture of Human Ventricular Myocytes Cultures of human ventricular rnyocytes were established as described in Appendix 3.33 1-333 Cells passaged 2 to 6 times, with a time fkom primary culture of less than 60 days, were utilized for this study.(Figme 18) Erperimental Design A detailed description of our in-viti-o technique of simulating "isch ernia" and <'rep in human ventricular myocytes is available in Appendix 4.)'*(Figure 18) Briefly, following 30 minutes of stabilization in 15 ml of normoxic PBS (including MgC12 0.49 mMM CaClz 0.68 mi and glucose 3.0 a;p02=150 mmHg), ischemia was simulated by placing the cells into a sealed plexiglass chamber flushed with 100% nitrogen to maintain anoxic conditions, while exposing the cells to a low volume (1.5 mL) of deoxygenated PBS @02=0 ma)for a period of 90 minutes. The volume of anoxic perfusate utilized was the minimum volume required to coat the ceilular monolayer for the prevention of cellular dehydration during the ischemic period. Reperfhion was accomplished by exposure to 15 mL of normoxic PBS for a period of 30 minutes. Preconditioning was simulated by exposing the cells to 20 minutes of ischemia and 20 minutes of repefision prior to proIonged (90 minute) ischemia. A smd sample of deoxygenated PBS (2 rnL) was placed in a centre dish within the sealed chamber to monitcr temperature and to confirm anoxic conditions at the end of each ischemic period. The temperature was maintained at 37'~ throughout the experiment. A pH of 7.40 +/- 0.05 and an osmolality of 290 +/- 20 mOsm/L was ensured with all solutions prior to use. Experimental Protocols Figure 20 summarizes the experimental protocols employed to evaluate the effects of varying preconditioning stimuli on cells undergoing prolonged ischemia and repefision, and the role of K+A7Pchannel opening in this process. Study I: Optimal dose and timing of pinacidil To determine whether human preconditioning can be reproduced by K*An channel opening, the K+*- channel agonist pinacidil (PIN; Sigma Chemical Co., St. Louis, MO) was utilized. A dose-response analysis was undertaken using varying doses (0-1 00 prnol) of pinacidil dissolved in normoxic PBS. Pinacidil was applied to the cells for 20 minutes following 30 minutes of stabilization, after which the cells were exposed to 20 minutes of repefision followed by prolonged ischemia and repefision. Once the optimal dose of pinacidil was determined (according to Trypan Blue exclusion), the protective effect was compared against that of other preconditioning stimuli; namely, ischemia and adenosine-mediated preconditioning. Treatment protocols for ischemic preconditioning and adenosine-mediated preconditioning were similar to those for pinacidil. Non-ischemic controls (NIC) undenvent stabilization in normoxic PBS for 30 minutes, followed by exposure to pinacidil or adenosine for 20 minutes followed by 140 minutes of reperfhion. Ischemic controls (IC) underwent stabilization for 70 minutes followed by prolonged ischemia and repefision. In both ischemic and non-ischemic controls, PBS solutions were replaced periodically in accordance with treatment times to enable maximal generalizeability between groups. (Figure 20) Study 2: Role of K+AP channel opening in human preconditioning To determine whether human preconditioning is dependent upon K+Apchannel opening, the channel antagonist glybenclamide (GLY; Sigma Chemical Co., St. Louis, MO) was utilized. A dose-response analysis was undertaken using varying doses (5-50 pmol) of glybenclamide dissolved in normoxic PBS. Glybenclamide was applied to the cells during 30 minutes of stabilization, during 20 minutes of pinacidil treatment, and during 20 minutes of pre- ischemic reperfhion. Once the KIAm inhibitory dose of glybenclarnide was determined (according to Trypan Blue exclusion), glybenclamide was applied to cells undergoing ischemic and adenosine-mediated preconditioning. Treatment protocols were similar to those previously outlined for pinacidil. Non-ischemic controls (NIC) were treated with glybenclamide for 70 minutes followed by 120 minutes of reperfusion. Ischemic control (IC) experiments were conducted with and without glybenclamide to rile out the existence of a negative (direct) effect other than that associated with K+ATPchannel inhibition. In both ischemic and non-ischemic controls, PBS solutions were replaced periodically in accordance with treatment times to enable maximal generalizeability between groups. (Figure 20) Assessment of Cellular Injuiy Cellular injury was assessed using non-confluent plates of cardiomyocytes (approximately 337,000 cells per 9 cm diameter culture dish) cultured for 4 to 5 days after the latest passage. Following the intervention of interest, cell plates were incubated with 0.4% Trypan Blue dye dissolved in normal saline (Sigma ChemicaI Co.; St. Louis, MO) and assessed for injury under an inverted light microscope (Nion Canada Instrument hc.; Mississauga, ON) at 200x magnification. Injured cells were unable to exclude the large molecular weight dye and stained blue.(Figure 21) The number of blue stained cells was counted from five standard locations on each plate and expressed as a percentage of the total number of cells. All counts were performed by a single observer who was blinded to the intervention. Biochemical Measurements Selected experiments involved biochemical assays for extracellular lactate concentrations and adenosine-triphosphate (ATP) content. Confluent cultures of cardiomyocytes (approximately 600,000 cells per culture dish) cultured for 5 to 10 days from the last passage were used for biochemical analysis. Following removal %om the culture dish, the extracellular fluid recovered f?om each intervention was analyzed for lactate using an enzymatic method described in Appendix 5 (Stat-Pack rapid lactate test kit, Behring Diagnostics; La Jolla, CA). The remaining cardiomyocytes were used to determine the concentrations of intracellular ATP following each intervention of interest. (Appendix 5) The specimens were flash frozen in liquid nitrogen and then freeze-dried. Specimens were analyzed by high performance liquid chromatography with the modifications described by Weisel, et aP34of the step gradient technique developed by Hull-Ryde, er al, and described in detail in Appendix 5 .335 The DNA in the cell extracts was recovered in 5% perchioric acid and quantified using a spectrophotometric, diphenylamine colour reaction, with calf thymus DNA as the standard (Appendix 5).336 Extracellular lactate and intracellular ATP values were then corrected for DNA content from each plate. Ischemic and non-ischemic control cardiornyocytes, although untreated, were subjected to similar protocols employing equivalent volumes of PBS for equal time periods with identical POz. Baseline biochemical measurements were made after removing the culture media and washing the cells with norrnoxic PBS, Statistical AnaCysis The SAS Statistical Package (SAS Institute, Cary, NC) was employed for analysis of all data. Data are expressed as the mean +/- standard deviation in the text and mean +/- standard error in the figures, with ten plates per group unless otherwise specified. Analysis of variance (ANOVA) was used to simultaneously compare continuous variables at different time periods. When statistically si@cant differences were found, they were specified by Duncan's multiple range test. Statistical significance was assumed for p RESULTS srudy I: Trypan-Blue dose-response experiments of pinacidil-mediated preconditioning revealed that 50 umol was the dose which provided maximal preconditioning when applied to human ventricular rnyocytes for 20 minutes followed by 20 minutes of pre-ischemic repefision (Figure 22.A) WC:lo+/-3, 100 umol:26+/-5, 50 umol:22+/-4, 10 umol:25+/-3, 1 umol:39+/-4, IC:38+/- 5 %Trypan Blue Uptake; ANOVA: p<0.00 1; differences between 50/10/100 pmol and 1 pol, IC, NIC pC0.05 by Duncan's multiple range test). When applied at this dose, pinacidil afforded significant protection against the injurious effects of ischemia and repefision; an effect which was similar in magnitude to that observed with ischemic preconditioning (PCO) and greater in magnitude than that observed with adenosine pretreatment (Pretreatment) (Figure 22.B) @TIC: lo+/-3, PCO:20+/-4, PIN:22+/-3, Pretreatment:25+/-4, IC:3 8+/-5% Trypan Blue Uptake; ANOVA: p multiple range test). Comparison between groups reveaIed that cells which were treated with pinacidil prior to prolonged ischemia and repefision experienced significant ATP preservative effects in comparison to ischemic controls (IC) (NIC: l.95+/-0.3, PCO: 0.81+/-0.3, PIN: l.73+/-0.3 Pretreatment: 1.go+/-0.4, IC: 0.75+/-0.4 mrnoVgDNA; ANOVA p<0.00 1; differences between PZNRretreatment and IC p<0.05 by Duncan's multiple range test). This ATP preservative effect was similar in magnitude to that observed with adenosine pretreatment.(Figure 23 .A) Unlike the case with anoxic preconditioning (PCO), ATP concentrations immediately following pinacidil treatment (or adenosine pretreatment) did not fall significantly in comparison to contro:s.(dATP post-preconditioning: PCO: 1.1+/-0.3, PIN: O.l+/-0.06, IC: 0.3+/-0.1 mrnoWgDNA; ANOVA p<0.01; differences between PC0 and PINK p<0.05 by Duncan's multiple range test.(Figure 23.B) Moreover, unlike the case with adenosine pretreatment, pinacidil did not have any detectable effect on supernatant lactate concentrations (in comparison to ischemic controls) at any period during the preconditioning cycie.(PIN: Stabilization: 0.6+/-0.2, Preconditioning: 0.8+/- 0.3, ischemia: 1A+/-0 -4, Repeesion: 0 -3+/-0 -2 moVgDNA; LC: StuMization: 0.6+/-0.2, Ischemia: 1-3 +I-0.3, ReperfLsion: 0.4+/-0.2 moVgDNA; p=NS) Trypan-Blue dose-response experiments of giybenclamide as a pinacidil antagonist revealed that 20 nmol was the lowest dose with which significant anti-preconditioning effects were demonstrated (Figure 24.A) (PIN+GLY: 5 nmol: 24+/-4, 10 nmol: 3 I+/-3, 20 nmol: 40+/- 4, 50 nmol: 43H-4, NIC: 8+/-3, IC: 40+/-5% Tlypan Blue Uptake; ANOVA: p<0.0 1; differences between IC, 20 nmol, 50 nmol and 5 nmol, 10 nrnol, NIC pC0.05 by Duncan's multiple range test). Glybenclamide applied at this dose effectively abolished the protective effects of pinacidil, anoxic preconditioning and adenosine pretreatment, as demonstrated by increased cellular injury and ATP depletion.(T.B.: MC+GLY:8+/-3, PCO+GLY:43+/-5, Pretreatment+GLY :37+/-6, PIN+GLY:40+/-4, IC+GLY:40+/-5% Trypan Blue Uptake; p=NS. ATP: NIC+GLY: 1.95+/-0.3, Pretreatment+GLY: 0.23+/-0.3, PIN+GLY: 0.46+/-0.4, IC: 0.62+/-0.2 mmoVgDNA; ANOVA p<0.01) (Figure 24.B, Figure 25) To rule out the possibility of a direct injurious effect, glybenclarnide was applied at varying doses during ischemia in the absence of pinacidil preconditioning.@igure 20) Under such circumstances, direct injurious effects (not solely due to K+*TPchannel inhibition) were observed only with glybenclarnide concentrations exceeding 20 nmol. (ISCHEI\IIA+GLY: 5 nmol: 40+/-4, 10 nmol: 37+/-5, 20 nrnol: 41+/-4, 50 nmol: 45+/-5, IC: 38+/-5% Trypan Blue Uptake; ANOVA: pcO.01; differences between 5 nrnol, 10 moly 20 nmol, IC and 50 nmol p CONCLUSIONS RAP channel opening with exogenous pinacidil effectively reproduced the beneficial effects of preconditioning without the need for an ischemic stimulus. Unlike the case with ischemic preconditioning, pinacidil preserved intracellular ATP following prolonged ischemia in comparison to controls. Moreover, inhibition of K+AIP channel opening with glybenclamide effectively abolished the protective effects of both ischemic and adenosine mediated preconditioning, suggesting its role as a rate limiting step in the preconditioning cascade. CMTERTHREE: MITOCHBNDFUAL-SPECIFIC CAPCHANNEL OPENER STUDIES Preconditioning is mediated via mitochondriai cATpchannel opening in human ventricular myocytes SITMlMARY OBJECTIVES: Although current data suggest a cardioprotective effect of potassium channel openers based on the opening of plasmaZemmal KrAp channels (Chapter I), such a hypothesis does not conform to our current model of quiescent human ventricular myocytes. In view of the previously demonstrated ATP-preservative effects of preconditioning, and due to the ubiquitous nature of the KCApchannel, we propose that the crucial site of K+An channel opening in human preconditioning occurs at the level of the mitochondria rather than at the level of the plasmalemma, as previously hypothesized. METHODS: To verify this hypothesis, the rnitochondrial-selective K+Av channel opener diazoxide (Dm)was utilized. After a dose response analysis, DZX was applied to human ventricular myocytes for 20 minutes followed by 20 minutes of pre-ischemic repelfusion, prior to prolonged ischemia and repetfusion. Certain plates were also exposed to 20 nmol of the K*An antagonist Glybenclamide (GLY) throughout the pre- ischemic period. Comparisons were made with plates exposed only to buffered saline prior to prolonged ischemia and reperfusion (Non-ischemic Controls; NIC). Cellular viability was assessed via % Trypan Blue exclusion and by measurement of cellular lactate release and intracellular ATP concentrations. RESULTS: Diazoxide at a dose of 20 pmol afforded significant protection against the injurious effects of prolonged ischemia and reperfirsion as expressed by a decrease in Trypan Blue uptake (NIC:lo+/-3, DZX:2 1+/-4, IC:3 9+/-4% Trypan Blue uptake; p<0.00 1). The degree of protection was similar in magnitude to that observed with ischemic and pinacidil mediated preconditioning, and greater in magnitude than that observed with adenosine mediated preconditioning. Glybenclamide effectively abolished the protective effects of diazoxide @ZX+GLY:4 1+/-5, IC:3 9+/-4% Trypan Blue Uptake; NS). Diazoxide had no effect on cellular lactate production however did demonstrate significant ATP preservative effects (NIC: 1.95+/- 0.3, DZX: 1.7 I+/-0.4,IC:0.75+/-0.4 mrnol/gm DNA; p<0.00 1). This ATP preservative effect was abolished with the addition of GLY (+GLY:0.53+/-0.4 mmoVgm DNA; p CONCLUSIONS: Mitochondrid specific Kkchannel opening effectively reproduced the protective effects of preconditioning by decreasing cellular injury and preserving intracellular ATP following simulated ischemia. In the absence of an effect on myocellular contractility, mitochondrid RATPopening may represent the find rate-limiting step in the human myocardiai preconditioning cascade. INTRODUCTION This chapter describes experiments designed to assess the role of mitochondria1 K+*IP channels in the preconditioning cascade. Current data suggests that the cardioprotective effects of pinacidil are related to the opening ofpZmalemntal KCAm channels.262,291-ws Charnel opening is believed to facilitate potassium extrusion thus leading to early membrane repolarization and action potential shortening. Such shortening may limit myocellular contractility thus enabling preservation of ATP stores which, in-turn, would account for cardioprotection during and following ischemia. Although this hypothesis may appear attractive at first, firrther examination reveals a lack of generalizeability to the current model. Since our human ventricular cardiomyocytes are quiescent (non-contractile) in nature, an alternate mechanism must have accounted for the protective effects observed with pre-ischemic pinacidil treatment. In view of the hypothesized ATP-preservative effects of preconditioning, and due to the ubiquitous nature of the KCAIPchannel, we propose that the crucial site of K*Ap channel opening in human preconditioning occurs at the level of the mitochondria rather than at the level of the plasmalema, as previously hypothesized. MATERIALS and METHODS Isolation and Culture of Human Ventricular Myocytes Cuttures of human ventricular myocytes were established as described in Appendix 3.33 1-333 Cells passaged 2 to 6 times, with a time from primary culture of less than 60 days, were utilized for this study.(Figure 18) Eqerimental Design A detailed description of our in-vitro technique of simulating ischemia and repefision in human ventricular myocytes is available in Appendix 4.332(Figure 19) Briefly, following 3 0 minutes of stabilization in 15 ml of normoxic PBS (including MgC12 0.49 mM- CaC12 0.68 mM, and glucose 3.0 m&I; po2=150 mmHg), ischemia was simuiated by placing the cells into a sealed plexiglass chamber flushed with 100% nitrogen to maintain anoxic conditions, while exposing the cells to a low volume (1.5 mL) of deoxygenated PBS @02=0 rnmHg) for a period of 90 minutes. The volume of anoxic perhsate utilized was the minimum volume required to coat the cellular monolayer for the prevention of cellular dehydration during the ischemic period. Reperfusion was accomplished by exposure to 15 rnL of normoxic PBS for a period of 30 minutes. Preconditioning was simulated by exposing the cells to 20 minutes of ischemia and 20 minutes of reperfitsion prior to prolonged (90 minute) ischemia. A small sample of deoxygenated PBS (2 mL) was placed in a centre dish within the sealed chamber to monitor temperature and to confirm anoxic conditions at the end of each ischemic period. The temperature was maintained at 37'~ throughout the experiment. A pH of 7.40 +I- 0.05 and an osmolality of 290 +/- 20 rnOsm/L was ensured with all solutions prior to use. Experimental Protocols To venfjl our hypothesis of a mitochondrial based mechanism, the mitochondriaI-selective K*AV channel opener diazoxide @W;Sigma Chemical Co., St. Louis, MO) was utilized (cardiac mitochondrial K+*TP channels have been demonstrated to be approximately 2000 times more sensitive to diazoxide than plasmaIemmal KIA- channels).289,328 A dose-response analysis was undertaken using varying doses of diazoxide dissolved in normoxic PBS. Non-preconditioned celis were treated with diazoxide for 20 minutes followed by 20 minutes of pre-ischemic reperhion prior to prolonged ischemia and reperfhion. Certain cells were simultaneously exposed to 20 MIOI of the RATpantagonist glybenclamide (GLY) during 30 minutes of stabilization, during preconditioning with diazoxide, and during pre-ischemic repefision. Non- ischemic controls were exposed to glybenclamide for 30 minutes, followed by glybenclamide with diazoxide for 20 minutes, followed by glybenclamide for 20 minutes, followed by 120 minutes of stabilization. Ischemic controls were stabilized in PBS for 70 minutes followed by prolonged ischemia and repefision. h- both ischemic and non-ischemic controls, PBS soIutions were replaced periodically in accordance with treatment times to enable rnkrnal generalizeability between groups. (Figure 26.A) Assessment of Cellular mjuty Cellular injury was assessed using non-confluent plates of cardiomyocytes (approximately 337,000 cells per 9 cm diameter culture dish) cultured for 4 to 5 days after the latest passage. Following the intervention of interest, cell plates were incubated with 0.4% Trypan Blue dye dissolved in normal saline (Sigma Chemical Co.; St. Louis, MO) and assessed for injury under an inverted light microscope (Niion Canada Instrument Inc.; Mississauga, ON) at 200x magnification. Injured cells were unable to exclude the large molecular weight dye and stained blue.(Figure 21) The number of blue stained cells was counted from five standard locations on each plate and expressed as a percentage of the total number of cells. All counts were performed by a single observer who was blinded to the intervention- Biochemical Memwements Selected experiments involved biochemical assays for extracellular lactate concentrations and adenosine-triphosphate (ATP) content. Confluent cultures of cardiomyocytes (approximately 600,000 cells per culture dish) cultured for 5 to 10 days from the last passage were used for biochemical analysis. Following removal fiom the culture dish, the extracellular fluid recovered fiom each intervention was analyzed for lactate using an enzymatic method described in Appendix 5 (Stat-Pack rapid lactate test kit, Behring Diagnostics; La Jolla, CA). The remaining cardiomyocytes were used to determine the concentrations of intracellular ATP following each intervention of interest. (Appendix 5) The specimens were flash frozen in liquid nitrogen and then freeze-dried. Specimens were analyzed by high performance liquid chromatography with the modifications described by Weisel, et a1334of the step gradient technique developed by Hull-Ryde, et al, and described in detail in Appendix 5."' The DNA in the cell extracts was recovered in 5% perchloric acid axid quantzed using a spectrophotometric, diphenylamine colour reaction, with calf thymus DNA as the standard (Appendix 9.)"Extracellular lactate and intracellular ATP values were then corrected for DNA content tiom each plate. Ischemic and non-ischemic control cardiomyocytes, although untreated, were subjected to similar protocols employing equivalent volumes of PBS for equal time periods with identical P01. Baseline biochemical measurements were made after removing the culture media and washing the cells with normoxic PBS. Statistical Analysis The SAS Statistical Package (SAS Institute, Cary, NC) was employed for analysis of all data. Data are expressed as the mean +/- standard deviation in the text and mean +/- standard error in the figures, with eight plates per group unless otherwise specified. Analysis of variance (ANOVA) was used to simultaneously compare continuous variables at different time periods. When statistically sigmficant differences were found, they were specified by Duncan's multiple range test. Statistical significance was assumed for p<0.05. RESULTS Trypan-Blue dose-response experiments of diazoxide-mediated preconditioning revealed that 20 pmol was the dose which provided maximal preconditioning when applied to humm ventricular myocytes for 20 minutes followed by 20 minutes of pre-ischemic reperfusion (Figure 26.B) (MC:8+/-4, 5 umol:37+/-4, 10 umol:27+/-4, 20 umol:21+/-4, 50 umol:23+/-5, 100 umol:30+/-5, IC:40+/-6 %Trypan Blue Uptake; ANOVA: p<0.001; differences between 20150 poland 100110 pol, 5 pmol/IC, NIC p 4, DZX:2 I+/-4, IC:39+/-5% Trypan Blue Uptake; ANOVA: p<0.00 1; differences between PCO/PIN/DZX, Pretreatment, NIC and IC p<0.05 by Duncan's multiple range test). Comparison between groups revealed that cells which were treated with diazoxide prior to prolonged ischemia and reperfitsion experienced significant ATP presemative effects in comparison to ischemic controls (IC). This ATP preservative effect was similar to that observed with pinacidil and adenosine pretreatment.(NIC: 2.0+/-0.3, PCO: 0.8+/-0.3, PIN: 1.7+/-0.3 Pretreatment: leg+/-0.4, DZX: 1.7+/-0.4, IC: 0.7+/-0.4 rnmoVgDNA; ANOVA pcO.001; differences between DZX/PIN/Pretreatment and IC p<0.05 by Duncan's multiple range test) (Figure 27.A) Moreover, unlike the case with ischemic preconditioning (PCO), ATP concentrations immediately following diazoxide treatment (or pinacidilladenosine pretreatment) did not fd in comparison to controls. Diazoxide did not have any detectable effect on supernatant lactate concentrations (in comparison to ischemic controls) at any period during the preconditioning cycle. (DZX: Stabilization: 0.6+/-0.2, Preconditioning: 0.4+/-0.1, Ischemia: l.l+/-0.3, Reperjksion: 0.8+/-0.3 moVgDNA; IC: Stabilization: O.6+/-0.2, Ischemia: 1.3+/-0.3, ReperJILsin: 0.4+/-0.2 rnoVgDN4 p=NS) Glybenclamide applied at a dose of 20 nmol effectively abolished the protective effects of diazodde, as illustrated by a reduction in both Trypan Blue exclusion and ATP preserwtion. (T.B.: DZX+GLY:41+/-5,IC:39+/-5% Trypan BIue Uptake; NS)(ATP: DZX+GLY:0.53+/-0.4 mmol/gm DNA; pC0.0 1 vs. NIC/DZX).(Figures 26.q 26.B) CONCLUSIONS Mitochondrial-specific channel opening effectively reproduces the protective effects of preconditioning without the requirement for an ischemic stimulus (which may be detrimentd). Although preconditioning may facilitate Kfm channel opening throughout the cell, the associated protective metabolic benefits may be linked primarily and specifically to the opening of mitochondrial CAPchannels. Moreover, in the absence of an effect on myocellular contractility, mitochondria K+ATPopening may represent the final rate-limiting step in the human myocardial preconditioning cascade. CHAPTER FOUR: PROTEIN-KINASE C STUDIES Prolein-kinase C mediates preconditioning via mitochondn'al firpchannel opening in human venhicular myocytes SUMRlARY OBJECTIVE3: We have previously demonstrated that protein-kinase C mediates both ischemic and adenosine mediated preconditioning. The aim of the following studies was to determine the sequence of protein kinase C (PKC)in the preconditioning cascade as well as its role (if any) in the activation of mitochondria1 ICATPchannels. METHODS: Isolated cultures of human ventricular myocytes (n=8 platedgroup) were stabilized in phosphate buffered saline for 30 minutes (S) followed by exposure to 90 minutes of simulated ischemia (I)and 30 minutes of reperfhion (R)(NIC: Non-ischemic controls; IC: Ischemic Controls). To determine the sequence of PKC in the preconditioning cascade, cells were treated with the PKC agonist PMA with or without the K+*-rPchannel inhibitor glybenclamide (GLY). To confirm the role of K+*v channel opening downstream to PKC activation in the preconditioning sequence, preconditioned cells (PCO, Pretreatment, PMA, PIN or DZX) were treated with the selective PKC antagonist Calphostin-C (Cal-C). To support the role of PKC in the activation of mitochondrid K-ATP channels during preconditioning, PKC activity was quantified and a standard slot-blot technique was utilized for an assessment of isoform-specific PKC translocation to mitochondrial membranes with various preconditioning stimuli. Finally, isoform-specific immuno-gold labelling of mitochondrial PKC was undertaken to demonstrate translocation of PKC to mitochondria via transmission electron microscopy of fixed cellular suspensions. RESULTS: PMA partially reproduced the protective effects of preconditioning (T.B.: NIC: lo+/-3, PCO: 19+/4, DZX:20+/- 4, PIN:22+/-3, Pretreatment:25+/-4, PMA:28+/4, IC38+/-5% Trypan Blue Uptake; ANOVA:p<0.001; differences between PCO/DZX/PIN, Pretreatment, PMA, and IC p<0.05 by Duncan's multiple range test; ATP: NXC:2.8+/-0.5, PMA:1.5+/-0.6, IC:0.5+/-0.4 mg/gDNA; ANOVA:p effects of PMA were abolished with the addition of GLY (PMA+GLY:36+/-3, IC:38+/-5% Trypan Blue Uptake; p=NS). Calphostin-C, while abolishing the protective effects of anoxic (PCO), adenosine (Pretreat), and PMA-mediated preconditioning, had no such effect on PIN or DZX-mediated preconditioning (T.B.: NIC: 1 I+/-2, PCO+CalC: 34+/-4, Pretreatment+CalC:36+/- 4, PMA+CalC:40+/-5, PIN+CalC:23+/-4, DZX+CalC:20+/-4, IC+CalC:39+/-5 %Trypan Blue Uptake; ANOVA:p<0.000 1; differences between PCO+CalC/Pretreat+CalC/PMA+CdC/ICfCalC and NIC/PIN+CalC/DZX+CalC p PMA+CalC:O. 7tAO.4, IC:O. 5+/-0.4 mg/gmDNA; difference between PMA and PMA+Cal-CAC p<0.0 1). PKC distributions to mitochondria1 membranes increased dramatically with either anoxic preconditioning, adenosine pretreatment, or treatment with PMA, as confirmed by digitized densitometry (PKC-a: PCO: 1.095, Pretreatment:0.920, PMA: 1.144, Control:0.3 27 stai~dirrd units; PKC-E: PCO: 1.153, Pretreatment:0.453, PMA: 1.109, Control:0.336 sfandbrd units). Assessment of PKC activity revealed enzymatic upregulation with various preconditioning stimuIi (Stabilization: 0.09+/-0.03, PCO: 0.24+/-0.04, Pretreatment: 0.39+/-0.05, PMA: 0-65+/-0.07; ANOVA: p<0.001; differences between Stab, PCO, Pretreat, and PMA p PKC-a to mitochondria with the various preconditioning stimuli. CONCLUSIONS: Protein kinase C exists as a crucial intermediary (second messenger) in the preconditioning cascade. Direct protein-kinase C activation reproduces the beneficial effects of preconditioning. Protein kinase C activation is necessary for the protective effects of anoxic preconditioning and adenosine preconditioning to be realized. Such protective effects are linked to channel opening, and Likely entail the translocation of protein kinase C to myocellular rnitochondrial membranes. rnTRODUCTX1ON We have demonstrated that brief anoxia and adenosine pretreatment confer significant protection to human ventricular myocytes by facilitating the opening of K+ATPchannels. Furthermore, we have shown this phenomenon to be mediated by the activation of extracellular adenosine receptors. However, the mechanism whereby such receptors yield K+ATPchannel activation remains to be elucidated. Extracellular receptors are often linked to intracellular effectors by a second messenger system. Protein kinase C represents such a second messenger system and may act as a key intermediary, ultimately resulting in the activation of KIATPchannels. Various studies, including our own, have suggested that PKC activation is necessary for preconditioning to take place. I 18.204- 209 Our previous studies of human ventricular myocytes revealed that both anoxic and adenosine preconditioning were abolished when preconditioned cells were treated with the PKC antagonists chelerythrine or ~al~hostin-~."~Nonetheless, other studies have questioned the role of PKC in preconditioning.210y211Moreover, no study has successfidly demonstrated a definitive linkage between PKC activation/translocation and KC.kTP channel opening in a human model. The purpose of the following studies was to determine the sequence of protein kinase C activation/translocation in the human preconditioning cascade as well as its role (if any) in the opening of mitochondrid KATPchannels. MATERIALS and METHODS Isolation and Culture of Humarr VentricularMyocytles Cultures of human ventricular myocytes were established as previously described.33 1-333 Cells passaged 2 to 6 times, with a time from primary culture of less than 60 days, were utilized for this study. AU celis were grown in 9.0 cm culture dishes and incubated under physiologic conditions (Appendix 3). Cells used for microscopic assessments were grown to non-coduence (approximately 223,000 cells per plate). Cells used for PKC activity and translocation studies were grown to confluence (approximately 600,000 cells per culture dish) by culturing for 5 to 10 days £?om the last passage. Experhen tal Design A detailed description of our in-vitro technique of simulating "ischemia" and "repefision" in human ventricular myocytes is available in Appendix 4.332(Figure18) Briefly, following 30 minutes of stabilization in 15 rnl of normoxic PBS (including MgClz 0.49 m,M CaClz 0.68 mM- and glucose 3.0 mM; pOz=150 mmHg), ischemia was simulated by placing the celIs into a sealed plexiglass chamber flushed with 100% nitrogen to maintain anoxic conditions, while exposing the cells to a low volume (1.5 mL) of deoxygenated PBS (p02=0 &g) for a period of 90 minutes. The volume of anoxic perfusate utilized was the minimum volume required to coat the cellular monolayer for the prevention of cellular dehydration during the ischemic period. Reperfhion was accomplished by exposure to 15 mL of nonnoxic PBS for a period of 30 minutes. Preconditioning was simulated by exposing the cells to 20 minutes of ischemia and 20 minutes of repefision prior to prolonged (90 minute) ischemia. A small sample of deoxygenated PBS (2 mL) was placed in a centre dish within the sealed chamber to monitor temperature and to confirm anoxic conditions at the end of each ischemic period. The temperature was maintained at 37'~ throughout the experiment. A pH of 7.40 +/- 0.05 and an osmolality of 290 +/- 20 mOsmL was ensured with dl solutions prior to use. Erpmimentd Rotocols Figure 28.A demonstrates the protocols utilized to determine the role of a protein base C (PKC) second-messenger pathway in KCADchannel mediated preconditioning. Studks of Protein Kinase-C (PKC) rnediatedpreconditioning To determine the role of protein kinase-C (PKC) stirnulafion/translocation in mitochondrial KCAv channel opening, 10 nmol of the PKC-stimulating phorbol ester PMA (4P- phorbol 12-myristate 13-acetate) (Sigma Chemical Co., St. Louis, MO) and 200 nmol of the selective PKC antagonist Calphostin-C dissolved in normoxic or anoxic PBS (Cal-C; Sigma Chemical Co., St. Louis, MO) were Non-preconditioned cells were exposed to PMA for 20 minutes followed by 20 minutes of pre-ischemic reperfbsion prior to prolonged ischemia and repefision. To determine the sequence of PKC activation/translocation in the preconditioning cascade, certain cells treated with PMA for 20 minutes followed by 20 minutes of pre-ischemic reperfbsion, were simultaneously treated with 20 nmol of the ICATPchannel inhibitor glybenclarnide (GLY) during 30 minutes of stabilization, during preconditioning with Ph/iq and during pre-ischemic reperfusion. Similarly, to confirm the role of RATPchannel opening downstream to PKC activation in the preconditioning cascade, preconditioned cells (FCO, Pretreatment, PMA or PIN/DZX) were treated with the selective PKC antagonist Cal-C during 30 minutes of stabilization, during preconditioning, and during pre-ischemic reperhion. Non- ischemic controls were exposed to Calphostin C or GLY for 30 minutes, followed by Calphostin C or GLY with adenosine, PMA, PIN or DZX for 20 minutes, followed by Calphostin-C or GLY for 20 minutes, followed by 120 minutes of stabilization. Ischemic controIs were stabilized in PBS for 70 minutes followed by prolonged ischemia and reperfusion. Studies of Protein Kinme C Activation/TrmIocafion Isoform specific translocation of protein kinase C (PKC)was demonstrated by performing a "slot blot" analysis on mitochondria1 membrane fractions using isoform-specific antibodies for both PKCa and PKC-E. Western blot analysis using chemiluminescent detection demonstrated that each antibody was specific for PKC with no evidence of non-specific background staining. Slot blots were then scanned using a commercially available software program (Molecular Images; Mississauga, ONT) and each band was assessed densitometricaUy, as outlined in detail in Appendix 6. PKC activity was measured by zn-situ phosphorylation of a PKC specific peptide substrate using a modification of a method previously reported by Heasley and Johnson and detailed in Appendix 5. 337-339 Measured phosphorylation rates were standardized for cell protein measured using the method of Lowry et The protein assay protocol is provided in Appendix 5. Isolation of Mitochondria1 Membranes Isolation of mitochondria1 membranes &om cultures of human ventricular myocytes was undertaken as described by Madden and colleagues34' and as outlined in Appendix 6. Immunogold Loculization of PKC Immunogold transmission electron microscopy (JEOL JEM I200 ExII transmission electron microscope) of ultra-thin sections was employed to demonstrate PKC translocation to mitochondria1 membranes. This technique as described by Ursell and colleagues342is outlined in detail in Appendix 6. Assessment of Cellular Injury Cellular injury was assessed using non-confluent plates of cardiomyocytes (approximately 337,000 cells per 9 cm diameter culture dish) cultured for 4 to 5 days after the latest passage. Following the intervention of interest, cell plates were incubated with 0.4% Trypan Blue dye dissolved in normal saline (Sigma Chemical Co.; St. Louis, MO) and assessed for injury under an inverted light microscope (Nikon Canada Instrument Inc. ; Mississauga, ON) at ZOOx magnification. Injured cells were unable to exclude the large molecular weight dye and stained blue-(Figure 21) The number of blue stained cells was counted from five standard locations on each plate and expressed as a percentage of the total number of cells. AU counts were performed by a single observer who was blinded to the intervention. Biochemical Measurements Selected experiments involved biochemical assays for extracellular lactate concentrations and adenosine-triphosphate (ATP) content. Confluent cultures of cardiomyocytes (approximately 600,000 cells per culture dish) cultured for 5 to 10 days from the last passage were used for biochemical analysis. Following removal from the culture dish, the extracelIu1ar fluid recovered fiom each intervention was analyzed for lactate using an enzymatic method described in Appendix 5 (Stat-Pack rapid lactate test kit, Behring Diagnostics; La Jolla, CA). The remaining cardiomyocytes were used to determine the concentrations of intracellular ATP following each intervention of interest. (Appendix 5) The specimens were flash fiozen in liquid nitrogen and then freeze-dried. Specimens were analyzed by high performance liquid chromatography with the modifications described by Weisel, el ap" of the step gradient technique developed by Hull-Ryde, et al, and described in detail in Appendix 5.))' The DNA in the cell extracts was recovered in 5% perchloric acid and quantified using a spectrophotornetric, diphenylamine colour reaction, with calf thymus DNA as the standard (Appendix 5).336 Extracellular lactate and intracellular ATP values were then corrected for DNA content fiom each plate. Ischemic and non-ischemic controI cardiomyocytes, although untreated, were subjected to similar protocols employing equivalent volumes of PBS for equal time periods with identical PO*. Baseline biochemical measurements Stdisticai Analysis The SAS Statistical Package (SAS Institute, Cq,NC) was employed for analysis of all data- Data are expressed as the mean +I- standard deviation in the text and mean +I- standard error in the figures, with eight plates per group unless otherwise specified. Analysis of variance (ANOVA) was used to simultaneously compare continuous variables at different time periods. When statistically sigdicant differences were found, they were specified by Duncan's multiple range test. Statistical significance was assumed for p RESULTS Studies of Protein Kinase-C (PKC) rnediatedpreconditianing To determine whether the protective effects of preconditioning can be reproduced via direct PKC stimulation, cells were treated with the PKC agonist PMA. PMA conferred a degree of protection to our human ventricular myocytes which although significant, was not as great as that observed with anoxic, adenosine-mediated, or PNDZX-mediated preconditioning. (T.B.: NIC: lo+/-3, PCO: 19+/-4, DZX:20+/-4, PIN:22+/-3, Pretreatment:25+/-4, PMA:28+/-4, IC38+/- 5% Trypan Blue Uptake; ANOVA:p<0.00 1; differences between PCOIDZXPIN, Pretreatment, PMA, and IC p ANOVA:pcO.000 1; differences between NIC, Pmand IC p 4, PMAtGLY: 36+/-3, IC:38+/-5 % Trypan BIue uptake; difference between PMA and PMA-tGLY/IC p<0.00 1)(Figure 28B) In a separate series of experiments, cells preconditioned with anoxia (PCO), adenosine pretreatment (Pretreatment) PMA, PIN or DZX were simultaneously exposed to the selective PKC antagonist Calphostin-C. As shown in Figure 29, Calphostin-C abolished the protective effects of anoxic preconditioning (PCO), adenosine pretreatment, and PMA, however had no effect on the protective properties of PIN or DZX &B-: NIC: 1I+/-2, PCO+Cd-C:34+/-4, Pretreatment+Cal-C:36+/-4, PMA+Cal-C:40+/-5, PIN+Cal-C:23+/-4, DZX+Cal-C:20+/-4, IC: 39+/-5,% Trypan Blue uptake; ANOVA; p<0.000 1; differences between PCO+Cal-C/Pretreat+Cal-C/PMA+Cal-Cand DZX+Cal-C/PIN+Cal-C p<0.05 by Duncan's Multiple Range test; ATP: PMA: IS+/-0.6, PMA+CalC:0.7+/-0.4, IC:0.5+/- 0.4 mg/gDNA; difference between PMA and PMA+CaI-C/IC p<0.01). Calphostin-C did not exacerbate ischemia when applied to ischemic controls during ischemia (IC+Cal-C:4ltI-4)- Stdies of Protein Kinase C activationhansIocarion In a separate series of experiments, both PKC translocation and activity were assayed. Figure 30 displays a representative slot blot analysis which shows an isoform specific translocation of PKC to mitochondria1 membranes of human ventricular myocytes preconditioned with either anoxia (PCO), adenosine (Pretreatment), or PMA. Results were compared to those of cells which underwent stabilization in nonnoxic PBS only (NIC). Densitometric analyses revealed no changes in PKC-a or PKC-E distributions with stabilization. However, there was a marked translocation to mitochondria1 membranes in cells exposed to anoxic preconditioning (PCO), adenosine pretreatment (Pretreat) or PMq as confirmed by digitized densitometry @?KC-a: PCO: 1.095, Pretreatment:0.920, PMA: 1.144, Control:0.3 f 7 standard units; PKC-E: PCO:1.153, Pretreatment:0.453, PMA: 1.109, Control:0.336 sfan&rd units)(Figure 30). In concomitant studies, we measured the effect of anoxic preconditioning (PCO), adenosine pretreatment (Pretreatment), PMA or stabilization in normoxic PBS on total PKC activity. Although both anoxic preconditioning and adenosine pretreatment stimulated PKC activity, the effect of PMA was far more potent (Stabilization: 0.09+/-0.03, PCO: 0.24+/-0.04, Pretreatment: 0.39+/-0.05, PMA: 0.65+/-0.07; ANOVA: p<0.00 1; differences between Stabilization, PCO, Pretreat, and PMA p<0.05 by Duncan's multiple range test, n=6/group). Finally, immunogold transmission electron microscopy of ultra-thin sections confirmed the intracellular localization of PKC with preconditioning. When anti-PKC-a linked to a 5 nm gold- conjugated Fab fragment was applied to cells preconditioned with either anoxia (PCO) or adenosine pretreatment (Pretreat), electron microscopy revealed a localization of PKC-ato the surface of intracellular organelles exhibiting an intricate internal membranous network.(Figure 3 1) Morphological examination demonstrated such organelles to resemble mitochondria. CONCLUSIONS The results of these studies suggest that both ischemic preconditioning and adenosine preconditioning are mediated by isofom-specific PKC translocation and activation. Protein kinase C exists as a crucial intermediary (second messenger) in the preconditioning cascade. Direct protein-base C activation partially reproduces the beneficid effects of preconditioning. Protein base C activation and translocation is necessary for the protective effects of anoxic and adenosine preconditioning to be realized. K*A~~channel opening exists downstream to PKC activation in the preconditioning cascade as evidenced by the ability of glybenclamide to abolish the protective effects of PMA and the ability of PIN/DZX to confer protection despite PKC inhibition with Cal-C. The protective effects of PKC are intimately linked to K*Ap channel opening, and ke1y entail the transIocation of protein kinase C to myocelldar mitochondrial membranes. CHAPTER FIVE: MIT.OCHONDRIAL STUDIES Preconditioning affords protection by facilitating mito& m&*dmetabolic potential in human ventricular myocytes SUMMARY OBJECTIVES: We have previously demonstrated that preconditioning is mediated via adenosine expression, PKC activatiodtrandocation, and mitochondrial KATPchannel opening. We have also demonstrated that such factors likely yield protection via the preservation of high energy phosphates (ATP). The aim of the following studies was to determine the mechanism whereby rnitochondrial KCAm channel opening affects mitochondrial metabolism resulting in ATF' preservation. METHODS: Isolated cultures ofhuman ventricular myocytes (n=8 plztes/group) were stabilized in phosphate buffered saline for 30 minutes (S) followed by exposure to 90 minutes of simulated ischemia (I)and 30 minutes of reperfusion @)(Ischemic ControIs; IC). To determine the effect of preconditioning on mitochondrial metabolism, cells (n=8 plates/group) preconditioned with anoxia (PCO), adenosine pretreatment (Pretreat) andlor diazoxide @ZX) were assessed for mitochondrial matrix volume and electron transport chain enzymatic activity. RESULTS: Analysis of respiratory chain enzymes revealed an increase in enzyme activity with anoxic preconditioning (PCO), adenosine pretreatment (Pretreatment), and DZX: COX: PCO:29+/-6, Pretreatment:20+/-5, DZX:27+/-5, IC: 12+/-3 Units/mg [p<0.05 vs. IC]; Com~lex -I+III: PCO: 134+/-28, Pretreatment: 118+/-17, DZX: 129+/-8, IC:46+/-12 Units/mg [p IC]; Comolex II+ttI: PCO: 14.9+/-2, Pretreatment: 12+/-3, DZX: 1I+/-0.7, IC: 4+/- 1 Unitshg [p<0.05 vs. IC]. Transmission electron microscopy of individual cell plates following adenosine pretreatment or diazoxide treatment revealed an increase in mitochondrial matrix volume in comparison to controls. CONCLUSIONS: Preconditioning afEords protection to human ventricular myocytes by maintaining mitochondrial electron transport chain flux during ischemia thereby enabling the preservation of ATP synthetic mechanisms. Such an effect may be accomplished by an increase in mitochondrial matrix volume which has previously been shown to drive electrons into the electron transport chain mechanism, thus facilitating forward flux. INTRODUCTION This chapter describes experiments designed to establish the mechanism whereby activation of mitochondria1 K+AIP channels via preconditioning yields protection against the detrimental effects of ischemia and repefision. We have previously demonstrated that preconditioning with adenosine pretreatment, PMA or RAP channel openers leads to preservation of intracellular ATP despite exposure of cells to prolonged ischemia and reperfhion. Although this phenomenon may explain the protective effects of preconditioning, the mechanism whereby this ATP-preservative effect is achieved has yet to be realized. Our aim was to determine a possible link to mitochondria1 metabolism and strcrctural characteristics. MATERIALS and METHODS Isolation and Culture of Human Ventricular Myocytes Cultures of human ventricular rnyocytes were established as previously described.33 1-333 Cells passaged 2 to 6 times, with a time from primary culture of less than 60 days, were utilized for this study. All cells were grown in 9.0 cm culture dishes and incubated under physiologic conditions (Appendix 3). Cells used for respiratory chain enzyme studies were grown to confluence (approximately 600,000 cells per culture dish) by culturing for 5 to 10 days from the last passage. Cells used for electron microscopic assessments were grown to non-confluence (approximateIy 223,000 cells per plate). Experimental Design A detailed description of our in-vztro technique of simulating "ischemia" and "repelfusion" in human ventricular myocytes is available in ~p~endix.~~~(Fi~ure18) Briefly, following 30 minutes of stabilization in 15 ml of normoxic PBS (including MgC12 0.49 rnM- CaC12 0.68 m,M and glucose 3.0 mi!; p0~150rnmHg), ischemia was simulated by placing the cells into a sealed plexiglass chamber flushed with 100% nitrogen to maintain anoxic conditions, while exposing the ceUs to a low volume (1 -5 mL) of deoxygenated PBS (pO~0mag) for a period of 90 minutes. The volume of anoxic perfhate utilized was the minimum volume required to coat the cellular monolayer for the prevention of cellular dehydration during the ischemic period. Repelfusion was accomplished by exposure to 15 mL of normoxic PBS for a period of 30 minutes. Preconditioning was simulated by exposing the cells to 20 minutes of ischemia and 20 minutes of repefision prior to prolonged (90 minute) ischemia. A small sample of deoxygenated PBS (2 mL) was placed in a centre dish within the sealed chamber to monitor temperature and to cob anoxic conditions at the end of each ischemic period. The temperature was maintained at 37'~ throughout the experiment. A pH of 7.40 +I- 0.05 and an osmolality of 290 +/- 20 mOsm/L was ensured with all solutions prior to use. Experimental Protocols Isolated cultures of human ventricular myocytes (n=8 plates/group) were stabilized in phosphate buffered saline for 30 minutes (S) followed by exposure to 90 minutes of simulated ischemia (I)and 30 minutes of repefision @)(Ischemic Controls; IC). To determine the effect of preconditioning on mitochondria1 metabolism and matrix volume, cells (n=8 platedgroup) preconditioned with anoxia (PCO), adenosine pretreatment (Pretreat) and/or diazoxide (DZX) were assessed for mitochondrial electron transport chain enrymatic activity immediately following the preconditioning stimulus. Ischemic and non-ischemic control cardiomyocytes, although untreated, were subjected to similar protocols employing equivalent volumes of PBS for equal time periods with identical POz. Studies of mitochondriaZ respiratory chain enzyme activity To determine the mechanism whereby preconditioning affects mitochondrial metabolism for high energy phosphate preservation, mitochondrial respiratory chain flux was studied by assessment of enzyme activity following various preconditioning stimuli. COX, Complex E+EI and Complex I+III activities were determined on whole cell extracts as described by Glerum el al.,'" Merante et and Pitkanen et a1.,345and described in Appendix 6. A.ll enzyme measurements were performed on a Cobas Fara analyzer (HoEhan LaRoche, N3). Srudies of mifochondriaI matrix vohe The effect of K+Aw channel opening on mitochondrial structural characteristics was evaluated via transmission electron microscopy (JEOL EM 1200 ExII transmission electron microscope) of fixed cellular suspensions afker treatment with adenosine and DZX.(Appendix 6) Morphometric assessments were undertaken using methods described by Schwerzmann and c~llea~ues.~~~(~~~endix6) Statistical Analysis The SAS Statistical Package (SAS Institute, Cary, NC) was employed for analysis of all data. Data are expressed as the mean +/- standard deviation in the text and mean +/- standard error in the figures, with eight plates per group udess otherwise specified. Analysis of variance (ANOVA) was used to simultaneously compare continuous variabIes at different time periods. When statistically signifkant differences were found, they were specified by Duncan's multiple range test. Statistical significance was assumed for p<0.05. RESULTS Analysis of respiratory chain enzymes revealed an increase in enqmatic activity with anoxic preconditioning (PCO), adenosine pretreatment (Pretreatment), and diazoxide (DZX): COX: PCO:29+/-6, Pretreatrnent:20+/-5, DZX:27+/-5, Control:12+/-3 Unitshg [p<0.05 vs. Cont]; Comdex I+IE PCO: 134+/-28, Pretreatment: 1 18+/-17, DZX: 129+/-8, Control:46+/-12 Unitdmg [p<0.05 vs. Cont]; Comolex II+n PCO: 14.9+/-2, Pretreatment: 12+/-3, DZX: 11 +/- 0.7, Control: 4+/-1 Unitdmg CpcO.05 vs. Cont] (Figure 32). Prolonged ischemia was found to have an overall inhibitory effect on enzymatic activity. Figure 3 3 displays electron micrographs which demonstrate the appearance of mitochondria1 swelling in human ventricular myocytes exposed to diazoxide in comparison to controls stabilized in normoxic PBS only. A similar phenomenon was observed in cells exposed to adenosine pre-treatment. (Mean M& Volume: DZX: 5.2+/-2.6;ADO: 3.9+/-3.1; CONT: 2. I+/-1.8 pLhgprotein; p=0.04, DZX vs CONT) CONCLUSIONS Preconditioning a&ords protection to human ventricuiar myocytes against the detrimental effects of ischemia and reperfusion by preserving the ability of mitochondria to synthesize ATP. This phenomenon is accomplished by upregulation of the enzymes responsible for maintaining forward flux of the electron transport chain. Such upregulation may be facilitated by changes in mitochondrial matrix volume resulting from K+*- channel-mediated ion influx. Figure 18: Representative photomicrographs of primary cultures of human paediatric (A) and adult (B) ventricular myocytes. (200X magnification) nap#1 Trap #2 Humidifier 37"C 4"C 37"C Culture dishes H, 0 Jacketed tubing (37°C) 4 Temperature probe Figure 19: Schematic diagram of simulated "ischemia" and "reperfision" model. Culture dishes of human ventricular cardiomyocytes are placed in an air-tight plexiglass chamber. To ensure anoxic conditions, 100% nitrogen (&) gas bubbled through two oxygen traps is utilized to continuously flush the sealed chamber, thereby displacing any ambient oxygen. Four culture dishes are placed in the chamber which is equipped with a central sampling dish to enable verification of anoxic conditions and to allow temperature monitoring with each ischemia/reperfusion experiment. / Normoxie PBS .Simulated Ischemia pO, = 0 mmHg I 30 20 20 90 30 minutes Figure 20: K-AT' Channel Opener Studies: In study 1) (after dose-response analyses), cells underwent either anoxic (PCO), adenosine mediated (Pretreat), or pinacidil mediated (PIN) preconditioning for a period of 20 minutes prior to prolonged ischemia and repefision. In study 2), cells treated with various preconditioning stimuli (PCO, Pretreat, PIN) were pretreated, simultaneously treated, and post-treated with the K-ATP channel inhibitor glybenclamide (GLY). All groups were compared to non-ischemic controls (NIC) which underwent 190 min. of stabilization, and ischemic controls (IC) which underwent 70 rnin. of stabilization followed by prolonged "ischemia" (90 rnin.) and "reperhsion" (30 rnin.). To rule out the possibility of a direct injurious effect, glybenclarnide was applied at varying doses during ischemia. (A=Adenosine) Figure 21: Light micrograph of cardiomyocytes stained with Trypan Blue. Left Pannel: cardiomyocytes stabilized in phosphate buffered saline for 30 minutes show little evidence of cellular injury. Middle Pannel: cardiomyocytes preconditioned with 20 minutes of "ischemia" followed by 20 minutes of "reperfusion" reveal relatively few injured cells (denoted by arrows) following prolonged ischemia and repefision. Right Pannel: non-preconditioned cardiomyocytes reveal large numbers of injured cells (denoted by arrows) following prolonged ischemia and reperfhion (200X magnification; scale bar=20 urn) (Reprintedfrom Ikonomidis et al. 30: 1995) *p<0.05 vs. NIC, 100, 1, IC MC 100 umol 50 umol 10 umoc I umol rc PIN Dose *p NIC PC0 PIN Pretreat IC Figure 22: A) Trypan blue dose response experiments revealed that 50 umol was the dose which provided maximal preconditioning when applied to human ventricular myocytes prior to prolonged ischemia and reperfusion. B) When applied at this dose, pinacidil afforded protection which was similar in magnitude to that observed with ischemic preconditioning (PCO), and greater in masitude+than that observed with adenosine pretreatment (Pretreat). (inC=non-ischemic control; IC=ischemic control) I * *p<0.05 vs. PCO, IC PC0 Pretreat PIN IC Stabilization Precond Ischemia Reperfusior Figure 23: A) Comparison between groups revealed that cells which were treated with pinacidil (PIN) prior to prolonged ischemia and reperfusion experienced significant ATP preservative effects in comparison to ischemic controls (IC). This ATP preservative effect was similar in magnitude to that observed with adenosine pretreatment (Pretreat). B) Unlike the case with anoxic preconditioning (PCO), ATP concentrations immediately following pinacidil treatment did not fall significantly in comparison to controls. (NIC=non-ischemic conho~;lC=ischernic control) V NIC 5nmol 10nmol 20nmol 50nrnol 1C GLY Dose Figure 24: A) Trypan Blue dose-response experiments of glybenclamide (GLY) as a pinacidil (PJN) antagonist revealed that 20 nmol was the lowest dose with which significant anti-preconditioning effects were demonstrated. B) Glybenclamide applied at 20 nm01 effectively abolished the protective effects of pinacidil, as well as those of anoxic preconditioning (PCO) and adenosine pretreatment (Pretreat). (nlC=non-ischemic control;IC=ischemic control) Stab+GL Y Precond+GL Y Ischemia Reperfusim Figure 25: Glybenclarnide (GLY) applied prior to, during and following preconditioning effectively abolished the ATP preservative effects of adenosine pretreatment (Pretreat) and pinacidi 1 (PIN). (Stab=stabilization; Precond=preconditioning; IC=ischemic contra[) 1 Normoric PBS .Simulated Ischemia pO = 0 mmHg I NIC I 1 minutes *p<0.05 vs NIC, 5/10/100umol and Nrc s U~O[ 10 urn01 20 urn01 50 unto1 100 ~m01 rc DZX Dose Figure 26: Diazoxide Studies: A) To determine the optimal dose of diazoxide (DZX), cells stabilized for a period of 30 minutes were treated with varying doses of diazoxide for a period of 20 minutes, followed by 20 minutes of pre-ischemic reperfision, and prolonged ischemia and reperfbion. Certain plates were simultaneously treated with the K-ATP channel antagonist glybenclarnide (GLY). All groups were compared to non-ischemic controls (NIC) which underwent 190 min. of stabilization, and ischemic controls PC) which underwent 70 rnin. of stabilization followed by prolonged ischemia (90 min.) and repefision (30 min.). B) Trypan Blue dose-response experiments of diazoxide mediated preconditioning revealed that 20 umol was the dose which provided maximal preconditioning when applied to human ventricular myocytes prior to prolonged ischemia and repefision. Sfabifizaf ion DZX Isch ernia Reperfmior *p<0.05 vs. NIC, Figure 27: A) Comparison between groups revealed that cells which were treated with diazoxide @ZX) prior to prolonged ischemia and repefision experienced significant ATP preservative effects in comparison to ischemic controls (IC). Glybenclarnide (GLY) effectively abolished such An-preservative effects. B) Trypan Blue experiments revealed that glybenclamide applied at a dose of 20 nrnol effectively abolished the protective effects of diazoxide. (NIC=non-ischemic control) I Nomoxie PBS Simulated Ischemia pO = 0 mmHg 1 30 20 20 90 30 min utes MC PMA Pm+GLY IC Figure 28: A) To determine the sequence of PKC in the preconditioning cascade, cells were treated with the PKC agonist PMA with or without the K-ATP channel inhibitor glybenclamide (GLY). To confirm the role of K-ATP channel opening downstream to PKC in the preconditioning cascade, preconditioned (PC) cells (PCO, Pretreatment, PMA, PIN or DZX) were simultaneously treated with the PKC antagonist Calphostin-C (Cal-C). Comparison was made with ischemic and non-ischemic controls (ICYNIC). B) PMA partially reproduced the protective effects of anoxic preconditioning, adenosine pretreatment, pinacidil and diazoxide. These protective effects were abolished with the addition of glybenclamide (GLY). *p<0.05 vs. NIC, PCO/Pretrea/PMA+CalC, IC Figure 29: Inhibition of PKC activation/translocation with Calphostin-C (CalC) abolished the protective effects of anoxic preconditioning (PCO), adenosine pretreatment (Pretreat) and PMA, but not the protective effects of pinacidil (PIN) or diazoxide (DZX), suggesting a K-ATP channel opener effect which exists downstream to PKC activation/translocation in the preconditioning cascade. (K:Non-ischemic Control; IC: Ischemic Control) PMA Pretreat NIC PC0 PKC a PKC E PMA Pretreat NIC PC0 Figure 30: Representative slot-blot analysis demonstrating an isoform specific translocation of PKC to mitochondria1 membranes of human ventricular myocytes preconditioned with either anoxia (PCO), adenosine (Pretreat), or PM. Digitalized densitometric values are also shown. (NIC:Non-ischemic Control; IC: Ischemic Control) Figure 31: Electron microscopy of human ventricular myocyte preconditioned with adenosine pretreatment. Immunogold localization revealed the intracellular localization of PKC-a to the surface of intracellular organelle resembling mitochondria. *pc0.05 vs. Control Jc 'rtC *p<0.05 vs. Control *p<0.05 vs. Control Pretreat DZX NIC Figure 32: Analysis of respiratory chain enzymes (COX, Complexes I+III, Complexes II+III) revealed an increase in enzymatic activity with anoxic preconditioning (PCO), adenosine pretreatment (Pretreat) and diazoxide (DZX) in comparison to controls. Figure 33: Electron micrographs of human ventricular myocytes following: A) stabilization in phosphate buffered saline (non-ischemic controls)), and B) treatment with 20 urn01 of diazoxide. Mitochondria (denoted by arrows) of cells treated with diazoxide appear swollen in comparison to non-ischemic controls. (N=Nucleus) CHAPTER 6: DISCUSSION DISCUSSION Despite recent advances in myocardial protection, the prevalence of low cardiac output syndrome following coronary bypass surgery (CABG) remains relatively high (approximately 7- 9%).' In the absence of intraoperative myocardial infarction, the development of low output syndrome following CABG represents inadequate intraoperative myocardial protection. Aortic crossclamping during coronary bypass surgery results in global myocardial ischemia.330 Although the detrimental effects of ischemia are lessened with cardioplegia, adenine nucleotides (ATP, ADP, and AMP) are degraded while being used to maintain myocyte integrity. The resulting nucleosides (including adenosine) wash out upon repefision, limiting nucleotide resynthesis and resulting in poor postoperative myocardial hction. . De novo purine synthesis is energetically costly, requiring 7 mol of either ATP or GTP per 1 rnol of AMP formed, and is very slow in organs such as the heart. 65,347 Canine studies have revealed that the depletion of cardiac ATP stores resulting £kom 15 min of ischemia requires approximately 1 to 2 weeks for repletion and full restoration of cardiac function.348,349 hi^ phenomenon may be secondary to ischemia-induced disruption of the mitochondrial electron transport chain, the mechanism by which cellular ATP is most efficiently produced.350 Thus ventricular dysfiinction secondary to myocardial stunning may reflect both ATP depletion as well as the low capacity for de novo purine synthesis.65 Accordingly, any efficient method of conserving ATP stores, repleting ATP stores, or facilitating purine synthesis would be both energetically and hnctionally advantageous. Adenosine kinase phosphorylation of adenosine is the most efficient method of salvaging purines, requiring 1 mol of ATP to form 1 mol of AMP. Unfortunately, the ability for direct purine salvage in heart muscle is rather limited. 65.88.348 Ischemic preconditioning is by far the most potent form of myocardial protection known. The cardioprotective effects of ischemic preconditioning have been shown in various species, including humans. 35 1-353 However, more recently, the protective effects of ischemic preconditioning in humans have been called into question. Perrauit et. al. reported that patients preconditioned with 3 minutes of crossclamping prior to institution of cardioplegia revealed increased levels of creatine kinase MI3 and lactate release at the end of cardioplegic arrest.'*' In addition, molecular biology data previously shown to be related to the preconditioning process (i-e. expression of m-RNA for both c-jos and heat shock protein 70) did not suggest a protective effect of preconditioning. Studies such as this along with the risks of repeated crossclamping (including aortic injury, intraoperative infarction and cerebral embolic disease) emphasize the need for identification of pharmacologic mediators that could safely and effectively harness the beneficial effects of ischemic preconditioning. Such mediators could be applied in the form of a simple additive to be administered in conjunction with cardioplegia during cardiac surgery. Adenosine may represent such an additive. Evidence in support of such a possibility was first introduced by Przyklenk and colleagues who reported that protection was dorded to non- ischemic myocardial regions adjacent to those which underwent ischemic preconditioning.'0 The authors suspected that preconditioning induced adenosine release which in turn initiated a sequence of cellular signalling events, resulting in protection from a subsequent prolonged ischemic episode. Using a microdialysis technique, van Wylen and colleagues found increases in adenosine and other soluble purines in canine myocardial interstitial fluid during the ischemic and repefision phases of preconditioning.354 In previous studies, our group confirmed the role of both endogenously released adenosine and exogenously administered adenosine in a human cardiomyocyte model of simulated cardioplegic arrest.L74 such a model permits an evaluation of preconditioning in human cardiornyocytes in the absence of alternate cell types (i-e. endothelial cells), and independent of the hernodynamic effects associated with phmacologic preconditioning preparations. Human cardiomyocyte cell culture model The cardiomyocytes employed in these studies have been extensively evaluated in previous reports. 35"56 Our cells were passaged 2-6 times and were cultured for up to 60 days &om the time of primary culture. These cardiomyocytes retain many characteristics of freshly isolated cells, but have distinct differences. Following enzymatic digestion and passaging, the cells change their shape, lose their striations, and become quiescent. Despite an abundant supply of mitochondria and contractile proteins, the sarcomeres become disrupted during division and do not re-establish their characteristic fbnctional format. The cardiomyocytes in culture are easily differentiated from other cell types. Endothelid cells are oval-shaped (15 X 20 p) and fibroblasts are spindle-shaped (4 X 80 pm), compared to the rectangular and much larger cardiomyocytes (40 X 80 p).In addition, endothelial cells grow poorly in the medium employed for cardiomyocytes, whereas fibroblasts have a much faster doubling time in culture and are easily identified as a spindle-shaped contaminant. The quiescent nature of our cardiornyocytes is likely the result of isolation techniques which cause a breakdown of myofibrillar organization. From a mechanical perspective, these cells may simulate the arrested heart encountered during cardiac surgery. The cellular concentrations of troponin I, troponin T and the MB isoform of creatine kinase are similar to those seen in- vivo.355 The metabolic response of these cells to ischemia also closely resembles our intraoperative findings during cardiac surgery. 15-17 Therefore, despite their quiescent state, we believe that these cells are phenotypically cardiomyocytes and provide a unique opportunity to evaluate the cellular response to ischemia and repefision as well as the effects of pharmacological additives such as adenosine. Our model of ischemia and reperhsion is similar to the effects of global ischemia on the myocardium. Although the volume overlying our cells during ischemia exceeds that found in the globally ischemic heart, reduction of the volume of ischemic PBS fiom 10 mL to 1.5 mL resulted in a marked increase in the products of ischemic metabolism, a decrease in the extracellular pH, and an increase in cell injury. Thus, our model may actually represent a form of low-flow ischemia analogous to limited cardioplegic pefision during cardiac surgery. The adenosine hypothesis The ability to precondition our cells against prolonged ischemia and repefision is similar to the effect seen in vivo. The degree of ischemia was crucial in regulating the protective effects of ischemic preconditioning. Anoxic preconditioning conferred greater protection than did hypoxic preconditioning as assessed via Trypan Blue exclusion. Thus the ischemic stimulus of ischemic preconditioning could not be minimized (in an effort to limit the detrimental effects of ischemia) without reducing the degree of protection afforded. Although lactate levels were elevated immediately after the ischemic preconditioning stimulus, lactate levels were similar in both preconditioned and ischemic control groups following both ischemia and reperfusion. Similarly, although intracellular ATP levels decreased significantly immediately following ischemic preconditioning, the rate and degree of ATP degradation during ischemia was significantly reduced in comparison to controls. Thus, despite an initial ATP deficit in the preconditioned group, both groups demonstrated similar degrees of ATP degradation following ischemia and repefision, implying some recovery of ATP levels or ATP synthetic capacity in the preconditioned group, and emphasizing the possible benefits of a pharmacological substitute which could presumably precondition without creating an initial ATP deficit ('ATP debt'). This hypothesis was substantiated when exogenous adenosine administration was found to preserve ATP levels compared to ischemic controls. We demonstrated that the protective effects of ischemic preconditioning could be transferred to non-preconditioned cells via the supernatant of preconditioned cells. To support our hypothesis that the crucial protective mediator was indeed adenosine, we demonstrated that the supernatants of anoxically preconditioned cells had the greatest concentrations of adenosine. Conversely, the supernatants of non-preconditioned cells had the lowest amounts of adenosine and confened no protection. The concentrations of adenosine recovered from the hypoxically preconditioned cells (6.7 nmoI) and f?om the non-preconditioned cells (1.1 nmol) were below the published dissociation constant (Kd) for the adult myocardial adenosine A* receptor (1.5 to 3.0 rnm~l).~~'In contrast, adenosine concentrations in the supernatant of anoxicdy preconditioned cells (16.3 nmol) greatly exceeded the reported Kd for the A1 receptor. These findings demonstrate once again that maximal ischemia is necessary for the greatest protection, and that the degree of ischemia and the degree of protection are both appropriately reflected by the amount of adenosine generated and released with preconditioning. To determine whether endogenous (ischemic) preconditioning fbnctions via an adenosine- mediated receptor pathway, cells undergoing supernatant preconditioning were simultaneously incubated with the non-selective adenosine receptor blocker SPT. In the presence of SPT, the protective effects of ischemic preconditioning were abolished, implying an adenosine-mediated receptor phenomenon. With exogenous adenosine, we were able to fbrther the concept of clinical preconditioning. To assess the benefits of exogenous adenosine, we treated our cells with varying doses of adenosine either prior to (Pretreatment), during (Ischemic treatment), or following (Reperfhion treatment) ischemia, or during aU three phases (Continuous treatment). We determined that adenosine was most protective when applied prior to ischemia (pre-treatment) and followed by pre-ischemic repefision. Administration of adenosine during ischemia (ischemic treatment) had a slight protective effect which was not as great as that seen with adenosine pretreatment. This discrepancy was likely due to the absence of a normodc reperfusion period (prior to ischemia) in the ischemic treatment group, a condition which seems to be necessary for the maximal effect of adenosine and the second messenger systems of preconditioning. Unlike previous reports in the literature, adenosine applied during repefision (repefision treatment) had no measurable effect. We suspect that adenosine pretreatment provided the maximum attainable protective effect since continuous treatment did not provide any additional benefits. The effects of adenosine pretreatment were receptor mediated since protection was afforded despite a period of pre-ischemic repefision (at which time no adenosine was present) and since the protective effects were abolished by simultaneous incubation with receptor antagonists. Using the same principle, we confirmed that the mild protective effects conferred with ischemic adenosine treatment were also secondary to receptor activation, and not secondary to a direct substrate-mediated effect as has been previously hypothesized (i.e. adenosine was not conferring protection by acting as a substrate for the production of high energy phosphates). Unlike the case with ischemic preconditioning, adenosine pretreatment resulted in a significant preservation of intracellular ATP levels following prolonged ischemia and repeifusion. This finding was likely due to the fact that no ATP deficit (ATP'debt') was incurred during the exogenous adenosine preconditioning process. Although adenosine pretreatment did not Sect final lactate concentrations (following prolonged ischemia and reperfusion) compared to controls, adenosine did increase extracellular lactate concentrations immediately following its appiication. This phenomenon (which occurred during both normoxia and anoxia) is consistent with the previously reported stirnulatory effect of adenosine on glycolytic flux (via upregulation of the rate-limiting eqme phospho-hcto kinase), as well as its effect on glucose uptake and utilization."* Such alterations may further facilitate ATP production. Protein kinase-C has been implicated as an important second messenger in animal studies of the ischemic preconditioning phenomenon. 338.359 Thus, we evaluated the hypothesis that human ischemic and adenosine preconditioning were meditated via this pathway. We found that the protective effects of ischemic preconditioning and adenosine pretreatment were dependent upon PKC stimulation, as protection was abolished in the presence of the PKC antagonist Calphostin- C. Moreover, the protective effects of preconditioning were partially reproduced by PKC stimulation using the selective agonist PMA Our slot-blot analyses confirmed that adenosine exposure results in the translocation of PKC-afiom the cytosolic to the membrane fiaction of human ventricular myocytes. Although the extent of translocation was similar between PMA and adenosine pretreatment, total PKC activity as measured by an in vitro phosphorylation assay was significantly higher following exposure to PMA Nonetheless, the protection aEorded by ischemic and adenosine preconditioning was greater than that seen with PMA. This finding suggested that preconditioning may act via more than one second messenger pathway. Although various mechanisms may contribute to the protective properties of adenosine in vivo, our model of isolated ventricular myocytes contimed a preconditioning effect of adenosine which was independent of alternate protective mechanisms and alternate cell types. Thus, adenosine was protective despite the absence of any effect upon coronary vasodilatation, adrenergic inhibition, and endothelid protection. Moreover, the isolated cell model and the short time course precluded any neovascularization-dependent effect. Mechanism of precondironiing 's pro fective effects As outlined above, we had previously successfblly demonstrated that human preconditioning was mediated via adenosine expression, receptor stimulation, and PKC activation and translocation. The goal of the current studies was to determine the final effector in this preconditioning sequence. As such, we aimed to define the processes, which when activated by PKC, afforded protection to human ventricular myocytes undergoing prolonged ischemia and reperfusion. Moreover, we endeavoured to determine the mechanism/s whereby such processes yielded their protective effects. KAIPchannel Opening: lhefinal emor Due to the ATP-preservative effects observed in our previous studies of human preconditioning, and in concert with previously published reports, 3 8,177 the ATP-mediated potassium channel (K1*n channel) represented an ideal model for the final effector of preconditioning. KCAw channels have long been known to open in response to cell ischemia and metabolic inhibition (pharmacologic or otherwise). The role of this final effector in the preconditioning sequence was confirmed by the current investigations. In our studies, preconditioning was reproduced with the KIATP channel agonist pinacidil (as determined via Trypan Blue exclusion), and all forms of preconditioning were abolished with glybenclamide-mediated K+ATP channel antagonism. Moreover, glybenclarnide exacerbated ischemia possibly by blocking inherent cellular auto-protective mechanisms. When compared to controls, K*Aw channel opening also facilitated ATP presenration despite exposure to prolonged ischemia and repefision. Unlike the case with adenosine or anoxia, no effect on intracellular lactate levels was observed. Despite the aforementioned studies, however, the mechanism by which such ion channel modulation is Wed to myocardial protection and ATP preservation remains unanswered. Previous reports have shown that KCApchannel activation catalyzes the outward transfer of K* ions, partly accounting for the early potassium loss and extracellular potassium accumulation seen in the ischemic myocardium. The addition of this outward K+ current to the myocardial action potential facilitates acceleration of membrane repolarization and action potentid shortening.291-293 Activation of less than 1% of the total myocyte KfATPchannel conductance has been shown to reduce action potential duration by at least one half"' This action potential (AP) shortening has served as the main basis for the hypothesized cardioprotective mechanisms inherent in K*Ap channel opening. The possible effects of such action potential shortening are twofold: 1. AP shortening may limit calcium entry (which normally takes place during the plateau phase of the action potential) which can serve to decrease the magnitude of twitch contractions, thereby consenring ATP; or 2. AE' shortening may markedly reduce the time for calcium influx via voltage-sensitive calcium channels while increasing the time during which the sodium-calcium exchanger may operate in forward mode to extrude calcium. Inhibition of intracellular calcium accumulation during ischemia can prevent cell injury or death secondary to activation of oxygen free radical production pathways, and may preserve energy stores which would otherwise have been utilized to maintziin normal calcium homeostasis.228*26~ws~296 Unfortunately, although these explanations represent plausible hypotheses for the protective effects of KeAn channel opening, our findings are inconsistent with such proposed mechanisms since the cardiomyocytes utilized in the aforementioned experiments are quiescent (non-contractile) in nature. Moreover, although the ATP preservation associated with K-p channel openers likely accounts for their cardioprotective effects, such ATP-preservative effects are not observed with anoxic (PCO) preconditioning (ATP 'debt') and thus cannot account for the protective effects of this phenomenon (i.e. PC0 confers protection despite the absence of an observable ATP preservative effect). As such, we hypothesize that the benefits afforded with preconditioning do not necessarily involve ATP preservation but rather a preservation of the capaczv to qmthesire ATP, both during and following prolonged ischemia and reperfusion. Such a possibility is supported by the fact that despite the initial fall in ATP with the ischemic stimulus of anoxic preconditioning, the ensuing temporal trend in ATP concentration follows that observed with the KrATpchannel openers pinacidil and diazoxide, such that the respective plots lay pardel to one another as demonstrated in Figure 23. This ATP-preservative effect of preconditioning was confirmed in our clinical studies of adenosine cardioplegia in patients undergoing coronary bypass surgery. The aforementioned ATP-synthetic fbnction is the role of the rnitochondrial electron transport chain. During the enzymztic reactions involved in glycolysis, fatty acid oxidation, and the TCA cycle, reducing equivalents are derived from the sequential breakdown of the initial metabolic In the case of glycolysis, NADH must be either reoxidized in the cytosol or transported to the rnitochondrial matrix in order to achieve the maximum energy yield from the oxidation of glucose. In order to transform this reducing power into utilizable energy, mitochondria have a system of electron carriers (associated with the rnitochondrial inner membrane) which convert reducing equivalents into utilizable energy by synthesizing ATP. This process is referred to as 'electron transport'. Unfortunately, the normal operation of the electron transport chain has been demonstrated to be markedly inhibited with prolonged ischemia in unprotected cells, likely due to the destruction of key transport chain enzymes.3s0 Thus, in view of the ATP-modulating effects of preconditioning, we proposed that the key determinant in this protective process primarily atfected the mitochondria rather than the cell membrane and thus involved rnitochondriaZ rather than plasmaZernrnai KrhTp channel opening. Indeed, Inoue and colleagues were able to estabiish the presence of KIATP channels in mitochondrial inner membranes using patch-clamp technology.321 Since mitochondria take up and extrude various inorganic and organic ions as well as larger substances such as proteins, the technique of patch cIamping could effectively provide real-time information on such transport and on energy transduction in oxidative phosphorylation. Using such technology, Inoue was able to demonstrate a resting mitochondrial inner membrane potential of -150 to -160 m~.~"Further studies would indicate that mitochondria generate an inward K' conductance when ATP in the inner matrix was deficient. This effectively led to a depolarization of the inner membrane (towards the 'equilibrium potential' of 29.5 mV) and a resultant change in mitochondrial water content. Although it was known that mitochondria in living cells would swell and contract by changing their water content, there was now a suggestion that such changes in matrix volume were related to respiratory chain fbnction. To hrther the above hypothesis, we employed mitochondrial-specific KtATP channel opening using diazoxide. Diazoxide is a potent va~odilator~~'and reduces insulin secretion362but has little effect on cardiac plasmalemmal KtA~P.289 TO determine whether mitochondria1 KtATPand plasmalemmal KCAp&om the same cell differ pharmacologically, Garlid and colleagues3" compared drug sensitivities of cardiac mitochondrial K+A~and plasmalemmal KATPreconstituted from beef heart. According to their findings, mitochondrial KtATPhrn heart and liver tissue did not differ significantly in their drug sensitivities (KIDvalues). Moreover, cardiac mitochondrid CATPand cardiac plasmalernmd KCAp exhibited similar sensitivities to benzopyran derivatives. However, cardiac mitochondrial was found to be approximately 2000 times more sensitive to diazoxide than cardiac plasrnalemmal KvAP. The low sensitivity of reconstituted cardiac plasrnalemmal K+AIPto diazoxide is entirely consistent with previously published reports. 289 our experiments, diazoxlde afforded similar protective properties as those observed with anoxic preconditioning, adenosine pretreatment, PMA and pinacidil. In addition, we established (using both western blot analyses and immunogold labelling) that protein kinase C translocates to mitochondrial membranes in response to the various preconditioning stimuli. To our knowledge, this is the first account of PKC translocation to the mitochondrial membranes of human ventricular rnyocytes. Despite such findings, one key question remained unanswered: that is, how is K-ATP channel opening linked to mitochondrial respiratory transport chain modulation? We have endeavoured to explain +&s phenomenon using two plausible hypotheses: 1) As previously noted, RAP channels are known to traverse the mitochondrial inner membrane.321 We hypothesize that protein base C, once activated by the preconditioning sequence, trandocates to the mitochondrial membranes of human ventricular myocytes. Here, PKC phosphorylates K+AP channels, thus converting such channels fiom the closed to the open state. Opening of these mitochondrial K+ATP channels has been shown by Halestrap and colleagues to shift the balance between potassium uniport and potassium/hydrogen antiport, causing a net transient potassium uptake and resultant mitochondrial inner membrane depolarization. As was noted by Inoue as well as Halestrap, such depolarization is intimately tied to mitochondrial matrix volume swelling. 321,363 Moreover, Halestrap demonstrated that increasing mitochondrial matrix volume over a fairly narrow range greatly activates electron transport at the point where electrons transfer into ~bi~uinone~~~(carrierwhich transports electrons kom Complex 11 to Complex m)(Figure 34)- Indeed, electron microscopy of our cells revealed an appearance consistent with mitochondria1 swelling following treatment with either adenosine or diazoxide. Thus, opening of mitochondrial K+ATPmay be a necessary component of the cellular signals calling, for example, for higher ATP production to support increased cardiac work or for inherent autoprotective mechanisms designed to increase tolerance to ischemia. Earlier studies by various investigators involving isolated mitochondria and cells or tissues in vitro revealed that similar changes can be produced in mitochondria by various experimental procedures.364 In these studies, normal mitochondria were often referred to as demonstrating an 'orthodox' configuration, whereas swollen mitochondria with a dense matrix were referred to as demonstrating a 'condensed' configuration. As early as 1971, Hackenbrock and colleagues revealed that transformation from the orthodox to the condensed state could be induced in all mitochondria in less than 6 seconds by the addition of 2-deoxyglucose which rapidly generates adenosine-diphosphate from adenosine triphosphate in the cell, thus inducing oxidative phosphorylation in mit~chondria.~~'Such findings support the idea that the transformation from an orthodox to a condensed configuration is linked to oxidative phosphorylation or forward flux of the electron transport chain; 2) Alternately, activation and maintenance of electron transport during ischemia and reperfhion may be achieved via ionic homeostatic mechanisms. Since opening of K+*TPchannels facilitates potassium entry and therefore a selective influx of positive charge, mitochondrial homeostatic mechanisms must act to balance this abnormality. One method of achieving such a balance would involve activation of the electron transport chain, which, when in forward flux, actively extrudes positive charges in the fom of H+ions. (Figure 34) To firther confirm such hypotheses, we demonstrated an increase in electron transport chain enzymatic activity with anoxic preconditioning, adenosine pretreatment and diazoxide treatment. Since ischemia is known to disrupt the normal hnction of the electron transport chain,350we believe that an increase or maintenance of electron transport chain fonvard flux during ischemia and reperfusion protects against any ischemia-related enzymatic disruption, and precludes any requirement for delayed recovery of ATP synthetic mechanisms. The ability to maintain normal ATP-synthetic capacity would account for the protection observed with anoxic (ischemic) preconditioning despite the absence of any observable ATP preservative effect. The ability to synthesize site specific K+Ap channel modulators bodes well for the eventual development of clinically applicable preparations. Such specific preparations would Likely have a greater therapeutic window than the compounds currently available. For example, K+ATPchannel opening using non-selective agonists such as pinacidil is plagued by numerous unwanted side- effects including arrhythmias and hypotension. Such effects may counteract or ultimately nullify the protective properties of these agents.326 An agent such as nicorandil would be ideal in this regard due to its mitochondria1 specific nature which would effectively preclude any hypotensive or pro-arrhythmic effects. The concomitant use of selective sarcolemmd K*ATPchannel antagonists (i-e. HMR 1883) may aIso be beneficial, since most such side effects are related to the modulation of sarcolemrnal rather than mitochondria1 channels. Moreover, these sarcolemmaI specific agents would not adversely affect preconditioning. Indeed, studies by BilIman and colleagues have shown that HMR 1883 has potent antifibrillatory effects in a canine model of chronic ischemia.366 Study Limitations Although the studies reported provide plausible explanations for the preconditioning phenomenon, certain limitations exist. The cultured human cardiomyocytes employed in our studies, although extensively evaluated in the past, have distinct differences from freshly isolated cells, which may limit their applicability to the in-vivo scenario. These cells become quiescent following enzymatic digestion and passaging. Despite an abundant supply of mitochondria and contractile proteins, the sarcomeres become disrupted during division and do not re-establish rheir characteristic functional format. Although such quiescent cells may simulate the mechanically arrested heart encountered during cardiac surgery, they differ in that they are not electrically depolarized as is the case with high potassium cardioplegic arrest. Moreover, the inability of these cells to contract during "reperfusion" may affect the interpretation of our metabolic results. The in vivo contracting heart may suffer &om a greater lactic acidosis and a greater depletion of high energy phosphates than observed in this study. Nonetheless, in comparison to a recent clinical review where myocardial levels of high energy phosphates in the region supplied by the left anterior descending artery were found to decrease by 22% following cardioplegic arrest, our experiments revealed a greater than 50% fall in ATP after prolonged ischemia and repefision. Finally, recent reports have suggested a loss of plasmalemrnal KtATp channel conductance with prolonged culture of rabbit right ventricular myocytes.367 This finding was believed to be secondary to the progressive loss of cytoplasmic T-tubules. Although we have no definitive evidence to mle out this phenomenon in our cell culture model, such a response has not been shown to affect mitochondrial KATP channels. Moreover, our use of controls as well as our policy of early culture use (within 4 to 5 days of primary culture) should preclude the contamination of our experiments due to channel downregulation. Our technique of simulated "ischemia" and "repefision" has been previously described in detail. Exposure of our cells to 90 minutes of simulated "ischemiay' resulted in sigficant cellular injury. Moreover, reduction of the pefisate volume f?om 10 to 1.5 mL resulted in an accumulation of the products of anaerobic metabolism and a reduction in extracellular pH, thus mimicking the effects of global ischemia on the heart. In addition to maintaining the anoxic environment, this low volume pexfbsate was necessary in our experiments to prevent lysis secondary to cellular dehydration. Unfortunately, the volume overlying our cells is somewhat greater than the volume to which cardiac cells are exposed during global ischemia, and thus may actually represent a form of low-flow ischemia analogous to limited cardioplegic infusion. Our experiments, although supportive, are mainly pharmacologic in nature. Although the agonists and antagonists utilized to demonstrate adenosine and potassium channel involvement are widely accepted, they lack specificity. For example, exogenous adenosine was utilized to demonstrate a preconditioning-mimetic effect on human cardiornyocytes, while SPT was employed to abolish preconditioning. While these results lend support to the hypothesized involvement of adenosine receptors in the preconditioning cascade, neither exogenous adenosine nor SPT is seIective enough to enable distinction between the adenosine Al and A3 receptor subtypes. Further experimems are necessary to delineate the contributions of these selective adenosine receptors to the preconditioning phenomenon. Similarly, adenosine and PMA incubation resulted in an increase in the phosphorylation of PKC-specific peptides, the effects of which were abolished with the application of Calphostin-C. However, due to the lack of specificity of the antibodies employed, we cannot comment with regards to which of the 11 isoforms of PKC were activated by preconditioning, or if these isoforms were different from those activated by adenosine. Moreover, definitive phosphorylation of the IC4*-p channel could not be demonstrated due to the inability to isolate such channels fiom human ventricular myocytes- Finally, although the KrAIp channel agonist diazoxide possesses a marked (2000 fold) ;rffilni~for mitochondria, it is not entirely rnitochondrial specific, and therefore may stimulate sarcolemmal in addition to rnitochondrial KIAP channels. Unfortunately, currently available sarcolemrnal-specific K+ATPchannel inhibitors (HMR 1883 and KMR 1098)~~~"~~were not available at the time of these experiments, such that observed effects may have been reIated to the activation of alternative receptor subtypes. The ECATPchannel inhibitor 5-hydroxydecanoate (5-HD), although previously available, was not postulated to be rnitochondrial specific until 1998,~" and as such, was not included in our investigations. Moreover, recent studies have questioned the selective nature of 5-HD due to such findings as action potential shortening and prevention of K+ efflux, effects generally postulated to be secondary to the inhibition of sarcolemmal KA-pchannels. Our studies involving the ultrastructural characteristics of preconditioned mitochondria were an attempt to provide evidence in support of our pharmacologic findings. Although our structural findings within cardiac mitochondria were compatible with previous reports, demonstration of a conclusive link to KrASpchannels cannot be accomplished without definitive identification and isolation of the human cardiac KC.- channel. Such an advancement will be crucial in confirming any inherent autoprotective effects of cardiac myocytes, and will aid researchers in the development of strategies aimed at stimulating and optimizing such autoprotective mechanisms. Finally, our studies are limited to the 'coronary bypass surgery' setting alone. However, if the preconditioning phenomenon is to realize its maximal potential in clinical medicine, any 122 protective effects of rATPchannel opening demonstrated in CABG must be expanded to alternate medical and surgical scenarios. To this end, we have undertaken studies aimed at demonstrating the benefits of mitochondria1 KfATPchannel opening within the transplant setting.(Appendix 2) In a model of porcine cardiac transplantation, diazoxide-enriched donor blood perfirsion of cardiac dografts stored for prolonged periods ex-vivo (8 hours) facilitated recovery of myocardial fimction following allograft implantation, and promoted preservation of high energy phosphates.(Appendix 2) Furthermore, in complementary studies involving globally ischemic rat hems, Kevelaitis and colleagues3" demonstrated that pre-treatment with diazoxide was an effective means of improving preservation during cold storage (as is the practice in contemporary cardiac transplantation). Future Studies Although our resuIts are consistent with recently published reports, 372-374 hrther studies may serve to confirm such findings. Our studies involving pharmacologic agonists and antagonists of sarcolemmal and mitochondria1 KfAp channels provide indirect evidence of channel augmentation. However, kture studies are necessary to demonstrate actual manipulation of channel conductance using patch clamping models of whoIe celIs or isolated mitochondria1 preparations. Such studies would enable definitive determination of channel responses to various preconditioning stimuli. In fact, baseline patch clamping assessments were undertaken by the author, yielding a consistent resting membrane potential of -60 to -70 mV. Unfortunately, due to the somewhat &agile nature of the ventricular myocytes involved, further studies involving voltage manipulation were unsuccessfLl. Future studies would also likely involve more specific pharmacologic agonists and antagonists. Although such specialized preparations were previously unavailable, recent studies have yielded specific antagonists for both the sarcolemmal ICtAm channel (HMR 1883, HMR 1098)~~'~~~as well as the mitochondria1 K+An channel (~-HD).~" In keeping with the channel opening hypothesis, mitochondrial membrane depolarisation produced by K+ entry would also be expected to reduce mitochondrial calcium ently via calcium uniport, thus limiting calcium overload.326 Studies of mitochondrial calcium homeostasis would be beneficial in fbrthering this hypothesis. In addition, Holmuharnedov and colleagues have demonstrated that preloaded mitochondria release calcium in response to KATP channel activation, suggesting a possible protective effect despite preceding calcium over~oad.~~~ Demonstration of such an effect may prove instrumental in the eventual application of KATP channel openers under reperfhion conditions, or following an ischemic insult for the purpose of preventing fbrther injury.326 Although a link to ischemia-reperfision injury was not ascertained in our studies, several studies have shown that sarcolemmal K'** channels open in response to f?ee radicals, suggesting some link to a protective response. 376,377 Future studies may aim to demonstrate a similar response of mitochondrial ECATPchannels. As was demonstrated in our studies of exogenous preconditioning, adenosine applied during ischemia conferred a mild protective effect to human ventricular myocytes undergoing prolonged ischemia and repefision. This finding was likely due to a mild 'mass effect' of adenosine in this model (i.e. adenosine acted as a substrate for the production of AMP via purine salvage pathways). In order to achieve the maximal response to such an effect, adenosine would have required much higher doses of administration. Unfortunately, such a benefit could not be exploited due to the inhibitory effect of high dose adenosine on the receptor mediated pathways of preconditioning. However, in the presence of diazoxide, such receptor mediated pathways are effectively bypassed, thereby enabling administration of high dose adenosine for its mass effects, while promoting preconditioning via its receptor effects. Thus, future studies would explore the possible additive effects of high dose adenosine and selective KrATp channel openers, effects which have recently been demonstrated in a rabbit model.j6* Similarly, studies to date are not unequivocal in favour of the sarcolemmal versus the mitochondria1 KrAm channel. As such, it is entirely possible that both channels have some involvement in preconditioning.326 Molecular cloning of the K+Av channel subunits Kir6.x and SUR has demonstrated a number of different subtypes possessing differential regulation and pharmacology.326 Future studies are necessary to determine the role of such varying channels and channel subtypes in preconditioning. ina ail^, in keeping with the modern day trend towards molecular biological experimentation, hture studies could involve the use of cloned channels and/or knockout mice bred to under-express the KCAv channel. Recent studies by Jovanovic and colleagues employed K+ATPdeficient COS-7 cells for such purposes. 3 15.3 16 When these cells were exposed to 3 minutes of chemical hypoxia using dinitrophenol, marked calcium loading was demonstrated (similar to that observed in non-preconditioned cardiomyocytes) irrespective of pinacidil administration. However, when subunits (SUR2A and Kir6.2) of the cardiac sa~colemrnalKhTp channel were co- transfected into these cells using viral vectors, calcium loading was attenuated in the presence of pinacidil. Further studies are warranted to examine the effect of mitochondrial-specific KIATP channel openers such as diazoxide in these cells, or to assess the ability of 5-HD to block the protective effect of pinacidil in such cells.3z6 The long anticipated development of a clone for the mitochondrial K+ATPchannel would be of significant benefit in this regard. Alternate Effector Mechanisms of Reconditioning The 'second window of protection' describes a phenomenon whereby the protection afforded hy preconditioning occurs up-to 24 hours following the initial stimulus.378 Although we did not investigate the possibility of such aa effect in our model of human ventricular rnyocytes, the existence of such a phenomenon may possess significant merit and clinical applicability. In fact, in patients undergoing elective coronary bypass surgery, administration of adenosine 24 hours pre-operatively may cord a degree of myocardial protection that is additive to that afforded by intraoperative adenosine administration, for an overall enhanced effect. Another hypothesized preconditioning effector involves the production of specific cardioprotective proteins known as 'heat-shock' proteins. Such proteins have been shown to be produced in response to both ischemic379and thermal2" stimuli, and in similarity to RAPchannel mediated preconditioning, are believed to be induced through G protein stimulation and protein kinase C a~tivation.~'' Udortunately, involvement of this effector in early preconditioning was eventually refbted when neither blockade of DNA transcription with actinomycin D, nor blockade of RNA translation with cyclohexamide were shown ta prevent preconditioning.380.381 In fact, this effector would eventually be hypothesized to mediate the delayed effects of preconditioning, or more specifically, the aforementioned second window of protection.378 Using polyclonal antibodies for HSP70s, Taggart and colleagues examined the induction and expression of heat shock proteins after an obligatory period of ischemia in patients undergoing CABG.'~~In four patients subjected to brief alternating periods of normothermic ischemia and reperfusion, the amount of myocardial HSP72 protein was increased several fold. This was accompanied by an increased expression of HSP72 messenger RNA. In contrast, the amounts of myocardial HSP72 protein and mRhlA were unchanged in patients exposed to prolonged ischemia, suggesting a response only to preconditioning-mimetic stimuli. Other lines of evidence, although controversial, suggest that preconditioning may afford its cardioprotective effects via production of inflammatory factors such as prostaglandins, which have also been shown to be mediated by adenosine release and protein kinase C activation2ZJ ,383 In rats, Vegh and colleagues demonstrated a reduction in the antiarrhythmic effects of preconditioning when hearts were pretreated with the cyclo-oxygenase inhibitor meclofen, suggesting a relationship to prostaglandin production.3" Other studies, however, have since refuted such Yet another possibIe effector mechanism involves the mediation or prevention of free radical production, a well known cause of myocardial ischemic repefision injury. Various animal studies have contributed to this hypothesis. In a rat model of global myocardial ischemia, Tosaki and colleagues demonstrated preconditioning to reduce the concentration of tissue malonyldialdehyde, a non-specific indicator of f?ee radical-induced tissue damage.386 Similarly, Osada and colleagues were able to abolish the protective effects of preconditioning by simultaneously exposing rat hearts to superoxide di~rnutase.)~~However, recent evidence seems to refbte such findings. Preconditioning may mediate its protective effects via modulation of intracellular calcium flux. Since G-protein stimulation has been shown to be linked to adenylate cyclase activity, preconditioning may lead to a fd in intracellular CAMP concentrations via activation of adenosine receptors. CAMP has been shown to facilitate activation of L-type calcium channels (via PKC- mediated phosphorylation) which leads to calcium influx into the cell.u7 Since calcium has long been known to mediate ischemic myocardial damage,388a reduction in myocardial calcium influx during ischemia may provide the effector mechanism for preconditioning. Such a hypothesis has been proposed by several authors, including Steenbergen and coIleagues who demonstrated a reduction in intracellular calcium concentrations using [32~]nuclear magnetic resonmce imaging when preconditioning was applied to globally ischemic rat hearts.38g Finally, in addition to the aforementioned KCATPand calcium channels, modulation of the Na'm exchanger has long been proposed to be involved in preconditioning. Since protons are produced in large amounts during ischemia, proton extrusion is accomplished by the cell via the Na'm exchanger. The resulting elevation of intraceUular NaC concentrations leads to intracellular calcium loading via the ~a'l~a~'exchanger. As previously noted, elevated intracellular calcium facilitates ischemia reperfbsion injury. Indeed, Inhibition of the Na'm exchanger during ischemia and/or reperfbsion has been shown to produce a substantial cardioprotective effect in various rnode~s.~" SUMMARY of ORIGINAL CONTRIBUTIONS The aforementioned series of experiments have attempted to define the mechanisms and benefits of myocardial preconditioning in a human model of simulated ischemia and reperfusion. In doing so, we have emphasized the importance of various pharmacologic substitutes for ischemic preconditioning. We have previously shown that: 1. Ischemic preconditioning protects human cardiomyocytes from prolonged ischemia and repefision through an adenosine-receptor, protein kinase-C mediated pathway. 2. A maximal ischemic stimulus is necessary for the maximal protective effects of ischemic preconditioning to be realized, resulting in the degradation of ATP prior to prolonged ischemia and repefision (ATP 'debt ') . 3. Exogenous adenosine applied prior to ischemia effectively mimics the protective effects of ischemic preconditioning through a receptor mediated pathway involving protein kinase C activation. In support of our hypothesis We have currently shown that: 4. The final rate-limiting step in this preconditioning sequence involves KrAn channel opening which effectively preserves intracellular ATP during prolonged ischemia without incurring an associated ATP 'debt'. 5. Although K+ATPchannels are ubiquitous in nature, the channels crucial to preconditioning are primarily those within the mitochondria as evidenced by the ability to reproduce preconditioning (despite myocyte quiescence) with the mitochondria-specific KtAp channel opener diazoxide. 6. Protein kinase C (PKC) exists as a crucial intemediaxy (second messenger) in the preconditioning cascade. When activated, protein kinase C translocates to mitochondrial membranes, thereby transforming mitochondrial K+An channels to the open position. 7. K+Aw channel opening induces an increase in mitochondrial matrix volume, which in turn facilitates forward flow of the electron transport chain, thus enabling the preservation of mitochondrial ATP synthetic capability. This phenomenon is supported by the observation of an upregulation in respiratory chain enzyme activity with preconditioning 8. Finally, as is demonstrated in Appendices 1 and 2, we have attempted to extend our work 'from bench to bedride'. Our experimental findings of carciiomyocyte protection and ATP presexvation were successfXy confirmed in both large animal models of cardiac transplantation (Appendix 2), as well as within the clinical scenario, in patients undergoing elective cardiac surgery (Appendix 1). The aforementioned experiments remain both novel and unique within the contemporary literature. Our model of human ventricular myocytes is the only one of its kind, and has yet to be reproduced by other groups. As such, the above findings have never before been demonstrated in human ventricular myocytes. In view of the author's surgical background, the emphasis on clinical applicability and reproducibility is paramount to this thesis. Indeed, recent studies have demonstrated similar findings in differing human in-vitro models. However, such studies have involved ahial trabeculae or atrial myocytes exclusively.372-374 Although these models are of interest, they lack the clinical significance attributable to a ventricular model such as that presented in this thesis. Should atrial preconditioning prove to be beneficial, such benefits would almost certainly be limited to anti-arrhythrnic effects. Whereas low output syndrome secondary to ventricular dysfinction is a major determinant of perioperative morbidity and mortality, atrial arrhythmias do not necessarily preclude a favourable outcome and are unlikely to result in death. As such, we believe that the benefits of atrial preconditioning are of limited importance in comparison to those attributable to ventricular preconditioning. CONCLUSIONS Preconditioning is the most powerfid endogenously mediated form of myocardial protection. The value of this phenomenon stems fkom its ability to preserve mitochondria1 ATP synthetic capability despite the exposure to profound ischemia and reperfhion. Essential to this process is ATP mediated potassium channel opening. The potential significance of ATP dependent potassium channels in modern clinical practice cannot be understated. The ubiquitous nature of this channel has prompted study into various applications of potassium channel openers including therapy for muscle diseases involving membrane depolarization (ie. Hypokalemic Periodic Paralysis), attenuation of neuronal death secondary to toxic glutamate release and calcium accumulation in brain ischemia, control of hypertension, and treatment of angina pectoris.u9 Unfortunately, due to a wide range of unwanted side effects associated with the administration of potassium channel openers, clinical application has been avoided. Although intensive efforts are underway to develop increasingly cardio-specific formulations, the pro-arrhythrnic properties of KtAlP channel openers currently limit their application to the intraoperative setting done. Fortunately, the surgical scenario provides a unique opportunity to manipulate and modify all facets of ischemia and reperfhion under controlled circumstances. Administration of KtATP channel openers either by a precrossclamp infusion or via cardioplegia offers an ideal application for this valuable resource, free of any arrhythmia related limitations. Moreover, our findings suggesting that KIAV mediated cardioprotection is not dependent on action potential shortening, and that the site of action is at the mitochondria rather than the cell membrane, may fixher promote clinical applicability by enabling the use of potassium channel openers specific for mitochondria, thus precluding any proarrhythmic tendencies. Nonetheless, despite such advances, certain questions remain unanswered. Although good evidence exists to suggest a protein kinase C mediated second messenger system, the PKC isoforms involved remain to be determined. Moreover, although multiple channel phosphorylation sites have been identified, actual channel phosphoryhtion has yet to be demonstrated. Presently, the Limiting factors remain the cloning of a human cardiac K+ATP channel along with production of a specific channel antibody for use in channel isolation and immunofluorescent staining. With such advancements along with improvements in patch-clamp technology, hture investigations should aim at repeating the aforementioned studies under patch- clamp conditions for codinnation of ion fluxes. Furthermore, since much of the data concerning K+An continues to be derived from animal studies where results are con£licting and interspecies differences are numerous, fbture clinical investigations are necessary. In deed, preconditioning effects demonstrated within the laboratory setting are of little use unless they can be reproduced within the clinical setting. Our studies of adenosine cardioplegia in patients undergoing coronary bypass surgery have contributed a great deal to this end- In summary, although our understanding of myocardial preconditioning is far from complete, identification and characterization of the K+*P channel may have brought us one step closer to our ultimate goal of optimidng intraoperative myocardial protection. Further studies involving human preconditioning are sure to improve our understanding of this valuable phenomenon. APPENDIX ONE Adenosine Cardioplegia in Contempormy Coronory Bypass Surgery: A Prospective-Randomized Trial SUMMARY OBJECTIVES: We endeavoured to further explore the possible protective effects of adenosine, and to determine the optimal mode of administration of exogenous adenosine by initiating a phase I1 prospective evaluation in patients undergoing elective coronary bypass surgery (CABG).~'" Since adenosine's protective effects were hypothesized to be both receptor and substrate mediated, and since late benefits could be related to a free radical-scavenging pathway, the effects of exogenous adenosine were evaluated both prior to and during the ischemic crossclamp period, as well as during reperfision. MZTHODS: Twenty-one patients undergoing elective CABG using tepid (29°C)4:l blood cardioplegia were assigned to receive adenosine, while 20 patients received no adenosine (control group). Patients randomized to the treatment group received a 10 minute precrosscIamp intravenous infirsion at 100 pmoVkg!min via the venous reservoir of the cardiopulmonary bypass circuit, followed by a 500 pol infusion via the first SO0 mL of high potassium cardioplegia (Low Dose). Control patients underwent similar interventions however received no adenosine. In a separate, non-randomized cohort, 12 patients with poor preoperative left ventricular hction (Ejection Fraction < 40%) received a 200 poVkg/min precrosscIamp and repefision adenosine infusion, in addition to a 2 mM cardioplegic infUsion throughout the crossclamp period (High Dose). Arterial and coronary sinus blood samples along with left ventricular biopsies were obtained prior to @re-crossclamp), during (crossc~nmp),and following (post-crossclamp) crossclamp to enable evaluation of adenosine levels, high-energy phosphate levels and rnetaboiic parameters. Postoperative hernodynamic parameters (pulse rate/rhythm, systolic/diastolic blood pressure, mean arterial pressure, pulmonary arterial pressure, cardiac output, cardiac index, systemic vascular resistance) were monitored to evaluate the clinical . benefit, if any, of adenosine administration. RESULTS: The pre-crossclamp intravenous adenosine ifision induced controllable hypotension in the high but not the low dose patients, although elevated serum adenosine levels were not measurable in either group. During the cardioplegic adenosine inibsions, serum adenosine levels increased dramatically in both groups (High Dose: pre-crossclam~1.49+/-0.14nmoVg serum, crosscZomp=ll82.~9+/-~.6nmoYg sewLow Dose: pre-crossclamp= 1.5+/-0.36 nmoYg serum, crossclmp=466.03+/-64.7 nmoVg serum; p<0.0 1). Similarly, markedly elevated tissue levels of adenosine were found in myocardial biopsy samples during the cardioplegic infusion only (Low Dose: pre-crossclamp=O. 19+/-0.11 pmo Vg; crossclnmp= 1-3 a+/-0 -24 pmoVg; p<0.0 1). Arterial-coronary sinus differences suggested myocardial metabolism of adenosine during the cardioplegic infusion. -Incomparison to controls where tissue ATP levels decreased by 25% during crossclamp, tissue ATP levels were preserved in both the low dose and high dose adenosine groups with crossclamping (Low Dose: pre- ~rossclamp= 2 1.7+/-3.5 pmoVg, post-crossclamp = 20.6+/-5.1 pmoVg; High Dose: pre- crosscImp = 26.8 +/-4 -2 prnol/g, post-crossclamp = 29.5+/-4.7; ControIs : pre-crosschp = 17.9+/-3 -2 pmoWg, post-crossclamp = 14.7+/-2.5 poVg; p<0.05). Patients receiving adenosine tended to produce more lactate during the pre- and early XCL periods in comparison to controls (Pre-crossclamp: Low Dose = -0.09tl-0.08 mmol/L, High Dose = -0.24+/-0.06 mrnol/L, Control = 0.16+/-0.1 mmoVL; Crossclamp: Low Dose = -0.3+/-0.06 rnmoVL, High Dose = -0.7+/-0.12 mmol/L, Conbd = 0.IS+/-0.1 mmoVL; p<0.05). No significant difference in coronary flow augmentation was noted with adenosine administration. Moreover, no metabolic or hernodynamic differences were noted between groups following XCL removal, and no clinical benefit was attributable to adenosine administration. CONCLUSIONS: Adenosine was sde for administration to patients undergoing coronary bypass surgery. Adenosine cardioplegia stimulated lactate production under normoxic conditions and facilitated preservation of high energy phosphates during ischemia. INTRODUCTION The contemporary results of coronary bypass surgery (CABG) are excellent. The following study was undertaken to determine the optimal dosage, route of administration, and timing of adenosine administration in patients undergoing elective coronary bypass surgery. Attempts were made to determine the mechanism of adenosine's effect, and to relate such effects to those previously described in our non-clinical models. METHODS Patient PopuIa fion Seventy-three patients undergoing isolated, elective, coronary bypass surgery consented to participate in a metabolic evaluation of adenosine enhanced cardioplegia. Patients with recent myocardial infarction, or previous cardiac operation were not eligible for entry into this study. All patients signed an informed consent form approved by our institutional ethics committee. The preoperative demographics of aJl patients by group are shown in Table Al-1. Operative Techniques All patients underwent coronary revascularization by one surgeon (RDW). The protocols for intraoperative patient management have been previously described in detail.3g' Briefly, cardiopulmonaxy bypass was established via ascending aortic cannulation and a single two stage right atrial cannula. During cardiopulmonary bypass, the hematocrit was maintained between 20% and 25%, pump flows between 2.0 and 2.5 ~/min/rn~,and mean arterial pressure between 50 and 60 rnmHg, with administration of sodium nitroprusside or phenylephrine hydrochloride as required. Patients were not actively cooled, and systemic temperatures were allowed to drift to 34"C+/-1 OC. Rewarming of all patients was commenced during construction of the third last anastomosis. The left internal mammary artery was anastornosed to the left anterior descending coronary artery as the last graft in all patients. Following anaesthetic induction and prior to initiation of cardiopulmonary bypass, patients were allocated to one of twc groups determined by a computer generated randomization table [Group 1: Low Dose group (N=21); Group 2: Placebo Controls (N=20)]. In a separate cohort, non-randomized patients with poor ventricular function (Ejection Fraction < 40%) received intraoperative adenosine at higher doses Fgh Dose group (N=12)]. Based upon our previous findings174and those of other investigators218,adenosine was administered at predetermined quantities either intravenously (via normal saline infusion) or via 8:l (arterial b1ood:crystalloid) cardioplegia. In the 'Low Dose' group, patients received a pre-crosscIarnp infUsion of adenosine at 100 umol/kg/min over a period of 10 minutes via the venous reservoir of the cardiopulmonary bypass circuit, followed by 500 umol via the initial arresting (high potassium) bolus of cardioplegia. In the High Dose group, patients received a pre-crossdamp infusion of adenosine at 200 umol/kg/rnin over a period of 10 minutes via the venous reservoir of the cardiopulmonary bypass circuit, followed by 2 mM via all cardioplegic infusions, followed by a repefision dose (delivered via the venous reservoir of the cardiopulmonary bypass circuit) of 200 umoI/kg/min during the first 15 minutes following crossclamp removal. Control patients undement similar interventions, however, received no adenosine. AU cardioplegic solutions contained equivalent concentrations of potassium chloride (27 mEqL for high potassium induction; 8 mEqL for low potassium maintenance), magnesium sulphate (6 rnEq/L for all solutions), tris(hydroxymet hy 1) amino methane (THAM; 125 rnrnol/L) and citrate-phosphate-dextrose (CPD; 4 mmoUL). AU individuals involved in conducting the operative procedure including surgeons, perfbsionists and nursing staff were blinded with respect to randomization groups. Following institution of cardiopulmonary bypass, all patients received an antegrade induction dose (approximately 1000 mL) of tepid (29'~) high potassium blood ~ardio~le~ia.~~' Upon achievement of successll cardioplegic arrest, patients received near-continuous retrograde delivery of via the coronary sinus. After completion of each distal anastomosis, the pro of the vein graft was connected to a cardioplegic manifold enabling simultaneous delivery of antegrade and retrograde blood cardiopelgia. Cardioplegic flow rates were maintained at 200 mL/minute, unless the coronary sinus pressure exceeded 40 mmHg, in which case flow rates were adjusted appropriately.3g2The flow rate was maintained above 100 rnL/min in all patients. " Following completion of all intraoperative biochemical assessments, patients were weaned from cardiopulmonary bypass and transferred to the cardiac intensive care unit. Postoperative intensive care unit management was uniform in all patients, and followed an intention to extubate within six hours of Serial hernodynamic measurements were obtained at 1, 2, 4, 8 and 24 hours following aortic cross-clamp removal, including heart rate, mean arterial pressure, pulmonary capillary wedge pressure, central venous pressure, and cardiac output. Cardiac index, left ventricular stroke work index, and systemic vascular resistance were calculated using standard formulae.391.392 Perioperative myocardial infarction and low cardiac output syndrome were determined using previously established criteria.394 Briefly, the presence of a new Q-wave or left bundle branch block on the postoperative electrocardiogram or a significant elevation of CKMB in the presence of a pre-existing EKG abnormality were employed to identify a myocardial infarction. Furthermore, low output syndrome was defined as the requirement for intraaortic balloon pump or sustained inotropic support for greater than 30 minutes in the intensive care unit. Patients who received low dose dopamine (<5 pg/kg!min) for renal perfirsion were not considered to have low output syndrome. Biochemical Assessments Arterial and coronary sinus blood samples were obtained at baseline, during each cardioplegic delivery, and at pre-specified intervals during reperfision. In addition, a coronary sinus catheter was left in-situ in order to enable sampling of coronary sinus blood at 2,4, 8 and 24 hours following aortic cross-clamp removal. Blood samples were assayed for oxygen content, acid, and lactate concentrations according to previously described protocoIs.391,392 Differences between arterial and coronary sinus blood samples enabled calculation of myocardial extraction or release of the aforementioned metabolic markers. During cardioplegic arrest, myocardial consumption or production was calculated after adjusting both extraction and release values for coronary flow. Due to adenosine's short half life within the clinical scenario (<6 seconds), specified blood samples and myocardial biopsies were also assayed for adencsine concentrations to enable confirmation of pharmacoIogic exposure. Left ventricular biopsies were obtained in all patients to enable determination of myocardial adenosine and high energy phosphate concentrations. Full thickness biopsies were snap-fkozen in liquid nitrogen and then homogenized in 250 rnL of phosphate buffered saline (PBS). Determination of adenosine and high energy phosphate levels was performed using a modification of a previously described technique.335 In a selected group of patients, measurement of coronary perfhion pressures was undertaken under varying flow conditions to enable determination of adenosine's effects on coronary vasodilatation. Baseline measurements of coronary pefision pressure were undertaken in all groups immediately upon achievement of cardioplegic arrest (prior to adenosine administration) and prior to cross-clamp removal (reperfbsion). In the Low Dose group, measurements were undertaken immediately following adenosine administration (via the initial arresting cardioplegic dose), as well as prior to cross-clamp removal. Measurements were conducted sequentially at flow rates of 100, 200 and 300 rnL/rninute, and were compared to controls. Sfatistical Analyses Statistical analysis was performed employing the SAS analytical program (SAS institute; Cary, NC). Categorical data were analyzed using chi-squared or Fisher's exact test as appropriate. Continuous data were analyzed using analysis of variance (ANOVA) and are expressed as mean+/-standard deviation, unless otherwise specified. Hernodynamic data were analyzed using analysis of covariance (ANOCOVA)examining the main effects of time, group, and preload. Left ventricular preload was estimated tiom measurements of pulmonary capillary wedge pressure. Statistical significance was assumed for a p value <0.05. RESULTS Operative Data Randomization groups were similar with respect to the number of distal anastomoses performed, the mean aortic cross-clamp and cardiopulmonary bypass times, and the volumes of cardioplegia delivered. There were no differences in the times of cardioplegic interruption expressed as a percentage of the total cross-clamp time @=0.47). The pre-crossclamp intravenous adenosine infusion induced controllable hypotension in the High Dose but not the Low Dose patients, although elevated serum adenosine levels were not measurable in either group. In contrast, during the cardioplegic adenosine infusions, serum adenosine levels increased dramatically in both groups (High Dose: pre-crossclamp = 1.49+/-0.14 nrnoYg serum; crossclamp = 1182.59+/-9.6 nmoVg serum. Low Dose: pre-crossc1amp = IS+/- 0.36 nmoI/g serum; crosscIamp: 466.03tL64.7 moYg serum; p elevated tissue levels of adenosine were found in myocardial biopsy samples during the cardioplegic hfbsion only (Low Dose: pre-crossclamp = 0.19+/-0.11 umoVg; crossclamp: 1.3 8+/- 0 -24 umol/g; p In keeping with the higher incidence of hypotension, patients in the High Dose group required sigmficantly higher doses of intravenous neosynephrine in comparison to both Low Dose and control patients in order to facilitate maintenance of adequate systemic pressures during adenosine administration (Mean dosage of neosynephrine, High Dose: 6.3+/-2.1 mg; Low Dose: 2.lW-1.6 mg; Controls: 1A+/-1 -7 mg; p=0.03). Biochemical OKtcomes Figure Al-1 demonstrates the effects of adenosine administration on myocardial lactate release during the surgical procedure in comparison to controls. Adenosine administration stimulated lactate release under both nomoxic and ischemic conditions. Patients receiving adenosine tended to produce more lactate during thepre- and early XCL periods in comparison to controls @re-crossclamp: Low Dose = -0.09+/-0.08 mmoVL, High Dose = -0.24+/-0.06 mmoVL, Conbol = 0.16+/-0.1 mmoVL; Crossdamp: Lav Dose = -0.3i-/-0.06 mmoVL, High Dose = - 0.7+/-0.12 rnmoI/L,, Control = O.lS+/-0.1 mmoVL; p<0.05). Figure A1-2 demonstrates the effects of adenosine administration on myocardial ATP concentrations in comparison to controls. In comparison to controls where tissue ATP levels decreased by 15% during crossclamp, tissue ATP levels were preserved in both the low dose and high dose adenosine groups with crossclamping (Low Dose: pre-crossclamp = 2 1.7+/- 3 -5 pmoVg, post-crosscZmp = 20.6+/-5.l pmoYg; High Dose: pre-crossclamp = 26.8+/-4.2 pmoVg, post-crosscZmp = 29.5+/-4.7; Controls: pre-crossclmnp = 1 7.9+/-3-2 prnoyg, post- crossclamp = l4.7+/-2.5 pmoUg; p<0.05). The presewative effects were not different between groups regardless of the dose of adenosine administered. Hernodynamic Outcomes Figures A1-3 and A14 demonstrate the hernodynamics for all three groups up to 24 hours postoperatively. Neither cardiac index nor left ventricular stroke work index were affected by adenosine administration, regardless of filling pressures at any time period. V'odilatory Effects Cardioplegic perfusion pressures were measured in all groups at varying flow rates. In the Low Dose group, perfhion pressures dropped immediately after adenosine administration at all flow rates (A perfusion pressures: 100 rnUmzn: -4.4+/-3 -5 mmHg; 200 mlJmin: - 1 1.4+/-9.1 mmHg; 300 mUmin: -9.6+/-6.2 mrnHg; p=0.037, effect of increasing flows). These changes were transient in nature in the Low Dose group, in that perfision pressures increased with hrther cardioplegic administration once adenosine was discontinued (100: 10.5+/-9.1; 200: 18.4+/- 16.9; 300: 16.6+/-11.8;p<0.05 vs post-cardioplegia pressures). In the High Dose group, perfusion pressures also dropped significantly (100: -6.1+/-5 -2; 200: - 19.3+/-8.7; 300: -28.6-W 13 -5; p=0.015, effect of increasing flows; p=0.42 vs Low Dose). In contrast to the Low Dose group, such changes were noted throughout the cross-clamp period due to the continuous administration of adenosine. In the control group, in contrast to adenosine treatment groups, cardioplegic pefision pressures increased linearly with increasing flow rates (100: l6.3+/-1 5.5; 200: 20.7+/- 18.5; 300: 3 1.7+/-27.2;p=0.022 effect of increasing flow rates). Finally, systemic hypotension was also noted in the high dose, but not the low dose group. Such hypotension, however, was easily controlled by simultaneous administration of systemic vasoconstricting agents (i-e. neosynephrine) via the venous reservoir. CONCLUSIONS Adenosine administration during coronary bypass surgery stimulated myocardial lactate production during both normoxic and ischemic conditions, likely via stimulation of glycolytic flux. Moreover, adenosine administration facilitated myocardial ATP preservation despite the prolonged ischemia associated with cardioplegic arrest. Although such differences were not affected by the dose of adenosine administered, high dose adenosine increased coronary vasodilatation to a greater extent than did low dose adenosine and necessitated administration of larger doses of neosynephrine in order to counteract associated decreases in systemic vascular resistance. Neither dose of adenosine had any significant effect on postoperative hernodynamics or patient outcomes. Table Al-1. Preoperative Characteristics by Group. I Characten'stic (NP) ( Low Dose (iV=2I) I High Dose (N=12) ( conhol (N=ZO) I Male Gender 19(95) 9(75) 20(100) Age > 70 years 9(45) 9(75) 12 (62) I L V Grade 3-4 0 12(100) 3(1 7) I L@t MMoin Disease I 1(8) I 3 (12) I >2 Vessel CAD 16(76) 12(100) 18(93) Diabetes 10(47) 2(16) T(25) Urgent Timing 0 0 0 APPENDIX TWO Dinoxide-Enhanced Donor Blood Perfusion for Prolonged Storage of Cardiac Allografts SUMMARY OBJECTIVES: Methods to extend allograft storage may deviate the shortage of donor hearts by enabling organ procurement form distant locales. METHODS: Yorkshire pigs (50-55 kg) were used to perform 16 orthotopic cardiac transplants. Storage time was doubled fiom current standards to 8 hours, during which hearts were either stored on ice (Control; n4) or perfbsed using intermittent retrograde normothennic (20°C)donor blood with (n=6) or without (n=6) the ATP-dependent potassium channel opener diazoxide @ZX). Donor blood (3 1lo+/-230 mL) was harvested, diluted in 2000 mL of crystalloid, and pefised via gravity at a pressure of 50 mmHg for 7 hours. A MiIlar micromanometer was used to measure left ventricular developed pressures at varying balloon induced end-diastolic volumes- Arterial and coronary sinus blood samples as we11 as myocardial biopsies were obtained to examine myocardial metabolism. RESULTS: All non-perfused hearts suffered severe ischemic contracture and could not be weaned from cardiopulmonary bypass. Following transplantation, 6 of 6 DZX pefised hearts were successfilly weaned fiom cardiopulmonary bypass in comparison to 5 of 6 non-DZX pefised hearts. Inotropic support was required to wean fiom cardiopulmonary bypass more frequently in the non-DZX group (3/6 vs. 1/6;p=O. 17). Similarly, more hearts in the non-DZX group required lidocaine therapy for the management of unretractable ventricular fibrillation (4/6 vs. 0/6;p4.04). There was no significant interactive effect between group and balloon volume either before or after transplantation. The average percent recovery of developed pressure was 76.7+/-18.1% in the non-DZX group and 87.8+/-27.3% in the DZX group (p=0.42). Similarly, there was no significant interactive effect between group and balloon volume before or after transplantation. Although hearts in both groups displayed diastolic dysfunction following transplant at ion, hearts in the DZX group appeared to be less profoundly Sected (% increase in diastolic pressures, non- DZX: I32.2+/-22.9%; DZX: 1 19.8+/-3 6.4%; p=0.27). Non-pefised hearts demonstrated the greatest reduction in baseline myocardial ATP concentrations (Pre-crossclamp: 65.9+/-17.3 umoWg; Post-crossclmp: 12.2+/-10.6 umoYg). Donor blood perfusion afforded significant ATP preservative effects in comparison to non-perfused controls. However, DZX perfusion afforded a greater degree of ATP preservation in comparison to the non-DZX group (DZY. Pre-crossclamp : 82.4+/-26.8 umol/g, Post-crossclamp: 53 +/-28.3 umol/g; non-DZX, Pre-crossclamp 106.9+/-2 1.1 umoWg, Post-crosscIamp: 73.7+/-31.5 umol/g). CONCLUSIONS: Donor blood pefision improved functional outcome following prolonged storage of cardiac allografts. DZX perfused hearts demonstrated fewer ventricular arrhythmias with weaning, and seemed to limit the development of diastolic dysfunction. Such effects may have been secondary to the ATP preservative effects of DZX. I2VTRODUCTION Orthotopic cardiac transplantation has proven to be an effective treatment for end-stage cardiac disease. Unfortunately, the number of patients awaiting transplantation greatly exceeds the number of available donor organs. Methods to extend the safe period of donor organ storage or to expand the use of marginal organs may help to alleviate this shortage. Presently, hearts are stored in ice after removal &om a donor. With this method, an ischemic time of up to four hours is normally well tolerated. Unfortunately, primary graft dysfbnction remains a major source of morbidity and mortality in the early postoperative period- Moreover, such a time limitation precludes procurement of donor organs fiom distant locales (i-e. procurement fiom Western Canada for transplantation in Eastern Canada, and vice versa), thus hrther contributing to organ shortage. Burt et aPg5demonstrated that the greatest loss of contractile function occurs during the period of hypothermic storage. Furthermore, See et atg6 demonstrated that hypothermic storage results in severe mitochondria1 dysfimction. Similarly, other investigators have found that hypothermia delays the recovery of myocardial metabolism. 397,398 Therefore, improved methods of allograft preservation are necessary to facilitate the recovery of both metabolism and hnction of donor hearts following cardiac transplantation. In an experimental animal modeI, we identified a very simpIe and practical technique that may double the safe storage time of donor hearts. This method makes use of the blood that is shed by the donor and normally discarded at the time of organ procurement. This blood, which is actually a mixture of arterial and venous blood as well as cardioplegic solution, can be collected and used to pehse the donor heart at room temperature. Our preliminary investigations have demonstrated that intennittent retrograde perfusion of the donor heart with shed donor blood during the period of hypothermic storage improves both finctional and metabolic recovery of the heart following transplantation. Using such methods, we successfilfy extended the currently accepted maximal storage time fkom four to eight hours.399 The aim of the following investigations was to determine the metabolic and functional benefits of the mitochondrial-specifk potassium channel opener diazoxide when administered via donor blood to cardiac allografts during normothermic storage. We hypothesized that perfision of porcine cardiac allografts with diazoxide-enhanced donor-blood would maintain myocardial viability during prolonged storage and would promote early recovery of aerobic metabolism and would preserve high energy stores, thus facilitating successll transplantation. IYiVOUT.EDGE TO DATE Continuous perfusion of cardiac allografts during hypothermic storage has been demonstrated to improve myocardial functional recovery by various investigators.400.40 I Unfortunately, the pefision systems employed in these studies are too elaborate and cumbersome to- permit portability and widespread use in non-tertiary medical care facilities. Often, such techniques require exogenous oxygenation or a roller pump mechanism to provide adequate fI ow rates. This necessary equipment adds to the bulk and complexity of the perfusion apparatus. To enable its use within remote settings, an ideal pefision circuit would have to be portable, safe and easy to assemble. In a pilot study we have developed a simple pefision circuit utilizing donor blood which is shed during organ procurement. This circuit consists of a standard blood transhsion apparatus, a 40 micron particulate filter, and a -el. Using this method, we are able to harvest donor blood, filter for particulate matter and initiate perfhion within ten minutes of donor heart extraction. Our model of 50-60 kg Yorkshire pigs provides blood volumes which are similar to those of adult humans and a coronary anatomy which is more clinically relevant than either canine or ovine models. Our experiments have shown that four hours of global ischemia in this model results in a significant depression of left ventricular hction in the transplanted heart when the standard method of cold static (non-pefised) storage is used for preservation. Donor blood perfhion improved both metabolic and functional recovery of transplanted hearts."2 Blood vs. Crystalloid Perfusate If the benefits of dograft pefision are reIated solely to the washout of toxic metabok intermediates, then perfusion with crystalloid solutions during storage may provide a degree of protection which is similar to that seen with blood perfusion. Although pefision of cardiac allografts with crystalloid solutions has been shown to be beneficial, 4OO,4O 1 there is considerable evidence to suggest that crystalloid pefision can result in severe endothelid damage.403,404 Moreover, preliminary studies conducted by our group have demonstrated crystalloid perfhion of donor organs past 4 hours to contribute to signrficant myocardial edema with an associated detrimental effect on ventricular fbnction following transplantation. Blood cardioplegia has proven to be superior to crystalloid perfbsion in reducing ischemic reperfhion injury. In a canine model of global myocardial ischemia, we found that blood cardioplegia significantly reduced irreversible myocardial injury.40s These findings were validated in two clinical trials at our institution comparing blood to crystalloid cardioplegia in patients undergoing coronary bypass surgery.406,407 Blood cardioplegia provides more oxygen carrying capacity, more buffering capacity against myocardial acidosis and a more physiologic pefisate. Therefore, despite practical advantages to the use of crystalloid solutions during remote procurement, a blood based perfhate should result in improved metabolic and functional recovery following transplantation. However, the blood volume that we are able to harvest in the pig is insufficient to provide allograft perfhion for longer than 6 hours. Although recirculation of the shed blood is technically feasible, a sigdicant lowering of pH levels with the second passage precludes the use of such an option. Therefore, partial dilution with a crystalloid solution is necessary to provide adequate pefisate volumes for the desired storage time of 8 hours. Our preliminary investigations have shown that diluting donor blood with 2 litres of crystalloid solution results in decreased hemoglobin concentrations (65-70 gdL) and oxygen saturations (80-85%). A significant degree of compensation ensues under such conditions whereby the oxyhemoglobin-dissociation curve shifts to the left, and the bIood increases its dissolved oxygen content. We believe that this compensation, along with the heart's decreased metabolic requirements under hypotherrnic or tepid conditions will ensure an adequate supply of oxygen to the myocardium despite hernodilution. Continuous vs. Intermittent Perfusion Due to Limited donor blood volumes (despite dilution) and the detrimental effects of recirculation, options for the delivery of blood are restricted to either a continuous low flow (10 cc/minute) infusion, or an intermittent high flow (200 cdminute) inhsion. Each method provides both benefits and drawbacks. Although continuous perfusion supplies blood to the heart in a continuous manner during storage, flow rates through the coronary vasculature are relatively low (I0 cdminute), which may result in the accumulation of toxic metabolites or an increase in coronary vascular resistance due to insufficient myocardial perfusion. Conversely, with int emittent perfksion, near physiological flow ra-tes and pressures are maintained. However, the donor heart is exposed to intermittent ischemic episodes during storage, with the possibility of detrimental effects. Nonetheless, previous studies by our group 399 have demonstrated that continuous pefision is poorly tolerated by the arrested heart due to the development of severe myocardial edema and complete loss of diastolic compliance. The susceptibility to edema is likely due to the non-contractile nature of the heart which seems to markedly increase fluid retention. As such, intermittent pefision was utilized for the current experiments. Normoihermic vs. HypothermmrcPerfusion and Storage Buckberg and colleaguesq8 demonstrated that cardioplegic-arrest resulted in a 95% reduction in myocardial oxygen consumption. Hypothermia resulted in a firrther reduction of only 2%. In addition, hypothermia led to a delay in the recovery of myocardial metabolism and hnction following cardioplegic arrest. Lichtenstein et ato9reasoned that normothermic cardioplegic arrest effectively reduces oxygen consumption while avoiding hypothermia-induced injury. However, normothermia does permit limited myocardial metabolism, with a resultant accumulation of metabolic end-products such as lactate and hydrogen ions. Thus, some form of pefision (intermittent or continuous) is required to support this metabolism and to wash away toxic metabolites. Unfortunately, maintaining normothermic (3 7°C) perfusion during storage would require a more complex system and may limit the portability and ease of implementation achieved with our current circuit. Our perfitsion circuit is ideally suited to deliver a room temperature (2 1-25°C) perfisate. Furthermore, recent evidence from our institution reveals that tepid (29°C) myocardial pefision may provide the optimal temperature during cardioplegic arrest.39~7410 Tepid cardioplegia avoids the delayed recovery of ventricular function seen with hypothermic ~ardio~le~ia~~~while preventing the accumulation of metabolic end-products seen with nonnothermic ~ardio~legia.~~~Earlier studies by en dry^" demonstrated that the myocardial temperatures achieved with immersion in ice resulted in significant damage to mitochondria1 structure and cellular function Preliminary observations in our laboratory indicate that collection of donor blood and perfhion at room temperature results in a left ventricular apical temperature of 21-28°C. We have demonstrated that our technique of tepid perfusion resulted in a rapid recovery of myocardial metabolism and function thus permitting the safe storage of donor organs for periods of up to 8 hours.399 Antegrade vs. Retrograde Delivev of Perjiusate In our previous studies, we employed an sonic cardioplegia tack which was left in place in the aortic root throughout the storage period. This cannula was used for blood perfusion during storage and for cardioplegic infusion during the implantation procedure. Theoretically, donor organs should be fiee of atherosclerotic disease and thus antegrade pefision should be excellent to all regions of the myocardium. However, successfbl antegrade delivery of perhate requires that the aortic valve remain closed during perfusion, a condition which is not necessarily present and cannot be confirmed in the arrested heart. Such a condition is not a pre-requisite for successfid retrograde pefision. Retrograde delivery of cardioplegia via the coronary sinus was first described in 1957~'~and reintroduced recently as a means of perfusing myocardium subtended by critically stenosed Carrier et upL6reported that the use of continuous retrograde delivery of cardioplegia during organ implantation was associated with improved outcomes following transplantation. Our studies have shown that in comparison to antegrade perfusion, retrograde perfusion reduced myocardial edema and prevented deterioration in diastolic AU experimental protocols were approved by our institutional animal care committee and conformed to the Guide for the care and use of hzboratoy animals ~Hpublicafionno 86-23, revised 1985). Donor Operation Animals were anaesthetized with intramuscular ketamine (3 0 mgkg) and isoflurane, intubated and ventilated with 100% oxygen to maintain normocarbia. Following a median sternotomy, the heart and great vessels were exposed. Umbilical tapes were placed around the superior and inferior venae cava to permit adjustment of cardiac preload by caval snaring. Systemic anticoagulation was achieved with an intravenous injection of 10,000 units of heparin. To prevent ventricular arrhythmias, all animals received a 100 mg bolus of lidocaine hydrochloride prior to cardiac instrumentation. Following baseline measurements, a cardioplegic tack was inserted into the aortic root, a cross-clamp applied across the ascending aorta and 1000 mL of high potassium crystalloid cardioplegia (composition in mmol/L: Na' 127, K- 20, M~"6, CI- 7, SO< 6, tris-hydroxyrnethyl aminomethane (THAM) 4, dextrose 135) delivered to achieve cardiac arrest. The inferior vena cava and the Iefi pulmonary vein were transected to prevent cardiac distension. The donor heart was then extracted and placed in a plastic bag for storage. Hmesting Donor Blood for Subsequent Perfsion Duri~gOrgan Reservation Following heparinization, cardioplegic arrest and donor heart extraction, remnant donor blood (appror 2500-3000 mL) was collected in a standard surgical suction receptacle and anticoagulated with an additional 10,000 units of heparin. To achieve adequate volumes and to maintain myocardial arrest during storage, the harvested donor blood was diluted with 2 litres of high potassium crystalloid cardioplegia. This diluted blood was then filtered with a 40 micron particulate filter and collected in a standard blood transfbsion apparatus. We have previously demonstrated that the composition of this pefisate is a mixture of cardioplegic solution, arterial and venous blood. The pefisate has a measured hemoglobin concentration of 7Wll g/dL with a pOt of 54*15 mmHg and an oxygen saturation of 84*16%. The perfusate was delivered by simple gravity at a rate of 200 dhinute once every 20 minutes. Despite perfirsion at room temperature (20-25"C), pIacement of the donor organ in a plastic bag immersed in donor blood resulted in left ventricular apical temperatures of 19-24°C. Such temperatures are not significantly different fiom those observed with static storage. Blood perfbsion was continued throughout a 7 hour storage period until a cardioplegic infusion was administered just prior to implantation of the allograft into the recipient. Recipient Operation Preoperative sedation and anaesthesia was similar to that described for the donor operation. In addition, a margind ear vein was used for intravenous access and maintained patent with a 50 mWhour 5% dextrose infusion. Continuous electrocardiographic monitoring was employed and a carotid artery line inserted to measure arterial pressures. Following median sternotomy, the heart and great vessels were exposed. Umbilical tapes were placed around the superior and inferior vena cavae. Systemic anticoagulation was achieved by the addition of heparin to the pump prime (10,000 U) as well as by administration of an intravenous dose of 10,000 U. Ascending aortic and bicaval cannulation was used to place the recipient on cardiopulmonary bypass. Flow rates were adjusted to maintain a systemic perfusion pressure of greater than 50 mmHg. No vasoactive medications were administered during cardiopulmonary bypass. Systemic pefision was maintained at 37°C. After aortic crossclamp, the recipient heart was extracted maintaining a cuff of right and left atrium. The left hemiazygous vein was ligated at its insertion into the coronary sinus. The anastornotic margins were inspected and trimmed accordingly, in preparation for orthotopic transplantation. Blood pefision of the allograft was thereafter discontinued and an initial 350mL blood cardioplegic dose infused in aU animals at a flow rate of 100 mUmin. The blood cardioplegia consisted of a 2: 1 mixture of blood:crystalloid and was delivered following the completion of each anastomosis. Following completion of all anastomoses, the aortic crossclamp was removed and all hearts were repefised for a period of 45 minutes. If ventricular fibrillation occurred during reperfusion, three attempts were made to defibrillate the heart. If unsuccessfbl, 100 mg of Lidocaine was delivered intravenously and defibrillation attempted again (in our previous four-hour studies, 2 of 8 control hearts remained in intractable ventricular fibrillation despite maximal efforts). If required, epicardial pacing was employed to maintain a heart rate of 80 beats per minute. After 45 minutes of reperfirsion, ig of calcium chloride was given to all animals and an attempt made to wean &om cardiopulmonary bypass. If required, an intravenous infusion of isoproterenol was initiated to assist weaning. Weaning from cardiopulmonary bypass was deemed successfbl ifthe animal maintained a mean systemic pressure of 65 mmHg for thirty minutes. The animal was thereafter euthanized under anaesthesia by intravenous potassium chioride injection and exsanguination. Biochemical Measurements of Myocardial Metabolic Recovery In our preliminary investigations, we found that normal aerobic myocardial metabolism was inhibited following 4 hours of hypothermic ischemia despite 45 minutes of repehsion. We hypothesized that by facilitating early recovery of aerobic metabolism via stimulation of the mitochondrial electron transport chain, improved knctional recovery could be achieved even following prolonged (8 hour) ischemia. As such, metabolic interventions were required to facilitate continued fonvard flux of the rnitochondrial electron transport chain in the face of ischemia. We assessed myocardial metabolism by obtaining arterial and coronary sinus blood samples as well as fill thickness left ventricular myocardial biopsies. Blood samples were analyzed for partial pressure of oxygen @02), partial pressure of carbon dioxide (pCOz), pw hemoglobin concentration (Kb), oxygen saturation (SaOz) and lactate concentration Oxygen content (OzCon) was calculated according to the formula: 1.39HbtSaOz+0.003*pO~ Blood samples for lactate determination were mixed with 2 measured volume of 6% perchloric acid. Lactate concentration was measured in the protein-fFee supernatant with a commercially available assay (Rapid Lactate Stat Pack, Calbiochem-Behring, La Jolla, Calif.) Samples were obtained at baseline, prior to donor organ arrest and at 3 0 minute intervals during organ storage. In addition, samples were obtained during each cardioplegic dose of the recipient operation, immediately prior to cross-clamp removal and at fifteen minute intervals during the reperfusion period. Myocardial biopsies were assayed for concentrations of high energy phosphates. Measurements of adenosine triphosphate were performed using the modifications described by Weisel et of the step gradient technique developed by Hull-Ryde el aL3" Briefly, 25-50 mg myocardial specimens were obtained after the intervention of interest and then flash frozen in liquid nitrogen. Following removal of blood and connective tissue, adenine nucleotides were extracted in 5% perchloric acid and measured by high performance liquid chromatography. Assessment of Ventricular Function Left ventricular function was assessed and compared for each heart before extraction from the donor animal and following implantation in the recipient. Prior to functional measurements, all animals were monitored electrocardiographically, and systemic blood pressures were directly transduced using carotid arterial catheters. A fluid Ued latex balloon connected to a Millar micromanometer catheter was inserted via the apex of the left ventricle to permit on-line measurements of heart rate and left ventricular systolic and diastolic pressures. Measurements of end-systolic (LVESP), end-diastolic (LVEDP) and developed pressures (DP) were performed at 5mL increments as the balloon volume was increased fkom 0 to 50 mL. The percent recovery of developed pressure was calculated as the ratio of the post-reperfusion DP to the baseline DP at the same balloon volume. The average percent recovery of developed pressure (%DP) was determined using the trapezoidal rule: %DP = (100)Jv,Vb PP(Posfreper_firsion)/DP (baseline) * dV/(Vb-V,)] where Va is the smallest matching postrepefision balloon volume and Vb is the largest matching postrepefision balloon volume- Similarly, the change in diastolic compliance following transplantation was calculated by determining the ratio of the integrated areas of the postreperfision and baseline LVEDP-balloon volume relationship: Mean LVEDP ratio = LVbIpR& * dV/(Vb-Val] where PB is the LVEDP at baseline and PR is the LVEDP postrepefision. The mean LVEDP ratio reflects the average change in LVEDP for a given change in balloon volume corrected for the pretransplant compliance. Therefore, a ratio of one indicates that the change in LVEDP for a given change in balloon volume was identical before and after transplantation with no change in diastoIic compliance. A ratio greater than one is indicative of decreased diastolic compliance. Statistical Analysis Statistical analysis was performed using the SAS program (SAS Institute; Cary, NC). Categorical data were analyzed using chi-squared or Fisher's exact test where appropriate. Continuous data are expressed as the mean+/-standard deviation. The left ventricular developed pressure-balloon volume relation was analysed with two-way repeated measures Analysis of Variance (ANOVA) to simultaneously evaluate the main eEects of group and balloon volume as well as the interactive effect ('group *baZZuon volume relation). If the F-statistic of the ANOVA was positive, differences between groups were specified using Duncan's multiple range test. Statistical significance was assumed at a=0.05. EXPERIMENTAL PROTOCOLS In order to determine the benefits of diazoxide-enhanced donor blood perfbion, the following experimental protocol was undertaken: Prior to donor heart extraction, allografts were randomized to receive intermittent retrograde donor blood pefision either with (N=6) or without (N=6) diazoxide @ZX) enhancement, during an 8 hour normothermic storage period. Both groups were compared to hearts stored on ice (N=4) using conventional non-pehsion methods. Diazoxide was administered at a dose of 20 uM in keeping with previous dose response findings in our model of human ventricular myocytes. To enable adequate pefision during the extended storage times and to ensure maintenance of mechanical arrest, the volume of blood perfixsate was increased by dilution with 2 litres of high potassium crystdoid cardioplegia. The total (diluted) harvested blood volume in the pefised groups was then divided into 24 equal boluses which were administered every twenty minutes during the 7 hours of storage. Thus, equal volumes of perfusate were delivered in both perfused groups. In the perfused groups, donor blood was delivered via gravity to achieve a flow rate of 200 mL/rnin, and a pefision pressure of approximately 60 mmHg. Our previous studies indicated that approximately 3000 mL of blood can be harvested from the donor's chest. During storage, pefised hearts were placed in a plastic bag at room temperature. Following the ischemic storage period, pefision was discontinued and all hearts received 350 mL of blood cardioplegia. In the diazoxide enhanced group, diazoxide was also given at similar quantities via the cardioplegic doses which were administered during organ implantation prior to cross-clamp removal. For this experiment, a valid control group would entail hearts that underwent cold static storage for 8 hours. However, our experience has shown that static storage of porcine hearts for greater than 4 hours leads to irreversible injury and an inability to wean from bypass following transplantation. In fact, in our previous experiments involving cold static storage of porcine hearts for 4 hours, only 3 of 8 hearts were successfidly weaned from bypass. Thus, to avoid an unnecessary waste of animals and resources, results of this experiment were compared with the currently accepted clinical 'gold standard' of hypothermic storage on ice for a period of 4 hours. The recipient operation was identical in all groups and is detailed above. RESULTS Donor blood (3 110+/-230 d)was harvested, diluted in 2000 mL of crystalloid cardioplegia, and pefised via gravity at a pressure of 50 mmHg for 7 hours. All non-perfused hearts suffered severe ischemic contracture and could not be weaned from cardiopulmonary bypass. Following transplantation, 6 of 6 DZX pefised hearts were successfblly weaned fiom cardiopulmonary bypass in comparison to 5 of 6 non-DZX perfused hearts (p=0.62). Inotropic support was required to wean fiom cardiopulmonary bypass more fkequently in the non-DZX group (3/6 vs. 1/6; p=0.17). Similarly, more hearts in the non-DZX group required lidocaine therapy for the management of unretractable ventricular fibrillation (4/6 vs. 0/6; p=0.04). Recovery of Left Ventricular Function Figure A2-1 illustrates the developed pressure-balloon volume relationship before and after transplantation in both groups. There was no significant interactive effect between group and balloon volume either before (group%oZume, F=1.22; p=0.37) or after (F=0.59; p=0.64) transplantation. The average percent recovery of developed pressure was 76.7+/-18.1 % in the non-DZX group and 87.8+/-27.3% in the DZX group (p=0.42), Figure A2-2 illustrates the LVEDP-balloon volume relationship before and after transplantation in both groups. There was no significant interactive effect between group and balloon volume either before (group+oZume, F=2.6; p=0.52) or after (F=1.8; p=0.22) transplantation. Although hearts in both groups displayed diastolic dysbction following transplantation (ratio>l), hearts in the DZX group appeared to be less profoundly affected (% increase in diastolic pressures, non-DZX: 2 32.2+/-22.9%; DZX: 119. a+/-3 6.4%; p=0.27) Recovery of Myocardial Metabolism In the pefised groups, lactate release increased significantly with storage, and then returned to baseline values following 45 minutes of reperfirsion (effect of time, F=2.03; p=0.02). There was no interactive effect between group and time (graup*time eflect, F=3.26; p=0.23), and there were no differences in lactate release between the two pefised groups @ZX versus non- DZX) at any time point. In contrast, the non-pefised hearts demonstrated marked lactate release upon reperfirsion, with no return to baseline values. Figure A2-3 demonstrates the effect on myocardid ATP preservation in the three groups. Non-pefised hearts demonstrated the greatest reduction in baseline myocardial ATP concentrations (Pre-crossclamp: 65.9+/-1 7.3 umollg; Post-crossclamp: 12.2+/- 10.6 urnol/g). Donor blood pefision afforded sigruficant ATP preservative effects in comparison to non- perfbsed controls. However, DZX perfbsion afforded a greater degree of ATP preservation in comparison to the non-DZX group (DW:Pre-crossclmnp : 82A+/-2 6.8 umol/g, Post-crossclamp : 63.I+/-28.3 um0Vg; non-DZy Pre-crossclamp 106.9H-2 1 1 umollg, Post-crossclamp: 6 1.7+/- 3 1 -5 umovg) (eflect of time, E8.12; p=0.00 1; group *time effect, F=2.04; p=0.0 1 5 8). CONC.USIONS Donor blood perfhion improved hctional outcome following prolonged storage of cardiac allografts. DZX perhsed hearts demonstrated fewer ventricular arrhythmias with weaning, and seemed to limit the development of diastolic dysfunction. Such effects may have been secondary to the ATP preservative effects of DZX. -Series 1 \ - - -a-- - Series2 40 I 20 0 I I 0 20 40 60 -Series 1 Figure A2-1. Developed pressure-balloon volume volume relationships in (A) the non-DZX group (76.7% recovery), versus @) the DZX group (87.8% recovery). (Series 1: preoperative; Series 2: Postoperative) ---+--Seriesl 14 **- Series2 *-4- -*- 1 ---+--Seriesl - - 0 I - -series2 Figure A2-2. Left ventricular end diastolic pressure-balloon volume relationships in (A) the non-DZX group (132.2% recovery), versus @) the DZX group (1 19.7% recovery). (Series 1: Preoperative; Series 2: Postoperative). efSect of time: p =0.001 group *time effect: p=0.0158 Figure A2-3. High energy phosphate degradation according to group (Pre-XCL: Precrossclarnp; Post-XCL: Postcrossclamp). APPENDIX mE Isolation and culture of human ventricular cardiomyocytes Technique of isolation and culture of human ventricular cardiomyocytes Briefly, 20 mg biopsies were obtained &om the right ventricular outflow tract of patients undergoing corrective surgery for tetralogy of Fallot. After washing the specimen in phosphate buffered saline (PBS; NaCl: 136.9 mmoVL, KCl: 2.7 mmoVL, Na2HP04: 8.1 mmoVL, -04: 1.5 mrnoUL; pH: 7.4) all connective tissue elements were removed and the remaining myocardial cells were separated by enzymatic digestion using a mixture of 0.2 % trypsin (Difco Laboratories; Detroit, MI) and 0.1 % collagenase (Worthington Biochemical Corp.; Freehold, NJ). The separated cells were seeded onto cell culture dishes and cultured at 37'~and 5% COz in Iscove's modified Dulbecco's medium (GBCO laboratories; Grand Island NY) containing 10% fetal bovine serum, 100 U/ml penicillin, 100 mglml streptomycin, and 0.1 r&l P-mercaptoethanol). Purification was achieved using a dilution cloning technique. Enzymatically isolated cells were seeded at a low density (50-100 cells per 9 cm diameter culture dish) to enable morphological identification of individual cardiomyocytes by their rectangular shape and large size (40x80 pm), and separation from alternate cell types such as fibroblasts and endothelial cells. Using a Pasteur pipette, single cardiomyocyte colonies were then transferred to a separate culture dish. Cell cultures were inspected daily, and any contaminated dishes were discarded. Culture purity of greater than 95% was demonstrated for each cell passage with fluorescent monoclonal antibody staining for actin (ENZO Biochemical Inc.; New York NY) and human ventricular myosin heavy chain (Rougier Bio-Tech Ltd.; Montreal, QUE). Cells passaged 2 to 6 times, with a time from primary culture of less than 60 days, were utilized for this study. APPENDIX FOUR Ischemia and Reperfusion Model Techniquefor cell culture ischemia and repe$usion The technique used for simulating cell culture ischemia and repefision was defined previously by Tumiati et aL3" Following stabilization in pefision PBS (phosphate-buffered saline as defined in Appendix 3 with the addition of MgClz, 0.49 mM, CaQ 0.68 mM and glucose 3.0 mM) at 37'~for 30 minutes, ischemia was simulated by placing the cells into an air- tight pleldglass chamber (Figure 19) flushed with 100% nitrogen and exposing them to a low volume (1.5 mL) of anoxic @O 2=0 mrnHg) or hypoxic @O ,=20 mmHg) perfbsion PBS at 37'~ for 90 minutes. Deoxygenated PBS was prepared in 100 mL quantities by degassing normoxic PBS with 5% CO2 and 95% & until the measured pOz reached 0 or 20 mmHg, and the measured pC02 reached 10 mmHg (Blood Gas Analyzer Model IL13 12, Instrumentation Laboratory, Milan, Italy). During this process, perfbsion PBS was passed through two oxygen traps including a 1% W/Vsolution of NaSO, in deionized water (Trap #I, Figure 19) followed by a bicarbonate buffer (Na2C03 20 rnM, NaHCOs 20 rnM, Trap #2, Figure 7). The solution pHwas adjusted to 7.40+/- 0.05 and the osrnolality corrected to 290+/-10 rnOsm/L using 1.0 M NaOH and NaC1, respectively. In order to venfL the desired conditions within the nitrogen chamber, 2 mL of anoxk perfusion PBS was also placed in an open dish within the chamber and tested to ensure the absence of oxygen 5 minutes &om the end of each ischemia experiment. Reperfusion was accomplished by exposure to 15 mL of normoxic 37'~perfusion PBS for 30 minutes. APPENDIX FIVE Biochemical Measurements (Lactate, ATP, adenosine, and PKC assays) Lactate Assays The lactate assays undertaken were performed using the "Stat-Pack" Rapid Lactate Test kit (Behring Diagnostics, SommerviUe, NJ). The assay procedure is based upon the following two reactions: LDH A) L-lactate + NAD Pyruvate + NADH ALAT B) Pyruvate + L-glutamate 3 L-alanine + a-KG where LDH = lactate dehydrogenase, NAD = nicotinamide adenine dinucleotide, NADH = nicotinamide adenine dinucleotide (reduced), ALAT = danine amhotransferase and a-KG = alpha-ketoglutarate. Lactate is oxidized to pyruvate in a reaction catalysed by LDH and a molar equivalent of NAD is simultaneously reduced. The py-vate formed in the reaction is converted to alanine in the ALAT reaction, thus forcing the LDH reaction to completion. The change in absorbance of NAD at 340 nrn is proportional to the concentration of lactate in the sample. The lactate kit is comprised of two solutions: Component Concentration Vial A: Tris (hydroxyrnethyl) aminomethane 220 mM Glutamate 21 mM Manine aminctransferase 2.4 x lo3U/L Lactate dehydrogenase 2.1 lo4 U/L Via2 B: Nicotinamide adenine dinucleotide 3.2 rnM The components of these two vials are mixed together and stored at 2OC. Following each intervention of interest, the supernatant was removed from the culture dishes and centrifbged at 2000 rpm and 4°C for 5 minutes. The fluid portion of the sample was then removed and stored at 4°C for a same day assay, or fiozen in liquid nitrogen for later use. A standard (STD) curve was constructed by spectrophotometric assay of the following lactate dilutions (made from a 3.0 mM stock solution): STD Stock (pL) Saline (yL) Concentration (rng/rnL) The assay was undertaken by preparing 1:30 samp1e:reagent dilutions and measuring the absorbance in a spectrophotorneter at 340 nrn. The standard curve was confirmed to linear and within the range of the sample values obtained. The sample lactate concentrations were then read from the standard curve in mg/mL, and corrected for the initial sampIe volume to yield a mrnol amount. IntracelluZur A TP and Adenosine Assays Following the intervention of interest, the cardiomyocytes (grown on 9 cm diameter culture dishes) were flash f?ozen in liquid nitrogen, lyophilised overnight, and maintained at -80°C until required. To begin the extraction, a solution of 80% methanol:20% Hfl was heated to 75°C. Four mL of this solution were added to the culture dish after which the cells were removed with a cell scraper and placed into a homogenizer. An aliquot of an internal standard (1.5 mM 2- 0-methyladenosine,OMA) was added to the homogenizer. The cells were then homogenized and transferred to a methanol rinsed tube maintained at Hfl. The homogenates were allowed to sit for 60 minutes followed by centrifugation of the tubes for 10 minutes at 2500 rpm and 4°C. The supernatant was saved for further processing and ATP determination. The pellet was stored at - 80°C for eventual DNA assay. The supernatants were evaporated to dryness under nitrogen gas. The residues were reconstituted with 0.5 L HzO and 0.5 rnL of a 4:1 solution of f?eon:heptane. Following centrifbgation for 10 minutes at 11000 rpm and 4"C, the high energy phosphates were separated into the upper aqueous layer which was, in turn, removed, lyophilised and stored at -80°C until used. The high pressure liquid chromatography @?LC) analysis for adenosine triphosphate (ATP) and adenosine is performed using a modified protocol described by Hull-Ryde et ~31."~ Briefly, samples are reconstituted in assay buffer consisting of 100 mM WLC grade M&.&P04 (acetyl nitrile; pH=5.705). Sample injection was accomplished with a manual injector (model U6K, Waters Associates, Mississauga, Canada). Step gradient delivery of solvents was performed with a reciprocating pump (models 501 and 510, Waters Associates). The chromatographic column (RadiaI-Pak Resolve C 18 column, Waters Associates) with a 5 pm internal particle size was seated in a radial compression module (model RCM 100, Waters Associates) set at 175 atmoshpheres. A programmable multiwavelength detector (model 490, Waters Associates) was used to monitor and integrate the peaks at the absorbance wavelength of 254 nm. Solvent flow was initiated isocratically with the assay buffer for IS minutes. The solvent was then converted to 40% methanol: &O in a continuous gradient fashion using curve 8 of the solvent programmer and was continued for a period of 10 minutes. Between injections, the column was equilibrated with assay buffer. The nucleotide and nucleoside peaks were quantitated against the OMA standard. The lower range detection capability of this system for high energy phosphates was 100 pmol. The results of the assay were standardized for sample DNA content as described below. DNA Analyses The aforementioned fiozen pelfet was reconstituted in 1.5 cc of 5% perchloric acid (PCA) on ice. The sample was centrifbged at 2200 rpm for 10 minutes, after which the supernatant was removed. The pellet was resuspended in 1.5 mL of cold 5% PCA and was left on ice for a 10 minute period. The samples were then placed into a 70°C water bath for a IS minute period. During this time, 0.2 rnL of DNA standard (calf thymus DNA) mixed with 0.2 mL of 10% PCA was also set into the 70°C water bath- The samples and standard were both centdkged at 2200 rpm for 10 minutes, and the supernatants transferred to new tubes. The DNA standard was diluted with diphenylamine reagent @PA) for creation of a standard curve with the desired concentrations of DNA standard (i-e. 1.667, 3.33, 6.667, 9.99,13.33, 16.667 pg/mL). The standard curve was confirmed to be linear and within the range of the sample values obtained. The samples were then added in 0.5 mL diquots to 1.5 rnL of the DPA reagent. AIl tubes were covered and allowed to sit at room temperature for 15 minutes. The optical densities of the samples and standards were read from a spectrophotometer at 600 nm. The optical densities were matched in order to determine the DNA concentrations via the standard curve. Sample vdues were read fkom the standard in &mL and were corrected for total sample volume to yield a final value in pg. Protein Kinase C Analyses Isoform specific translocation of protein kinase C (PKC) was demonstrated by performing a 'slot blot' analysis on cellular cytosolic and membrane fractions. Following the intervention of interest, cells were washed, scraped, and resuspended in 50 pL of 50 pmoVL TRIS-buffered saline (150 mmol/L NaCl in 50% glycerol, pH=7.2). Cells were then sonicated and centrifbged at 14,000 rpm for 5 minutes. Following removal of the cytosolic soluble supernatant fraction, the pellet was resuspended in 50 pL of TRIS-baered saline to yield the membrane enriched £?action. Both fractions were then divided into equal aliquots of 25 pL each. One aliquot was employed for determination of protein concentrations after which the equivalent of 20 pg of protein for each sample was placed in the slot blot apparatus. Following protein transfer to nitrocellulose, each blot was exposed to an isoform-specific antibody for PKC-a and PKC-E. Western blot analysis using chemiluminescent detection demonstrated that each antibody was specific for PKC with no evidence of non-specific background staining. Slot blots were then scanned using a commercially available software program (Molecular Images; Mississauga, ONT) and each band was assessed densitornetricdy. Protein kinase C activity was measured using a modification of a previously reported assay.339 Confluent cultures of cardiomyocytes were exposed to the treatment of interest for 20 minutes. The cells were then rinsed, scraped and resuspended in 50 pL of 50 pmoVL TRIS- buffered saline. Following sonication, 10 @ of each cell extract was added to 15 pL of reaction buffer for 60 minutes. The reaction buffer consisted of equal concentrations of a Iissarnine rhodamine B-labelled peptide containing a PKC-specific phosphorylation site (epidermal growth factor receptor, RKRnRRL), an activating solution (Phosphatidyl-L-serine, lmg/mL), and a buffer containing 10 mmol/L ATP, 50 mrnoVL MgC12, 0.5 mmoVL CaCl z, 0.01% Triton X-100 and 100 mmol/L TRIS(hydroxyrnethy1)-amino methane, pH=7 -4 (PIERCE Biotechnology; Rockford, L). Following incubation, the reaction mixture was fractioned through a DEAE-sep harose colum equilibrated with 20 mmoYL HEPES (pH=7.9 at ~OC),20% glycerol and 1 mmoVL EDTA After binding to the positively charged column, phosphoryIated peptide was eluted with a 2 rnmoVL NaC1-HEPES buffered saline soIution. The absorbance of the eluted fluid was then measured using a spectrophotorneter (Beckmann Ltd.; Fullerton, CA) at 570 nm. Reaction buffer that had not been exposed to any cell extracts was also placed on the column, and the eluate used as a negative control. Cell extracts exposed to 20 nmol/L PMA (4P-phorbol 12-myristate 13- acetate) (Sigma Chemical Co., St. Louis, MO), a PKC stimulating phorbol ester, were employed as positive controls. Absorbance was subsequently corrected for protein content and expressed in relative units for absorbancehg protein. APPENDIX SIX Mitochondrid Studies Isolation of Milohondn'al Membranes from Human Venlrieular Myocytes Although several methods exist for the isolation of mitochondria from animal tissues (i-e. - differential centrifugation, sucrose-density gradient centrifugation), such methods have not been applied successllly to tissue culture preparations. The strategy employed for the aforementioned studies is based upon that described by Madden and colleagues whereby the ability of PercoU to separate plasma and microsomal membranes fiom mitochondria and lysosomes is combined with the ability of metrizamide to separate mitochondria from lysosomes341: Mitochondria were isolated fiom semi-confluent cellular aggregates (5 plates per treatment group; approximately 1x10~human ventricular myocyteslplate). All procedures were performed at 4'~. Cells were disrupted by nitrogen cavitation and further homogenized by four strokes with a Potter-Elvehjem homogenizer. The homogenate was centdkged at 1300g, for 5 minutes. The supernatant was decanted and placed on ice. The nuclear pellet was resuspended in 0.25 M sucrose and centrifuged at 1300g, for a further 5 minutes. This step was repeated and the supernatants fiom each wash were pooled accordingly to create a total perinuclea. sample (PNS)- In order to separate mitochondria fiom the PNS, a hybrid Percoll/metrizarnide discontinuous gradient was prepared by overlaying two rnL of 3 5% metrizamide (Nyegaard; Oslo, Norway) with two mL of 17% metrizamide followed by 5 mL of 6% Percoll (Sigma Chemical Co., St. Louis, MO) in multiple sample tubes. The tubes were then gently filled with PNS (-4.75 mL). Centfigation was performed for 15 minutes at 50,500gm (20,000 rpm) using an ultracentrifuge. The material recovered at the 17/35% metrizamide interface was resuspended in 100 pL of isolation buffer (0.25 mM sucrose, 20 Mm Tris, I mM EDTA; pH 7.0). An aliquot was removed and used to determine protein concentration. The remzining mitochondria were solubilized in 2x gel loading bder (125 rnM, Tris-HC1 pH 6.5, 4% SDS, 20% glycerol 573 pM B-mercaptoethanol) and used for Western analysis.(28) Assessment of Electron Transport Chain Envmatic Activity: To determine the mechanism whereby preconditioning affects mitochondria1 metabolism for high energy phosphate presenration, mitochondrial respiratory chain flux was studied by assessment of enzyme activity. COX, Complex IItm, and Complex I+III activities were determined on whole cell extracts as described by Glerum el ~l.,~'~Merante et al.,'" and Pitkanen et ~1.~~'All enzyme measurements were performed on a Cobas Para analyzer (HoBnan LaRoche, NO- Immunogold Labelling of PKand Transmission EM: Transmission electron microscopy and immunogold labelling methods were undertaken as described by Ursell et ~1."~Cells in confluent cultures were removed £?om their respective plates by gentle triturition, centrifuged at 5000 rpm for 5 minutes, and washed three times in phosphate buffered saline (PBS). The cells were fixed for 2 hours at room temperature in 0.001% gluteraldehyde and 2% paraformaldehyde in PBS,pelleted at 1000 rpm for 5 minutes, and washed three times in PBS. Pellets of cells were reacted with primary antibody to PKC-a for 75 minutes, and then immunolabelled by incubation with either gold-conjugated goat anti-rabbit IgG or gold- conjugated goat anti-mouse IgG. Both secondary antibodies were conjugated with 5 nm gold particles. CeUs were then post-fixed in 1% osmium tetroxide in Sorensen's buffer, pH 7.20, for 1 hour, dehydrated in an ascending series of ethanols, infiltrated and embedded in Embed 8 12 via propylene oxide. Following polymerization, ultra-thin sections were cut on a diamond knife with an ulaamicrotome exhibiting a pale gold interference colour. The sections were stained with ethanolic uranyl acetate and lead citrate, viewed, and photographed in a JEOL EM 1200 ExII transmission electron microscope. Morp hometric Assessments Purified mitochondria were filtered through Millipore filters (pore size 0.22 p)using positive air pressure. Post-fkation was undertaken as outlined above, after which the filters were dissolved in propylene-oxide. This resulted in a mitochondria1 pellicIe which could be embedded as a flat disk. The estimation procedures were undertaken at two suitably gradated IeveIs of magnZcation. The low magdication was used to estimate mean pellicle thickness and total volume, where as the higher magnification was used to estimate the volume and number of mitochondria in the pellicle. The volume density was estimated using 25 test points, while 9 subquadrants were used to count rnitochondrial profiles. I. Naylor CD, Slaughter PM: Cardiovascular health and services in Ontario. An ICES atlas. 1999;16-1 98(Abstract) 2. Lear S, Stachenko S: 1997 Canadim Cardiovascular Society Consensus Conference. 1997;(Abstract) 3- Takaro T, Peduzd P, Detre KM, Hultgren HN, Murphy ML, van der Bel-Kahn I, Thornsen J, Meadows WR: Survival in subgroups of patients with left main coronary disease: Veteran's Administration Cooperative Study of surgery for patients with coronary arterial occlusive disease. CircuIafion1982;66: 14-22 4. Varnauskas Ea: Twelve year follow up of sunival in the randomized European Coronary Surgery Study. AEhW 1988;3l9:332-337 5. Califf RM, Harrel FEJ, Lee KL, Rankin KS, Hlatky MA, Mark DB, Jones RH, Muhlbaier LH, Oldham HN, Pryor DB: The evolution of medical and surgical therapy for coronary artery disease: A 15 year perspective. JAMA l989;26 1:2077-2086 6. Myers WO, Gersh BJ, Fisher LD, Mock MB, Holmes DR Schaff HV, Gillespie S, Ryan TI, Kaiser GC, and other Cass investigators. Medical versus early surgical therapy in patients with triple vessel disease and mild angina pectoris. Ann Thorac Surg l987;44:47 1-486 7. Brown AC, Hutter AM", DeSanctis RW: Prospective study of medical and urgent surgical therapy in randomizable patients with unstable angina pectoris: Results of in- hospital and chronic mortality and morbidity. Am Hecrt J 198 1; 102:959-964 8. Parisi AF, Khuri S, Deupree RH, Sharma GVRK, Scott SM, Luchi RJ: Medical compared with surgical management of unstable angina: 5 year mortality and morbidity in the Veteran's Administration Study. Circulation 1989;80: 1176- 1 1 89 9. Christakis GT, Birnbaum PL, WeiseI RD, Ivanov J, David TE, Salerno TA, and the Cardiovascular Surgeons at the University of Toronto. The changing pattern of coronary bypass surgery (CABG).CirmIafion 1989;80:151-161 10. Maharajh GS, Masters RG, Keon WJ: Cardiac operations in the elderly: who is at risk? Ann Borac Surg l998;66:1670-1 673 11. Tsai-Tsung-Po., Nessim SS, Kass RM, Chaw A, Gray R, Khan SS, Blanche C, Utley C, MatlofFJM: Morbidity and mortality afler coronary artery bypass in octogenerians. Ann Thorac Surg 1991;51:983-986 12. Mohan R, Amsel BJ, Walter PJ: Coronary artery bypass grafting in the elderly: a review of studies in patients older than 64, 69, or 74 years. Cardiology l992;8O:215-225 13. Fremes SE, Christakis GT, Weisel RD, et al. A clinical trial of blood and crystalloid cardioplegia. J Zhorac Cardiovasc Surg 1984;88:726-741 14. Teoh KH, Christakis GT, Weisel RD, et al. Accelerated myocardial metabolic recovexy with terminal wam blood cardioplegia (hot shot). J 23oruc Cardiovasc Surg 1997;91:888-895 15. Hayashida N, Ikonomidis JS, Weisel RD, et al. The optimal cardioplegic temperature. Ann 7horac Surg 1994;58:961-971 16. Hayashida N, Ikonomidis JS, Weisel RD, et al. Adequate distribution of warm cardioplegic solution. J Thorac Cmdiovasc Surg 1995;110:800-812 17. Rao V, Cohen G, WeiseI RD, et aI. Optimal flow rates for integrated cardioplegia. J norac cara?ovarc Surg 1998;1 15:226-23 5 18. Horirnoto H, Gaudette GR, Saltman AE, Krukenkamp IB: The role of nitric oxide, K- ATP channels, and cGMP in the preconditioning response of the rabbit. J Surg Res 2000;92:56-63 19. Baines CP, Wang L, Cohen MV, Downey JM: Myocardial pr-otection by insulin is dependent on phosphatidyl-inositol3-kinasen but not protein kinase C or K-ATP channels in the isolated rabbit heart- Basic Res Cardioi 1999;94:188-1 98 20. Elliot GT: Monophosphoryl Lipid A: a novel agent for inducing pharmacologic myocardial preconditioning. J 7hrornb 77irornbolysis 1996;3 :225-23 7 21. Aitchison KA, Baxter GF, Awan MM, Smith RM, Yellon DM, Opie LH: Opposing effects on infarction of delta and kappa opioid receptor activation in the isolated rat heart: implications for ischemic preconditioning. Basic Res CardioZ2000;95 :1 - 10 22. Liang BT, Gross GJ: Direct preconditioning of cardiac myocytes via ~pioidreceptors and K-ATP channels. Circ Res 2000;84: 1396-1400 23. Wang Y, Ashraf M: Activation of alpha-1 adrenergic receptor during calcium preconditioning elicits strong protection against calcium overload injury via protein kinase C signalling pathway. J Mu[ Cell Cardoll998;3 O:2423 -243 5 24. Lochner A, Genade S, Tromp E, Podzuweit T, Moolrnan J: Ischemic preconditioning and the beta-adrenergic signal transduction pathway. Cirdation 1999;100:958-966 25. Light PE: Cardiac K-ATP channels and ischemic preconditioning: current perspectives. Can J CardioZ1999; 15: 1123- 1130 26. Toller WG, Gross ER, Kersten Page1 PS, Gross GJ, Wadtier DC: Sarcolemmal and mitochondria1 ATP-dependent potassium channels: mechanism of desflurane induced cardioprotection. Anaesthesiology 2000;92: 173 1 - 173 9 27. Ito H, Shimojo T, Fujisaki H, Tamamori M, Ishiyarna S, Adachi S, Abe S, Marumo F, Hiroe M: Thermal preconditioning protects rat cardiac muscle cells fiom doxorubicin induced apoptosis. Lzye Scz l999;64:755-76 1 28. Mahgoub h4.& Guo JH, Gao SPYTaher MM, Salter DD, Wechsler AS, Abd-Elfattah AS: Hyperdynamic circulation of arteriovenous fistda preconditions the heart and limits infarct size. Ann Zhurac Surg 1999;68:22-28 29. Chirac P: De Motu Cordis. Adversaria Analytica 1698; 121 30. Hemck JB: Clinical features of sudden obstruction of the coronary arteries. JAMA 1912;59:2015 3 1. Blumgart HL, Gilligan DR, Schlesinger MJ: Experimental studies on the effects of temporary ocdusion of the coronary arteries: The production of myocardial infarction. Am Heart J 1941;21:374 32. Jennings RB, Schaper J, HilI ML, Steenbergen CJ, Reimer KA: Effect of reperfision late in the phase of ischemic injury: changes in cell volume, electrolytes, metabolites and ultrastructure. Circ Res 1985;56:262-278 33. Reimer KA, Hill ML, Jennings RB: Prolonged depletion of ATP and of the adenine nucleotide pool due to delayed resynthesis of adenine nucleotides following reversible ischemic myocardial injury in dogs. JMolec CeN CardioZl98 1; 13 :229-239 34. Swain JL, Sabina K, McEMe PA, Greenfield JCJ, Hohes EW: Prolonged myocardial adenine nucleotide depletion after brief ischemia in the open chest dog. Am J Physiol1982;242:H8 18-H826 35. Braunwald E, Kloner RA: The stunned myocardium: proIonged postischemic ventricular dyshnction. Circulation 1 982;66: I 146- 1 149 36. Marban E, Korestune Y, Correti M, Chako VP, Kusuoka H: Cdcium and its role in myocardial cell injury during ischemia and reperfusion. CzrcuZation 1989;80:17-22 37. Bolli I, Jeroudi MO, Pate1 BS, Aruoma 01, HalliwelI B, Lai EK, McCay PB: Marked reduction of free radical generation and contractile dyshnction by antioxidant therapy begun at the time of repefision: evidence that myocardial stunning is a manifestation of repefision injury. Circ Ees 1989;65 :607-622 38. Reimer KA, Murry CE, Yarnasawa I, Hill ML, Jennings RB: Four brief periods of myocardid ischemia cause no cumulative ATP loss or necrosis. Am J PhysioZ 1986;251:H1306-HI315 39. Muny CE, Jennings RB, Reimer KA: Preconditioning with ischemia: a delay of lethal cell injury- in ischemic myocardium. Circz(Zafion1986;74: 1 124- 1 13 6 40. Przyklenk K, Bauer B, Ovize M, Kloner RA, Whittaker P: Regional ischemic "preconditioning" protects remote virgin myocardium f?om subsequent sustained coronary occIusion. CircufafionI993;87:893-899 41. Thannhauser SJ, Bommes A: ExperirnentelIe Studies Ober den Nucleinstofhechsel. II. Mitteilung. Stoffwechselversuche mit Adenosin und Guanosine. J PhysioZog Chem 1914;9:336-343 42. Drury AN, Szent-Gy0rgyi.A The physiological activity of adenine compounds with especial reference to their action upon the mammalian heart. J Physiiol Lond 1929;68:213 - 237 43. Lindner FJRigler R: Uber die Beeinflussung der Weita der Herzteranzgefasse durch Produkte des Zellkern stofiechsels. Pfleug Arch 193 1;226:697-708 44. Bemet DW, Drury AN: Further observations relating to the physiological activity of adenine compounds. JPhysioZ Lond 193 1 ;72:288-320 45. Fieisch A, Weger P: Die gefasenveitemde Wirlcung der phosphorylierten Stofiechselprodukte. Pfleug Arch 193 8;23 9:362-369 46. Honey Ritchie WT,Thompson WAR: Action of adenosine upon the human heart. Q JMed l93O;23:485-489 47. Jezer 4 Oppenheimer BS, Schwartz SP: The effect of adenosine on cardiac irregularities in man. Am Heart J 1934;9:252-258 48. Rothrnan H: Der Einfluss der Adenosin phosphosaure auf die Hentatigteit. Archive Erperimenta Pathologika PharamakoZogica 1 93 1 ;1 55 :129- 13 8 49. Wayne EJ, Goodwin JF, Stoner HB: The effect of adenosine triphosphate on the electrocardiogram of man and animals. Br Hearz J 1949;1 155-67 SO. Wedd AM: The action of adenosine and certain related compounds on the coronary flow of the perfused heart of the rabbit. J PhmmacoZ &uer lher 193 1 ;4 1 :3 55-366 51. Wedd AM, Fern WO: The action on cardiac musculature and the vagomimetic behaviour of adenosine. JPhmmacol mer Ther 1933;47:365-375 52. Berne RM: Cardiac nucleotides in hypoia: possible role in regulation of coronary blood flow- Am JPhysiol1963;204:317-322 53. GerIach E, Becker F: Anonymous. Topics and perspectives in adenosine research. Berlin, S pringer-Verlag, 19 87, 54. Olsson RA: Changes in content of purine nucleoside in canine myocardium during coronary occlusion. Circ Res l970;26:30 1-3 06 55. Bardenheuer H, Schrader I: Supply to demand ratio for oxygen determines formation of adenosine by the heart. Am JPhysiol1986;250:H173-HI80 56. Berne RM: The role of adenosine in the regulation of coronary blood flow. Circ Res l980;47:807-813 57. Olsson Patterson RE: Adenosine as a physiological regulator of coronary blood flow. Prog Molec SubceN Bid 1976;4:227-248 58. Sattin 4 Rall TW: The effect of adenosine and adenine nucleotides on the cyclic adenosine 3',5'-phosphate content of guinea pig cerebral cortex slices. Molec Phurmacol 1970;6:13-23 59. Londos C, Wow J: Two distinct adenosine-sensitive sites on adenylate cyclase. Proc National Acad Sci USA 1977;74:5482-5486 60. Belardinelli L, Isenberg G: Isolated atrial myocytes: adenosine and acetylcholine increase potassium conductance. Am J PhysioZ l983;244:H734-H737 61. Kurachi Y, Nakajima T, Sugimoto T: On the mechanism of activation of muscarinic K+ channels by adenosine in isolated atrial cells: involvement of GTP-binding proteins. Pfireug Arch 1986;407:264-274 62. Schrader J: Metabolism of adenosine and sites of production in the heart, in Berne RM, RaU TW, Rubio R (eds): Regulatory function of adenosine. The Hague, MartinudIViijhoff, 1983, pp 133-156 63. Bertolet BD, Hill JA: Adenosine: diagnostic and therapeutic uses in cardiovascular medicine. Chest 1993;104: 1860- 1871 64. Bardenheuer H, Schrader I: Supply to demand ratio for oxygen determines formation of adenosine by the heart. Am JPhysioll980;H389-H398 65.Olsson Rq Pearson JD: Cardiovascular purinoceptors. Physiol Rev 2 990;70:761-845 66. Holian A, Owen CS, Wilson DF: Control of respiration in isolated mitochondria: quantitative evaluation of the dependence of respiratory rates on [ATP], [ADP] and [Pi]. Arch Biochem Biophys 1977;18 1 :1 64- 17 1 67. Gamble WJ, La Farge CG, Fyler DC, Weisul J, Monroe RG: Regional coronary venous oxygen saturation and myocardial oxygen tension following abrupt changes in ventricular pressure in the isolated dog heart. Circ Res 1974;34:672-681 68. Holtz J, Grunewdd WJ, Manz q RestofiWV, Bassenge E: Intracapillary hemoglobin oxygen saturation and oxygen consumption in different layers of the left ventricular myocardium. Pfleug Arch 1977;370:253-258 69. Sparks HV, Bardenheuer H: Regulation of adenosine formation in the heart. Circ Res l986;58:193-201 70. Lloyd HGE, Schrader J: The importance of the transmethylation pathway for adenosine metabolism in the hearts, in Gerlach E, Becker BF (eds): Topics md perspectives in adenosine research. Berlin, Springer-Verlag, 1987, pp 1 99-207 71. Achterberg PW, De Tombe PP, Harmsen E, De Iong JW: Myocardial S- adenosylhomocysteine hydrolase is important for adenosine production during normoxia. Bzochim Biophys Acta 1985;840:393-400 72. Hershfield MS, Kredich NM: S-adenosylhomocysteine hydrolase is an adenosine- binding protein: a target for adenosine toxicity. Science Washngon DC 1 978;202:757- 760 73. Olsson RA: Ligand binding to the adenine analogue binding protein of the rabbit erythrocyte. Biochemistry 1978;1 7:3 67-375 74. Olsson Rq Saito D, Steinhart CR: Compartmentalization of the adenosine pool of dog and rat hearts. Circ Res l982;50:617-626 75. Kwong FYP, Davies 4 Tse CM, Young ID, Henderson PJF, Baldwin SA: Purification of the human erythrocyte nucleoside transporter by imrnunoaffinity chromatography. Biochem J 1988;255:243-249 76. Paterson ARP, Kolassa N, Cass CE: Transport of nucleoside drugs in animal cells. Phannucol Ther 1981;12:515-536 77. Plagemann PGW,Wohlheuter RM: Permeation of nucleosides, nucleic acid bases, and nucleotides in animal cells. Current Topics in Membrane Transport 1980;14:225-23 0 78. Plagemann PGW, Wohlheuter RM: Nucleoside transport in mammalian cells and interaction with intracellular metabolism, in Berne Rall TW, Rubio R (eds): ReguZutoryfirnction of adenosine. The Hague, Nijhoft; 1983, pp 179-201 79. Olsson Gentry MK, Snow JA: Steric requirements for binding of adenosine to a membrane carrier in canine heart. Bzochim Biophys Acta 1973;3 11 :242-250 80. Paterson ARP, Oliver IM: Nucleoside transport: Inhibition by p- nitrobenzylthioguanosine and related compounds. Can JBiochern 1971;49:271-274 81. Heller LJ, Sherin JC, Mohrman DE, Dole WP: Adenosine receptor blockade potentiates cardiac interstitial adenosine during metabolic stimulation in isolated rat rearts. CircuIation 1987;76:N-327(Abstract) 82. Mckenzie JE, Steffen RP, Haddy FJ: Effect of theophylline on adenosine production in the canine myocardium. Am JPhysiol l987;252:H204-,EI2lO 83. Paterson ARP, Jakobs ES, Ng CYC, Odgard RG, Adjei AA: Nucleoside transport inhibition in-vitro and in-vivo. in Gerlach E, Becker BF (eds): Topics and Perspectives in Adenosine Research- Berlin, Springer-Verlag, 19 87, pp 89- 10 1 84. Hopkins SV, Goldie RG: A species difference in the uptake of adenosine by heart. Biochem Pharmacol1971;20:3359-3365 85. Parkinson FE, Clanachan AS: Subtypes of nucleoside transport inhibitory sites in heart: a quatitative autoradiographic analysis. Eur J Pharmacol1989; 163:69-75 86. Achterberg PW, Strove RJ, De Jong IW:Myocardial adenosine cycling rates during normoxia and under conditions of stimulated purine release. Biochem J l986;23 5: 13-1 7 87. Bontemps F, Van Den Berghe G, Hers HG: Evidence for a substrate cycle between AMf and adenosine in isolated hepatocytes. ProcNatl AcadSci USA 1983;80:2829-2833 88. Maguire MH, Lukas MC, Rettie IF: Adenine nucleotide salvage synthesis in the rat heart. Pathways of adenosine salvage. Biochim Biophys Acta l972;262: 108- 1 15 89. Deussen & Moser G, Schrader J: Contribution of coronary endothelid cells to cardiac adenosine production. Pfleug Arch 1986;406:608-6 14 90. Gorman MW, Bassingthwaighte JB, Olsson RA, Sparks HV: Endothelial cell uptake of adenosine in skeletal muscle. Am JPhysiol1986;250:H482-H489 91. Kubler W, Spieckeman PG, Bretschneider W: Iduence of dipyridamole on myocardial adenosine metabolism. J Mol Cell Cardiol 1970; 1 :23 -28 92. Catravas JD: Removal of adenosine fiom the rabbit pulmonary circulation, in vivo and in vitro- Circ Res 1984;54:603-611 93. Liu MS, Feinberg H: Incorporation of adenosine-8-14C and inosine-8-14C into rabbit heart adenine nucleotides. Am J Physiol 1971;220: 1242- 1248 94. Becker BF, Gerlach E: Uric acid, the major catabolite of cardiac adenine nucleotides and adenosine, originates in the coronary endothelium. in Gerlach E, Becker BF (eds): Topics and Perspectives in Adenosine Research. Berlin, Springer-Verlag, 198 7, pp 209- 22 1 95. Ronca-Testoni S, Borghini F: Degradation of perfused adenine compounds up to uric acid in isolated rat heart. JMol Cell CardioI 1982; 14: 177-180 96. Arnold ST, Cysyk RL: Adenosine export £?om the Liver: oqrgen dependency. Am J Physiol 1986;251 :G34-G39 97. MdediP, Holmes EW: Purine salvage pathways in myocardium. Anm Rev Physiol l985;47:691-705 98. Chechik BE, Baumal R, Sengupta S: Localization and identity of adenosine deaminase positive cells in tissues of the young rat and calf. Hisrochem J 1983; 15:373-387 99. Geisbuhler T, Altschuld R4, Trewyn RW, Ansel AZ, Larnka K, Brierly GP: Adenine nucleotide metabolism and compartmentalization in islated adult rat heart cells. Circ Res l984;54:S36-546 100. Bardenheuer H, Whelton B, Sparks HV: Adenosine release by the isolated guinea pig heart in response to isoproterenol, acetylcholine, and acidosis: the minimal role of vascular endothelium. Circ Res 1987;61:594-600 101. Burnstock G, Buckley NJ: The classification of receptors for adenosine and adenine nucleotides. in Paton DM (ed): Methob in Pharmacology. Methods Used in Adenosine Research. New York, Plenum, 1985, pp 193-2 12 102. Van Calker D, Muller M, Hamprecht B: Adenosine regulates via two different receptors, the accumulation of cyclic AMP in cuitured brain cells. J Neurochem 1979;33:999-lOO5 103. Fain IN, Malbon CC: Regulation of adenylate cyclase by adenosine. Mol CeN Biochem 1979;25:143-151 104. Cam CS, HilI RT, Masamune H, et al, Evidence for a role for both the adenosine A1 and A3 receptors in protection of isolated human atrial muscle against simulated ischemia. Cardiovasc Res 1997;36:52-59 105. Liang BT, Jacobson KA: A physiological role of the adenosine A3 receptor: sustained cardioprotection. Proc Natl Acad Sci USA 1998;95:6995-6999 106. Tracey Magee W, Masamune H, Kennedy SP, Knight DR Buchholz RA, Hill RJ: Selective adenosine A3 receptor stimulation reduces ischemic myocardial injury in the rabbit heart. Cardiovasc Res 1997;33:NO-4 15 107. Berne RM, Rubio R: Coronary Circulation. in Berne RFcl, Sperlakis N, Gieger SR (eds): Handbook of Physi~Zogy.~Washington DC, American Physiology Society, 1979, pp 873-952 108. Hori M, Kitakaze M: Adenosine, the heart, and corornary circulation. Hypertension 1991; 18~565-574 109. BeIardinelIi L, Isenberg G: Actions of adenosine and isoproterenol on isolated mammalian ventricular myocytes. Circulation Research 19 83 ;5 3 :287-297 110. Sato H, Hori M, Kitakaze M: Endogenous adenosine blunts beta-adrenoreceptor mediated inotropic response in the hypoperfbsed canine myocardium. Circulation 2 992;85: 1594-1603 11 1. Cronstein BN, Levin RI, BelanoE J, Weissmann G, Hirschhorn R: Adenosine: an endogenous inhibitor of neutrophil-mediated injury to endothelial cells. J CZin Invest 1986;78:760-770 112. Cronstein BN, Kramer SB, Weissmann G, Hirschhorn R: Adenosine: a physiological modulator of superoxide anion generation by human neutrophils. J Exp Med 1983;158:1160-1177 113. Newman WH, Becker BF, Heier M, Nees S, Gerlach E: Endothelium-mediated coronary dilitation by adenosine does not depend on endothelial adenylate cyclase activation: studies in isolated guinea pig hearts. Pfleug Arch 1988;413:1-7 114. Kitakaze M, Hori M., Sato H: Endogenous adenosine inhibits platelet aggregation during myocardial ischemia in dogs. Circ Res 199l;69: 1402- 1408 115. Mainwaring q Lasley R, Rubio R, Wyatt Dq Mentzer RM: Adenosine stimulates glucose uptake in the isolated rat heart- Surgery 1988;13:445-449 116. Wyatt DA, Edmunds MC, Rubio Berne RM, Lasley RD, Mentzer RM: Adenosine stimulates glycolytic flux in isolated pefised rat hearts by Al-adenosine receptors. Am J Physiol1989;257:Hl952-H1957 1 17. Berne EW: Adenosine: a cardioprotective and therapeutic agent. Cardiovasc Res l993;27:2 118. Ikonomidis JS : Preconditioning Human Ventricular Cardiomyocytes. 1995;3O(Thesis/Dissertation) 119. Hechter 0: Concerning possible mechanisms of hormone action. Vit Horn 1955; 13:293-346 120. Lochner A, Genade S, Tromp E, Opie L, Moolman J, Thomas S, Podzuweit T: Role of cyclic nucleotide phosphodiesterases in ischemic preconditioning. Mol Cell Biochem 1998; 186:169-175 121. Sandhu Thomas U, Diaz RJ,Wdson GJ: Effect of ischemic preconditioning of the myocardium on CAMP. Circ Res Z996;78: 137-147 122. Graziano MP, Gilrnan AG: Guanine nucleotide-binding regulatory proteins: mediators of transmembrane signaling. Trenrls PhmcolSci 1987;8:478-48 1 123. Neer EJ, Clapham DE: Roles of G protein subunits in transmembrane signalling. Nature Lond 1988;333:129-134 124. Rodbell M: Programmable messengers: a new theory of hormone action. Trends Biochem Sci 1985; 10:461-464 125. Kitakaze Hori M, Kamada T: Role of adenosine and its interaction with alpha adrenoreceptor activity in ischemic and repefision injury of the myocardium. Cardiovasc 126. Thomton JD, Liu GS, Downey IM: Pretreatment with pertussis toxin blocks the protective effects of preconditioning: evidence for a G-protein mechanism. J Mol Cell Curdiol1993;25:3 11-320 127. Lasley RD, Mentzer RM: Pertussis toxin blocks adenosine A1 mediated protection of the ischemic rat heart. JMol Cell Cardioi 1993;25:815-821 128. Liu Y, Downey JM: Preconditioning against infarction in the rat heart does not involve a pertussis toxin sensitive G protein. Cardiovasc Res l993;27:608-6Il 129. Szilvassy 2, Ferdinandy P, Bor P, Jakab I, Lonovics J, Koltai M: Ventricular overdrive pacing induced anti-ischemic effect: a conscious rabbit model of preconditioning. Am J Physol1994;266:H203 3-EX2041 130. Iwase T, Murakami T, Tomita T, Miki S, Nagai K, Sasayama S: Ischemic preconditioning is associated with a delay in ischemia induced reduction of beta-adrenergic signal transduction in rabbit hearts. CircuZatratron1993;88:2827-2837 13 1. Fu LX, Kirkeboen KA, Liang QM, Sjogren KG, Hjalmanon 4 nebekk A: Free radical scavenging enzymes and G protein mediated receptor signalling systems in ischemically preconditioned porcine myocardium. Cardiovasc Res 1993;27:6 12-6 16 132. Peralta EG, Ashkenazi 4 Winslow JW, Ramachandran J, Capon DJ: Differential regulation of PI hydrolysis and adenylyl cyclase by muscarinic receptor subtypes. Nature Lond 1988;334:434-437 133. Jochem G, Nawrath H: Adenosine activates a potassium conductance in guinea-pig atrial heart muscle. Eqerienfia Basel 1983;39: 1347- 1349 134. fsenberg G, Serbai E, Klockner U: Ionic channels and adenosine in isolated heart cells. in Gerlach E, Becker BF (eds): Topics and Perspectives in Adenosine Research. Berlin, Springer-Verlag, 1987, pp 323-3 3 5 135. Kurtz A: Adenosine stimulates guanylate cyclase activity in vascular smooth muscle * cells. JBioZ Chem 1987;262:6296-6300 136- Dolphin AC, Forda SR, Scott RH: Calcium dependent currents in cultured rat dorsd root ganglion neurones are inhibited by an adenosine analogue. J Physiol Lond 1 986;3 73 :47-61 137. Dolphin AC, Prestwich A: Pertussis toxin reverses adenosine inhibition of neuronal glutamate release. Nature Lmd l985;3 16:148- 150 138. Londos C: On multiple targets for fat cell receptors. in Gerlach E, Becker BF (eds): Topics and Perspectives in Adenosine Research. Berlin, Springer-Verlag, 1987, pp 239- 248 139. Barret EJ, Schwartz RG, Francis CK, Zaret BL: Regdation by insulin of myocardial glucose and fatty acid metabolism in the conscious dog. J CZin Invest l984;74: 1073-1079 140. Eckel J, Pandalis G,Reinaur H: Insulin action on the glucose transport system in isolated cardiocytes f?om adult rat- Biochem J l983;Z l2:385-392 141. Long CJ, Stone TW: Adenosine reduces ago& induced production of inositol phosphates in rat aorta. JPhmm Pharmacal1988;39:1010-1014 142. Streb H, Irvine RF, Berridge MJ, Schulz I: Release of calcium from a non- mitochondrial intracellular store in pancreatic acinar cells by inositol 1,4,5-triphosphate. Nature 1983;306:67-69 143. Takai Y, Kishirnoto A, Kikkawa U, Mori T, Nishizuka Y: Unsaturated diacylgIycerol as a possible messenger for the activation of calcium-activated, phospholipid dependent protein kinases system. Biochem Biophys Res Comrnun l979;9 1: 12 18- 1224 144. Newton AC: Protein base C: Structure, function and regulation. J Biol Chem 1995;270:28495-28498 145. Coussens L, Parker PJ, Rhee L, Yang-Feng TL: Multiple, distinct forms of bovine and human protein kinase C suggest diversity in cellular signaling pathways. Science 1986;233:859-866 146. House C, Kemp BE: Protein kinase C contains a pseudosubstrate prototope in its regulatory domain. Science I987;23 8: 1726-1728 147. Taylor SS, Radzio-Andzelrn E: Three protein kinase structures define a common motif Sfruclure l994;2:36-3 55 148. houe M, Kishimoto A, Takai Y, Nishizuka Y: Studies on a cyclic nucleotide- independent protein kinase and its proenzyme in mammalian tissues. 11. Proenzyme and its activation by calcium-dependent protease fiom rat brain. J Biol Chem l977;252:76 10- 7616 149. Goto h4, Cohen MV, Downey JM: The role of protein base C in ischemic preconditioning. Ann NY Acad Sci l996;793 :177- 190 150. Kemely PI, Krebs EG: Consensus sequences as substrate specificity determinants for protein kinases and protein phosphatases. JBiol Chem 1991;266:15555-15558 151. Mochly-Rosen D, Koshland DE: Domain structure and phosphorylation of protein kinase C. J Bid Chem 1987;262:229 1-2297 152. O'Brian C, Ward NE: Stimulation of the ATPase activity of rat brain protein kinase C by phospho acceptor substrates of the enzyme. Biochemistry 1991;3 O:2549-2554 153. Orr JW,Newton AC: Intrapeptide regulation of protein kinase C. J Bzol Chem 1994;269:8383-8387 154. Limas CJ, Limas C: Involvement of microtubules in the isoproterentol induced down regulation of myocardial B-adrenergic receptors. Biochim Biophys Acta 19 83; 73 5 :1 8 1- 184 155. Kraft AS, Anderson WB, Cooper HL, Sando JJ: Phorbol esters increase the amount of calcium, phospholipid-dependent protein kinase associated with plasma membrane. Nature 1983;301:621-623 156. Miura T, Tsuchida A: Adenosine and preconditioning revisited. Clin Erper PhacolPhysiol1999;26:92-99 157. Shukla SD, Halenda SP: Phospholipase D in cell signalling and its relationship to phospholipase C. Life Sci 1991;48:851-866 158. Cohen MV, Liu Y, Liu GS, et al. Phospholipase D plays a role in ischemic preconditioning in rabbit heart. Circulation 1 996;94:1 7 1 3 - 1 7 1 8 159. Vahlhause C, Schultz 3& Post H, Onallah Heusch G: No prevention of ischemic preconditioning by the protein kinase C inhibitor staurosporine in swine. Circ Res l996;79:407414 160. Przyklenk K, Sussman MA, Simkhovich BZ, Kloner RA: Does ischemic preconditioning trigger translocation of protein kinase C in the canine model? Circulation 1995;92:1546-1557 161. Miura T, Kawamura S, et al. Effect of protein kinase C inhibitors on cardioprotection by ischemic preconditioning depends on the number of preconditioning episodes. Circ Res 1996;79:407414 . 162. Vahlhause C, Schultz R, Post H, Rose J, Heusch G: Prevention of ischemic preconditioning only by combined inhibition of protein kinase C and protein tyrosine kinase in pigs. JMol Cell Cardiol lW8;3O:197-209 163. Baines CP, Cohen MV, Downey JM: Signal transduction in ischemic preconditioning: the role of kinases and mitochondria1 K-ATP channels, J Cardiovasc Electrophysiol 1999; I O:741-754 164. Neet Y Hunter T: Vertebrate non-receptor protein tyronsine kinase families. Genes cells 1996;1:147-169 165. Van der Geer P, Hunter T, Lindberg RA: Receptor protein tyrosine kinases and their signal transduction pathways. Anrm Rev Cell Biol1994; 10% 1-337 166. Fagerstrom S, Pahlman S, Nanberg E: Protein kinase C dependent tyrosine phosphorylation of p 13 Ocas in differentiating neuroblastoma cells. J Biol Chem 1998;27312336-2343 167. Levi NL, Hanoch T, Benard 0,et al. Stimulation of Jun N-terminal kinase (JNK) by gonadotropin releasing hormone in pituitary alpha T3-1 cell line is mediated by protein kinase C, c-Src, and CDC-42. Mol Endacrinol 1998;12:8 15-824 168. Ping P, Zhang J, Zheng YT, et al. Demonstration of selective protein kinase C- dependent activation of Src and Lck tyrosine hases during ischemic preconditioning in conscious rabbits. Circ Res 1999;85:542-550 169. Jaffe MD, Quinn NK: Wmup phenomenon in angina pectoris. Lancet 1980;2:934- 936 170. Wiarns DO, Bass T& Gerwitz H, Most AS: Adaptation to the stress of tachycardia in patients with coronary artery disease: insight into the mechanism of the warm up phenomenon. Cirnrlafion 198 5;7 1 :687-692 171. Muller DWM, Topol EJ, Califf RM: Relationship between antecedent angina pectoris and short term prognosis after thrornbolytic therapy for acute myocardial infarction. Am Heart J 1990;1 19:224-23 1 172. Kloner Rq Shook T, Przyklenk K: Previous angina alters in-hospital outcome in TIMI 4. Circulation 1 993 ;8 8 :I-49 173. Walker DM, Walker JM, Pattison C, Pugslet W, Yellon DM: Preconditioning protects isolated human muscle. Circulation l993;88:I- 13 8 174. Cohen G, Shirai T, Weisel RD, Rao V, Merante F, Tumiati LC, Mohabeer MK, Borger MA, Li RK, Mickle DAG: Optimal myocardial preconditioning in a human model of ischemia and reperfusion. Circulation 1998;98:137-143 175. Kerensky RA, Kutcher Mq Braden GA: The effects of intracoronary adenosine on preconditioning during coronary angioplasty. Clin Cordol 1995;18:9 1-96 176. Deutsch E, Berger Kussmaul WG, Hirshfeld JW,Hemnann HC, Laskey WK: Adaptation to ischemia during percutaneous translumind coronary angioplasty. Clinical, hernodynamic and metabolic features. Circulation 1990;82:205 1 177. Alkhulaifi AM, Yellon DM, Pugsley WB: Preconditioning the human heart during aorto-coronary bypass surgery. Eur J Cardiothor Surg l994;8:270-276 178. Jenkins DP, Pugsley WB,Alkhulaifi AM, Kemp M, Hooper J, Yellon DM: Ischemic preconditioning reduces troponin T release in patients undergoing coronary artery bypass surgery. Heart 1997;77:3 14-3 1 8 179. Jacobsohn E, Young CJ, Aronson S, Ferdinand FD, Albertucci M: The role of ischemic preconditioning during minimally invasive coronary artery bypass surgery. J Cardiothorac Vmc Anaesrh 1997;11:787-792 180. Barbash GI, White HD, Modan h4, Van de Werf F: Antecedent angina pectoris predicts worse outcome after myocardial infarction in patients receiving thrombolytic therapy: Experience gleaned from the International Tissue Plasminogen Activator I Streptokinase Mortality Trial. J Am Coil Cardiol l992;20:36-41 181. Denys BG, Lee J, Reddy PS, Uretsky BF: Is there adaptation to ischemia during PTCA? Eur Heart J 1992; 13:246 182. DeBruyne B, Vantrimpont PJ, Goetua SM, Stockbroch J, Paulus WJ: Can coronary angioplasty serve as a model of myocardial preconditioning? Circulation 1992;86:1-3 78 183. Perrault LP, Menasche P, Be1 de Chaumaray T, Peynet J, Mondry 4 Olivero P, Emanoil-Ravier R, Moalic J: Ischemic preconditioning in cardiac surgery: a word of caution. J Thorac Cardiovusc Surg 1996;112: 2378-1386 184. Malkowski MT, Kramer CM, Parvid ST, Dianzumba S, Marquez J, Reichek N, Magovern JA: Transient ischemia does not limit subsequent ischemic regional dysfimction in humans: a transesophageal echocardiographic study during minimally invasive coronary artery bypass surgery. J Am CoZZ Cardiol l998;3 1: I03 5- 1039 185. Liang BT, Gross GJ: Direct preconditioning of cardiac myocytes via opioid receptors and K-ATP channels. Circ Res 1999;84: 1396-1400 186. Tomai F, Crea F, Gaspardone A, Versaci F, Ghini AS, Ferri C, Desideri G, Chianello L, Giofie PA: Effects of naloxone on myocardial ischemic preconditioning in humans. J Am Coll Cardiol 1999;33:1863-1869 187. Parikh V, Singh M: Possible role of cardiac mast cells in norepinephrine-induced myocardial preconditioning. Method Find Exp Clin PhamacoZ 1999;21 :269-274 188. Musters RJ, van der Meulen ET, Zuidwijk M, MuIler A, Simonides WS, Banejee 4 van Hardeveld C: PKC-dependent preconditioning with norepinephrine protects sarcoplasmic reticulum function in rat trabecdae following metabolic inhibition. J Mol Cell Cardioll999;3 1: 1083-1094 189. Horimoto &I,Saltman AE, Gaudette GR, Krukenkamp IB: Nitric oxide generating beta adrenergic blocker nipradilol preserves post-ischemic cardiac fimction. Ann norac Surg 1999;68:844-849 190. Toller WG, Montgomery MW, Pagel PS, Hettrick Dq Warltier DC, Kersten JR: Isoflurane-enhanced recovery of canine stu~edmyocardium: role for protein kinase C? AmesthesioIogy l999;g 2 :713-722 191. Novalija E, Fujita S, Karnpine IP, Stowe W: Sevoflurane mimics ischemic preconditioning effects on coronary flow and nitric oxide release in isolated hearts. Anaesthesiohgy 1999;91:701-712 192. Zhao L, Elliot GT: Pharmacologic enhancement of tolerance to ischemic cardiac stress using monophosphoryl Lipid A Ann N Y Acad Sci 1999;874:222-235 193. Lerman BB, Belardinelli L: Cardiac electrophysiology of adenosine: basic and clinical concepts. Circulation 1991$3 :1499- IS09 194. Belardinelli L, West A, Crarnpton Berne RM: Chronotropic and dromotropic effects of adenosine. in Berne RM, Rall TW, Rubio R (eds): Regulatory firncfion of adenosine. Boston, Martinus Nijhoff, 1983, pp 3 77-3 96 195. Song Y, Thedford S, Lerman BB, Belardinelli L: Adenosine sensitive afterdepolarizations and triggered activity in guinea pig ventricular myocytes. Circ Res 1992;70:743-753 196. Fox AC, Reed GE, Glassmand E, Kaltman AJ, Silk BB: Release of adenosine f?om human hearts during angina induced by rapid atrial pacing. J CZin Invest 1974;53:1447- 1457 197- Fox AC, Reed GE, Meifman H, Sifk BB: Release of nucleosides fiom canine and human hearts as an index of prior ischemia. Am J Cardid 1979;43:52-58 198. Sylven C, Jonzon B, Edlund A: Angina pectoris-like pain provoked by i.v. bolus of adenosine: relationship to coronary sinus blood flow, heart rate and blood pressure in healthy volunteers. Eur Heart J 1989;10:48-54 199. Sylven C, Beermann B, Jonzon BIBrandt R: Angina pectoris-like pain provoked by intravenous adenosine in healthy volunteers. BMJ l986;293227-230 200. Baggioni I, Olafsson B, Robertson RM, Hollister AS, Robertson D: Cardiovascular and respiratory effects of adenosine in conscious man: evidence for chemoreceptor activation. Circ Res l987;61 379-786 201. Verani MS, Mahmarian JJ, Hixson JB, Boyce TM, Staudacher RA: Diagnosis of coronary artery disease by controlled coronary vasodilitation with adenosine and thallium- 201 scintigraphy in patients unable to exercise. Circulation 1990;82:80-87 202. Smits P, Lenders Thien T: Caffeine and theophylline attenuate adenosine induced vasodilitation in humans. Clin Pharmacol i%er 1990;48:4 10-418 203. Watt AH, Bayer A, Routledge PA Swift CG: Adenosine induced respiratory and heart rate changes in young and elderly adults. Br J CIin Phmacol 1 989;27:165- 1 71 204. Ytrehus K, Liu Y, Downey JM: Preconditioning protects ischemic rabbit heart by protein kinase C activation. Am J Physiol 1994;266:H1145-HI152 205. Armstrong S, Downey M, Ganote CE: Preconditioning of isolated rabbit cardiomyocytes: induction by metabolic stress and blockade by the adenosine antagonist SPT and Calphostin C, a protein kinase C inhibitor. Cordovasc Res 1994;28:72-77 206. Armstrong S, Ganote CE: Preconditioning of isolated rabbit cardiomyocytes: effects of glycolytk blockade, phorbol esters and ischemia. Cmdiovasc Res 1994;28:1700- 1706 207. Liu Y, Ytrehus K, Downey JM: Evidence that translocation of protein kinase C is a key event during ischemic preconditioning of rabbit myocardium. J Mol Cell Cardiol 1994;26:66l-668 208. Speechly-Dick ME, Mocanu MM, Yellon DM: Protein base C. Its role in preconditioning in the rat. Circ Res 1994;75:586-590 209. Kohl C, Linck B, Schmitz W, Scholz H, Scholz J, Toth M: Effects of carbachol and (-)-N6-phenylisopropyladenosine on myocardial inositol phosphate content and force of contraction. Br J Pharmacol1990; 10 1:829-834 210. Ikeda U, Arisaka H., Takayasu T, Takeda K, Natsume T, Hosoda S: Protein base C activation aggravates hypoxic myocardial injury by stimulating Na+/H+ exchange. JMol cell cardiol1988;20:493-500 21 1. Yuan S, Sunahara FA, Sen AK: Turnour promoting phorbol esters inhibit cardiac finction and inhibit redistribution of protein kinase C in perfbsed beating rat heart. Circ Res Z987;61:372-378 212. Lee HT, Lafaro RT, Reed GE: Pretreatment of human myocardium with adenosine during open heart surgery. JCard Surg 1995;10:665-676 213. Cohen G, Feder-Elituv R, Iazetta J, Fremes SE: Phase 2 studies in adenosine cardioplegia. CircuZation 1998;98:I1225-II233 214. Cohen G, Shirai T, Weisel RD, Rao V, Merante F, Tumiati LC, Mohabeer MK, Borger Mq Li RK, Mickle DAG: Optimal myocardial preconditioning in humans. Ann NY Acd Scz 1999;874:306-319 215. Houltz E, Ricksten SE, Milocco I, Gustavsson T, Caidahl K: Effects of adenosine infusion on systolic and diastolic left ventricular hction afler coronary artery bypass surgery: Evaluation by computer assisted quantitative 2-D and doppIer echocardiography. Anaesrh Adg 1995;80:47-53 216. Owall 4 Ehrenberg J, Brodin LA, Juhlin-Dannfelt A, SoIlevi A: Effects of low dose adenosine on myocardid performance after coronary artery bypass surgery. Acta Anaesthesiol Scandin 1993;37:140-148 217. Anonymous. Effects of acadesine on the incidence of myocardial infarction and adverse cardiac outcomes after coronary artery bypass graft surgery. Multicentre Study of Perioperative Ischemia Research Group. Anaesthesiology 1995;83 :658-673 218. Mentzer RM, Birjiniuk V, Khuri S, Lowe JE, WoPS, Weisel RD, Wellons H& Barker ML, Lasley RD: Adenosine myocardial protection: preliminary results of a phase I1 clinical trial. Ann Surg 1999;229:643-649 219. Currie RW,Karmazyn M, Kloc Mailer K: Heat shock response is associated with enhanced postischemic ventricular recovery. Circ Res 1988;63543-549 220. Domelly TJ, Sievers RE, Wssern FLJ, Welch WJ, Wolfe CL: Heat shock protein induction in rat hearts. A role for improved myocardial salvage after ischemia and repefision? Circulation 1992; 8 5 :769-778 221. Magghwar SB, Dhanraj DN, Somani SM, Ramlaunar V: Adenosine acts as an endogenous mediator of the cellular antioxidant defense system. Biochern Biophys Res Commun l994;2O 1:508-5 15 222. Keyser DO, Alger BE: Arachidonic acid modulates calcium current via protein kinase . C and oxygen radicals. Neum 1990;5:545-553 223. Bianchi G, Calvani AB, Mantovani M, Prino G: Enhancement by defibrotide of prostanoid neosynthesis &om arachidonic acid: an adenosine receptor mediated effect? Sern Bromb Hemost 1991;17:399-403 224. Ab A, Ford-Hutchinson AW, Nicholson DW: Activation of protein base C downregdates leukotriene C4 synthase activity and attenuates cysteinyl leukotriene production in an eosinophilic substrain of HL-60 cells. JImmunol 1994;153 :776-788 225. Vergauwen L, Wespel P, Richter EA: Adenosine receptors mediate synergistic stimulation of glucose uptake and transport by insulin and by contractions in rat skeletal muscle. J CZin Invest 1994;93:974-98 1 226. Vander Heide RS, Reimer KA, Jennings RB: Adenosine slows ischemic metabolism in canine myocardium in vitro: relationship to ischemic preconditioning. Cardiovasc Res 1993;27:669-673 227. Ikonomidis JS, Salerno Tq Wittnich C: Calcium and the heart: an essential partnership. Can J Cardiol 1 990;6:305-3 16 228. Gross GJ, Auchampach JA: Role of ATP dependent potassium channels in myocardial ischemia. Cardiovasc Res 1992;26:10 1 1 - 1 0 16 229. Eckert R, Utz J, Trautwein W, Mentzer RM: Involvement of intracellular calcium release mechanism in adenosine induced cardiac calcium current inhibition. Surgery 1993;114:334-342 230. Whalley DW, Wendt DJ, Grant AO: Basic concepts in cellular cardiac electrophysiology. Pace 1995; 18: 1556- 1574 23 1. Carmeliet E: Cardiac transmembrane potentids and metabolism. Circ Res 1978;42:577-587 232. Noma A: ATP regulated potassium channels in cardiac muscle. Nature l983;3OS: 147-148 233. Kukuljan M, Labarca P, Latorre R: Molecular determinants of ion conduction and inactivation in potassium channels. Am JPhysiol 1995;268:C535-C556 234. Jan YN, Jan LY, Dennis M: Two mutations of synaptic transmission in Drosophilia. Proc Royal Soc Lond 1977;B198:87-108 235. Cook NS: Potassit(rn channels: structure, ~Iasszjication,function and threrapeutic - potentia2. New York, Chicester publishers, 1990, pp 23 236. Rehm H, Lazdunski M: Purification and subunit structure of a putative potassium channel protein identified by its binding properties for dendrotoxin I. Proc National Acad SCZUSA 1988;85:4919-4923 237. Pongs 0,Kecskemethy N: Shaker encodes a family of putative potassium channel proteins in the nervous system of Drosophilia. EM30 J 1988;7:1087-1 096 23 8. Darniano RJ: The electrophysiology of ischemia and cardioplegia: implications for myocardial protection. J Cardiac Surg 1995;1 OA45-453 239. Lazdunski M: ATP-sensitive potassium channels: an overview. J Cmdiovasc Pharmacol 1994;24:SI-S5 240. Cook DL, Hales CN: Intracellular ATP directly blocks potassium channels in pancreatic Beta cells. Nature 1984;3 1 1 :271-273 241. Spruce AE, Standen NB: Voltage dependent ATP sensitive potassium channels of skeletal muscle membrane. Nature 1985;3 l6:73 6-738 242. Standen NB, Quayle TM: Hyperploarizing vasodilators activate ATP sensitive potassium channels in arterial smooth muscle. Science l989;245 :177- 180 243. Bernardi H, DeWeille JR, Epelbaum J, Mourre C: Proc Nafionai Acad Sci l993;gO:1340-2344 244. Misler S, Gee WM: A metabolite regulated potassium channel in human pancreatic isZet cells, Diabetes l989;3 R422-427 245. Rorsman P, Hellman B: Voltage activated currents in guinea pig pancreatic alpha cells. J Gen Physiol l988;g 1 :223-242 246. Thornton JD, Thornton CS: Blockade of ATP sensitive potassium channels increases infarct size but does not prevent preconditioning, in rabbit hearts. Circ Res 1993;72:44-49 247. Gross GI, Auchampach JA: Blockade of ATP sensitive potassium channels prevents myocardial preconditioning in dogs. Czrc Res 1992;70:223-233 248. Nichols CG: Molecular biology of the K-ATP channel. Where's the clone? curdiovasc Res l994;28:6 249. Ashford MLJ, Bond CT, Blair T& Adelman 1P: Cloning and knctiond expression of a rat heart K-ATP channel. Nature 1 994;370 :456-459 250. Ashford MLJ, Bond CT, Blair TA, Adelman JP: Cloning and finctional expression of a rat heart K-ATP channel. (Retraction of Nature, 1994; 370:456459)- Nature 1995;378:792 251. Ho K, Nichols CG, Lederer WJ, Lytton J: Cloning and expression of an inwardly rectifying ATP regulated potassium channel. Nature l993;362:31-3 7 252. Inagaki N, Tsuura Y, Namba N, Masuda K: Cloning and fbnctional characterization of a novel ATP-sensitive potassium channel ubiquitously expressed in rat tissues, including pancreatic islets, pituitary, skeletal muscle, and heart. JBioC Chem 1995;270:5691-5694 253. Inagaki N, Gonoi T, Clement JP, Namba N: Reconstitution of I K-ATP: an inward rectifier subunit plus the sulfonylurea receptor. Science l995;27O:1 166-1 169 254. De Weille 3R, Schmid-Antomarchi Y Fosset h4, Lazdunski M: Regulation of ATP- sensitive potassium channels in insulinoma cells. Activation by somatostatin and kinase C, the role of c-AMP. Proc National Acad Sci USA 1989;86:2971-2975 255. Ito H, Vereecke J, CarmeIiet E: Mode of regulation of the ATP-sensitive potassium channel in guinea pig ventricular cell membrane. J Physiol 1994;478: 10 1 - I 07 256. Bernardi H,De Weille JR, Epelbaum J: ATP modulated potassium channels sensitive to antidiabetic suEionylureas are present in adenohypophysis and are involved in growth hormone release. Proc National Acad Sci USA 1993 ;90:1340- 13 44 257. Noma A, Shiasaki T: Membrane currents through adenosine triphosphate regulated potassium channels in guinea pig ventricular cell. JPhysol1985;3 63 :463480 258. Nichols CG,Lederer WJ: Adenosine triphosphate-sensitive potassium channels in the cardiovascular system. Am J PhysioZ 199 1 ;261 :HI 675-H1686 259. Misler S, Fake LC: A metabolite regulated potassium channel in rat pancreatic Beta cells. Proc National Acad Sci USA 1986;83:7119-7123 260. Findlay I: The effects of magnesium upon adenosine triphosphate sensitive potassium channels in a rat insulin secreting cell line. J Physiol l987;39 1 :61 1-629 26 1. Horie M, Irsawa H: Voltage -dependent magnesium block of ATP sensitive potassium channels in guinea pig ventricular cells. J Physiot l987;387:25 1-272 262. Findley I: The ATP sensitive potassium channel of cardiac muscle and action potential shortening during metabolic stress. Cardiovasc Res 1994;28:760-761 263. Spruce AE, Standen NB: Studies of the unitary properties of ATP regulated potassium channels of fiog skeletal muscles. J Physzol l987;382:2 13-286 264. Kakei M, Noma A: Properties of AT?? regdated potassium channels in guinea pig ventricular cells. J Physzol1985;363:44 1-462 265. Quayle JM,Standen NB: The voltage dependent block of ATP sensitive potassium channels of frog skeletal muscles by caesium and barium ions. J Physiol 1988;405:677- 697 266. Ashcroft FM: ATP sensitive potassium channels. Ann Rev Neurosci 1988;1 1 :W-ll8 267. Terzic A, Tung RT, Kurachi Y: Nucleotide regulation of ATP sensitive potassium channels. Cardiovasc Res 1W4;28 : 746-753 265. Takano M, Qin D: ATP dependent decay and recovery of potassium channels in guinea pig ventricular myocytes. Am J PhysioI 1990;258:H45-H50 269. Furukawa T, Virag L, Furukawa N, Sawanobori T:Mechanism for reactivation of the ATP sensitive potassium channel by MgATP complexes in guinea pig ventricular rnyocytes. J Physioll994;479:95-107 270. Takano M, Noma A: The ATP sensitive potassium channel. Prog Neurobiol 1993;42:21-30 271. FaWer B, Brandle U, Glowatzki E, Zenner HP: Kir2.1 inward rectifier potassium channels are regulated independently by protein kinases and ATP hydrolysis. Neuron 1994;13:1413-1420 272, McNicholas CM, Wang W, Ho K, Hebert SC: Regulation of ROMK-1 potassium channel activity involves p hosp horylation processes. Proc National Acad Sci USA 1994;91 18077-8081 273. Wollheim CB, Dume MJ: Activators of protein kinase C depoiarize insulin secreting cells by closing potassium channels. EiWO 1 988;7:2443-2449 274. Dume MJ, West-Jordan JA: The gating of nucleotide sensitive potassium in insulin secreting cells can be modulated by changes in the ratio of ATP/ADP and non- hydrolyzable derivatives of both ATP and ADP. Mernb Bioll988;104: 1 65- 1 77 275. Corkey BE, Deeney JT: Regulation of steady state fkee calcium levels by the ATPIADP ratio and orthophosphate in permeabilized insulinoma cells. J Biol Chem 1988;263:4247-4253 276. Elliot AC, Smith GL: Simultaneous measurement of action potential duration and intracellular ATP in isolated ferret hearts exposed to cyanide. Circ Res 1989;64:583-592 277. Coetzee WA: ATP sensitive potassium channels and myocardial ischemia: why do they open? Cardiovasc Drugs Ther l992;6:2O 1-208 278. Benz I, Kohihardt M: Differential sensitivity of cardiac K-ATP channels to guanine nucleotides: evidence for a heterogeneous channel population. Europ Biophys J l992;21:299-302 279. Misler S, Gillis K: Modulation of gating of a metabolically regulated ATP dependent potassium channel by intracellular pH in Beta cells of the pancreatic islet. J Memb Biol 1989;109:135-143 280. Cuevas J, Basset AL, Cameron JS, Furukawa T: Effect of H+ on ATP regulated potassium channels in feline ventricular myocytes. Am J Physiol 199 1;261:H755-H76 1 281- Koyano T, Kakei M, Nakashima H, Yoshinaga M: ATP regulated potassium channels are modulated by intracellular Ht in guinea pig ventricular myocytes. J Physiol 1993;463 1747-766 282. Fan Z, Makielski JC: Intracellular H+ and calcium modulation of trypsin modified ATP sensitive potassium channels in rabbit ventricular myocytes. Am J Physiol 1996;322:H233-H246 283. Challinor-Rogers JL, McPherson GA: Potassium channel openers and other regulators of K-ATP channels. CIin Exper Phannacol Physiol1994;Z 1 583-597 284. Edwards G, Weston AH: The pharmacology of ATP sensitive potassium channels. Ann Rev Pham2acol Toxicof 1993;33 597-637 285. Findlay I: Sulphonylurea drugs no longer inhibit ATP sensitive K+ channels during metabolic stress in cardiac muscle. J Pharmacol Exper merap 1993;266:456-467 286. Grover GJ, Dzwonczyk S, Sleph PG, Sargent CA: The ATP sensitive potassium channel blocker glybenclamide does not abolish preconditioning in isolated ischemic rat hearts. JPharmacalwer merap 1993;265:559-564 287. Auchampach JA, Cavero I, Gross GJ: Nicorandil attenuates myocardial dysfimction associated with transient ischemia by opening ATP dependent potassium channels. J Cardiovasc Pharmacol l992;20:765-77 1 288. Venkatesh N, Stuart JS, Lamp ST, Alexander LT: Activation of ATP sensitive K+ channels by cromakalim: Effects on cellular K+ loss and cardiac function in ischemic and repefised mammalian ventricle. Circ Res 1992;7l: 1 324- 1333 289. Fake JF, Findlay I: Effects of tolbutamide, glybenclamide and diazoxide upon action potential recorded &om rat ventricular muscle. Biochim Biophys Acta l989;984: 1-5 290. Sato T, Wu B, Nakamura S, Kiyosue T: Cibenzoline inhibits diazoxide and 2,4- dinitrophenol activated ATP sensitive K+ channels in guinea pig ventricular cells. Br J Pharmacul1993;108:549-556 29 1. Coetzee WA: ATP sensitive potassium channels and myocardial ischemia: why do they open? Cardiovasc Drug merap l992;6:ZO 1-208 292. Cavero I, DjelIas Y, WonJM: Ischemic myocardial cell protection conferred by the opening of ATP sensitive potassium channels. Cardovmc Drug Therap 1 995; 9:245- 255 293. Grover GJ: Protective effects of ATP sensitive potassium channel openers in experimental myocardiai ischemia. J Cmdiovasc PharmacoZ 1 994;24: S 18-S27 294. Findley I, Faivre JF: ATP sensitive Kt channels in heart muscle: spare channels. FEBS 1991;279:95-97 295. McPherson CD, Pierce GN, Cole WC: Ischemic cardioprotection by ATP sensitive K+ channels involves high energy phosphate preservation. Am J Physiol l993;265 :H1809- HI818 296. Cole WC, McPherson CD, Sontag D: ATP regulated K+ channels protect the myocardium against ischemia reperfirsion damage. Circ Res 199l;69:571-58 1 297. Grover GJ, McCullough JR, Henry DE: Anti-ischemic effects of the potassium channel activators pinacidil and cromakalim and the reversal of these effects with the potassium channel blocker glyburide. JPhamacolm Therap 1989;251:98-104 298. Grover GJ, McCullough D'Alonzo AJ, Sargent CA: Cardioprotective profile of the cardiac selective ATI? sensitive potassium channel opener BMS- 180448. J Cardiovasc Pharmacol l995;25:40-50 299. Auchampach JA Maruyama M, Cavero I: Pharmacological evidence for a role of ATP regulated potassium channels in myocardial stunning. Circulation 1992; 86:3 11-3 19 300. Imai N, Liang CS, Stone CK: Comparative effects of nitroprusside and pinacidil on myocardial blood flow and infarct size in awake dogs with acute myocardial infarction. Circulation l988;77:705-7 1 1 301. Grover GJ, Dzwonczyk S, Parham CS: The protective effects of cromokdirn and pinacidil on reperfhion function and infarct size in isolated perfused hearts and anaesthetized dogs. Cardiovasc Drug Therap 1990;4:465-474 302. Gross GJ, Auchampach JqMaruyama M: Cardioprotective effects of nicorandil. J Cardiovusc Phmacol l992;20:522-528 303. Daut JW,Maier-Rudolph W, Von Beckerath N: Hypoxic dilitation of coronary arteries is mediated by ATP sensitive potassium channels. Science 1990;247: 1341 - 1344 304. Samaha FF, Heineman FW, Ince C: ATP sensitive potassium channel is essential to maintain coronary vascular tone in vivo. Am J Physiol l992;262:C 1220-C1227 305. Kirsch GE, Codina JB: Coupling of ATP sensitive K+ channels to A1 receptors by G proteins in rat ventricular myocytes. Am JPhysiol1990;259:H820-H826 306. Nakhostine N, Lamontagne D: Adenosine contributes to hypoxia induced vasodilitation through ATP sensitive K+ channel activation. Am J Physzd 1993;265:H1289-HI293 307. Van Winkle DM, Chien GL: Cardioprotection provided by adenosine receptor activation is abolished by blockade of the K-ATP channel. Am J Physiol 1994;266:H829- H839 30 8. Ikonomidis IS: Preconditioning human ventricular myocytes. ~esi'issertation. 1995;134 309. Ito H, Vereecke J, Carmeliet E: Mode of regulation by G protein of the ATP sensitive K+ channel in guinea pig ventridar cell membrane. J Physiol l994;478: 10 1-1 07 3 10. Grover GJ, Sleph PG, Dmonczyk S: Role of myocardial ATP sensitive potassium channels in mediating preconditioning in the dog heart and their possible interaction with adenosine A1 receptors. Circulation 1992;86:13 10-13 16 3 1 1. Speechly-Dick ME, Grover GJ, Yellon DM: Does ischemic preconditioning in the human involve protein kinase C and the ATP dependent K+ channel? Studies of contractile action after simulated ischemia in an atrial in vitro model- Circ Res 1995;77:1030-1035 3 12. Auchampach JA, Grover GI, Gross GJ: Blockade of ischemic preconditioning in dogs by the novel ATP dependent potassium channel antagonist sodium 5- hydroxydecanoate. Cardiovasc Res 1992;26:1054-1 062 3 13. Wore CL, Sievers RE: Loss of myocardial protection after preconditioning correlates with the time course of glycogen recovery within the preconditioned segment. Circulation 1993;87:881-892 314. Yao Z, Gross G: Activation of ATP sensitive potassium channels lowers the threshold for ischemic preconditioning in dogs. Am J Physiol l994;267:Hl88 8-H1894 3 15. Jovanovic A, Jovanovic S, Lorenz E, Temc A: Recombinant cardiac ATP-sensitive K+ channel subunits confer resistance to chemical hypoxia re-oxygenation injury. Circulation 1986;74:I124-1136 316. Jovanovic 4 Jovanovic S, Carrasco AJ, Tenic A: Acquired resistance of a mammalian cell line to hypoxia re-oxygenation through cotransfection of Kir6.2 and SURl clones. Lab hvesf l998;78:1 10 1-1 107 317. Schulz R, Rose J, Heusch G: Involvement of activation of activation of ATP- depe~dentpotassium channels in ischemic preconditioning in swine. Am J Physiol 1994;267:H1341-HI352 3 18. Yao 2, Gross G: Effects of the K-ATP channel opener bimakalim on coronary blood flow, monophasic action potential duration and infarct size in dogs. Circulation 1994;89:1768-1775 3 19. Grover G, D'Alonso A, Dmonczyk S, Parham CS, Darbenzio RB:Preconditioning is not abolished by the delayed rectifier K+ blocker dofetilide. Am J Physiol 1996;271 :H1207-HI214 320. Armstrong S, Liu G, Downey J, Ganote CE: Potassium channels and preconditioning of isolated rabbit cardiomyocytes: effects of glyburide and pinacidil. J Mot CeN Cardiol l995;27:1765- 1774 321. Inoue I, Nagasi H, Kishi K, Higuti T: ATP-sensitive K+ channel in the rnitochondrial inner membrane- Nature 199 1;3 52:244 322. Paucek P, Mironova G, Mahdi F, Beavis A, Woldegiorgis G, Garlid KD: Reconstitution and partial purification of the glibenclarnide sensitive ATP dependent K+ channel from rat liver and beef heart mitochondria. JBiol Chem 1992;267:26062-26069 323. Okuyama Y, Yamada M, Kondo C, et al. The effects of nucleotides and potassium channel openers on the SUR2AKir6.2 complex K+ channel expressed in a mammalian cell line, HEK 293T cells. PfIugers Arch 1998;435:595-603 324. Liu Y, Sato T, O'Rourke B, Marban E: Mitochondria1 ATP dependent potassium channels: novel effectors of cardioprotection? Circulation 1998;97:2463-2469 325. Sato T, Sasaki N, O'Rourke B, Marban E: Nicorandil activates mitochondrial ATP dependent potassium channels in rabbit ventricular cells. Circulation 1998;98 :I- 3 43(Abst~act) 326. Gross GJ, Fryer RM: Sarcolemmal versus mitochondrial ATP sensitive Kt- channels and myocardial preconditioning. Circ Res l999;84973-979 327. Garlid KD, Paucek P, Yarov-Yarovoy V, et al. Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP sensitive Kt channels: possible mechanism of cardioprotection. Circ Res 1997;81 : 1072-1082 328. Garlid KD, Paucek P, Yarov-Yarovoy V, Sun X, Schindler PA: The mitochondrial K-ATP channel as a receptor for potassium channel openers. J Biolog Chem l996;27 1:8796-8799 329. Gogelein H, Hartung J, Englert H, SchoIkens B: HMR I 883, a novel cardioselective inhibitor of the ATP-sensitive potassium channel. J Phamtacol Fxp 7her 1998;286:1453- 1464 330. Hill JA, Utterback DB, Keim SG, Dugger D, Mayfield WR, Shryock JC: Validation of a new method for sampling human plasma adenosine in vivo. Cor Art Dis 1992;3:963- 97 1 33 1. Ikonomidis JS, Tumiati LC, Weisel RD, Mickle DAG, Li RK: Preconditioning in human ventricular myocytes with brief episodes of simulated ischemia. Curdiovasc Res l994;28: 1285-1291 332. Turniati LC, Mckle DAG, Weisel RD, Williams WG, Li RK: An in-vitro model to study myocardial ischemic injury. J Tissue Cult Metho& 1994; 16: 1-9 333. Li RK, Shaikh N, Weisel RD, WilIiams WG, Mickle DAG: Oxyradical-induced antioxidant and Lipid changes in cultured human cardiomyocytes. Am J Physiol 1994;266:H2204-HZll 334. Weisel RD,Mickle DAG, Finkle CD, Tumiati LC, Madonik MM, Ivanov J: Delayed myocardial metabolic recovery after blood cardioplegia. Ann Zhorac Surg 1989;48:503- 507 335. Hull-Ryde Eq Lewis Veronee CD, Lowe JE: Simple step gradient elution of the major high energy compounds and their metabolites in cardiac muscle using high performance liquid chromatography. J Chromatogr l986;3 77: 165-174 336. Burton K: A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J 1956;62:315-323 337. Heasley LE, Johnson GL: Regulation of protein kinase C by nerve growth factor, epidermal growth factor, and phorbol esters in PC12 pheochrornocytoma cells. J Biol Chem 1989;264:8646-8652 338. Heasley LE, Johnson GL: Detection of nerve growth factor and epidermal growth factor-regulated protein kinases in PC12 cells with synthetic peptide substrates. Mol PharmacoZ1989;35:331-338 339. Ikonomidis JS, Shirai T, Weisel RD, Ivanov J: Endogenously released adenosine mediates human ventricular cardiomyocyte preconditioning through protein kinase C. Am JPhysiol1996; 340. Lowry Ow Rosebrough NJ, Farr AL, Randall RJ: Protein measurements with the folin-phenol reagent. J Biol Chem 195 1; 193 :265-275 341. Madden E4 Storrie B: Tne preparative isolation of mitochondria fiom chinese hamster ovary cells. AnaIyt Bzochem 1 987;1 63 :3 50-357 342. UrseU McLaurin I, Wood DD, Ackerley C4MoscareUo MA: Localization and partial characterization of a 60 kDa citrulline-containing transport form of myelin basic protein fiom M03-13 cells and human white matter. JNeurosci Res 2 995;42:41-53 343. Glem M, Robinson BH, Spratt C,Wilson I, Patrick D: Abnormal kinetic behaviour of cytochrome oxidase in a case of Leigh disease. Am JNman Genet 1987;41:584-593 344. Merante F, Petrova-Benedict q MacKay N: A biochemically distinct form of cytochrome oxidase (COX) deficiency in the Saguenay-Lac-S aint-Jean region of Quebec. Am JHurnan Genet 1993;53:481-487 345. Pitkanen S, Merante F, Mcleod DR, Applegarth D, Tong T, Robinson BH: Familial cardiomyopathy with cataracts and lactic acidosis: a defect in complex I (NADH- dehydrogenase) of the mitochondria1 respiratory chain. PedRes 1996;39:513-52 1 346. Schwerzmann K, Cruz-Orive LM, Eggrnan R, Sanger 4 Weibel ER: Molecular architecture of the inner membrane of mitochondria Erom rat liver: A combined biochemical and stereological study. J Cell Biol 1986;1 O2:97-103 347. Mauser M, Hofbeister Neinaber C, Schaper W: Influence of ribose, adenosine and "AICAR" on the rate of myocardial adenosine triphosphate synthesis during repefision after coronary artery occlusion in the dog. Czrc Res 1985;56:220-230 348. Harmsen E, Detombe PP, Dejong JW,Achterberg PW: Enhanced ATP and GTP synthesis fiom hypoxanthine or inosine after myocardial ischemia. Am J PhysioZ 1984;246:H37-H43 349. Kloner RA, De Boer LWV, Braunwald E: Recovery from prolonged abnormalities of canine myocardium salvaged from ischemic necrosis by coronary reperfusion. Proc Natl Acad Sci USA 198 I;78:7152-7156 350. Merante F, MickIe DAG, Weisel RD, Li RK, Tumiati LC, Rao V, Wdliams WG, Robinson BH: Myocardial aerobic metabolism is impaired in a cell culture model of cyanotic heart disease. Am J PhysioZ 1998;275:H1673-HI681 35 1. KIoner RA, Yellon DM: Does ischemic preconditioning occur in patients? J Am Call cardiol I994;24:1 133-1 142 352. Lawson CS: Does ischemic preconditioning occur in the human heart? Cardiovasc Res 1994;28:146 1-1466 3 53. YelIon DM, Alkhulaifi AM, Pugsky WB: Preconditioning the human myocardium. Lancet 1993 ;342:276-277 354. Van Wylen DGL: Effect of ischemic preconditioning on interstitial purine metabolite and lactate accumulation during myocardial ischemia. Circulation 1994;89:2283-2289 355. Li RK, Weisel RD, Wiams WG, MickIe DAG: Methods of culturing cardiornyocytes from human pediatric ventricular myocardium. J Tiss Cult Meth 2992;14:93-100 356. Li RK, Mickle DAG, Weisel RD, et al. Human pediatric and adult ventricular cardiomyocytes in culture; assessment of phenotypic changes with passaging. Cardiovasc Res 1996;32:362-373 357. Arend LJ, Somenberg WK, Smith WL, SpieIman WS: A1 and A2 adenosine receptors in rabbit cortical collecting tubule cells: modulation of hormone stimulated c- AMP. Jch Invest 1987;79:710-714 358- Mentzer RM,Bunger R, Lasley RD: Adenosine enhanced preservation of myocardial bction and energetics. Possible involvement of the adenosine A1 receptor system. Cardiovasc Res 1993;27:28-35 359. Liu GS, Richards CS, Olsson RA, Mullane K, Walsh RS, Downey JM:Evidence that the adenosine A3 receptor may mediate the protection af5orded by preconditioning in the isolated rabbit heart. Cardovase Res l994;28 :105% 106 1 360- DaIy JM: Bioenergetics and Oxidative Metabolism. in DevIin TM (ed): Text Book of Biochemistry with CZinical CorreIations. New Yo& Wiley-Liss Inc. 1992, pp 270 361. Quast U, Cook NS: In vitro and in vivo comparison of two K+ channel openers, diazoxide and cromakalim, and their inhibition by glibenclamide. J Pharmnacol Exp Ther l989;2SO:26 1-271 362. Trube G, Rorsman P, Ohno-Shosaku T: Opposite effects of toIbutamide and diazoxide on the ATP-dependent K+ channel in mouse pancreatic beta cells. Pflugers Arch 1986;407:493-499 363. Halestrap AP: Regulation of mitochondrial metabolism through changes in matrix volume. Biochem Soc Trans l994;22: 522-529 364. Ghadially FN: AnonymousUZfrasfruc~ralPathology of the Cell Matrix. New York, Pendant Publ. 1995, pp 242 365. Hackenbrock CR, Rehn TG, Weinbach EC, Lemasters JJ: Oxidative phosphorylation and ultrastructural transformation in mitochondria in the intact ascites tumor cell. J Cell Bid l97l;l: 123-137 366. Billman GE, Engiert HC, Scholkens BA: HMR 1883, a novel cardioselective inhibitor of the ATP sensitive potassium channel. J Phannacd Eip Ther 1998;286: 1465- 1443 367. Christe G: Localization of Kt channels in the tubules of cardiomyocytes as suggested by the parallel decay of membrane capacitance, IK-1 and IK-ATP during culture and by delayed response to barium. JMolec Cell CardioZ 1999;3 1 :2207-22 13 368. Sato T, Sasaki N, O'Rourke B, Marban E: Adenosine primes the opening of mitochondrial ATP-sensitive potassium channels. CircziZafion2000; 102:800-8 13 369. Jung 0, Englert HC, Jung W, Gogelein H, Scholkens BA, Busch AE, Linz W: The K-ATP channel bIocker HMR 1883 does not abolish the benefit of ischemic preconditioning on myocardid infarct mass in anaesthetized rabbits. Nmnp Schrniedebergs Arch PharmacoI 2000;361:445-45 1 370. Jaburek M, Yarov-Yarovoy V, Paucek P, Garlid KD: State-dependent inhibition of the mitochondria1 K-ATP channel by glyburide and 5-hydroxydecanoate. J Biol Chem l998;273: 13578-13582 371. Kevelaitis E, Oubenaissa A, Peynet J, Mouas C, Menasche P: Preconditioning by mitochondria1 ATP-sensitive potassium channel openers: an effective approach for improving the preservation of heart transplants. Circulation 1999; 100 :IT-3 45 372. Carroll Yellon DM: Myocardial adaptation to ischemia: the preconditioning phenomenon. Int JCardioZ 1999;68:S93-S 101 373. Cleveland JC, Meldrum DR, Rowland RT, Cain BS, Meng Gamboni-Robertson F, Banejee 4 Harken AH: Ischemic preconditioning of human myocardium: protein kinase C mediates a permissive role for alpha 1-adrenoreceptors. Am J Physiol l997;273 :H902- H908 374. Ghosh S, Standen NB, GaIinanes ha: Evidence for mitochondrid K-ATP channels as effectors of human myocardial preconditioning. Cardovosc Res 2000;45:934-940 375. Holrnuhamedov E, Jovanovic S, Dzeja P, Jovanovic 4 Terzic A: Mitochondrial ATP-sensitive K+ channels modulate cardiac mitochondria1 function. Am J PhysioZ 1998;275:H1567-HI576 376. Tokube K, Kiyasue T, Arita M: Opening of cardiac K-ATP channel by oxygen fkee radicals produced by xanthine oxidase reaction. Am J PhysioI1996;271 :H478-H489 377. Ichinari K, Kokei M, Matsuoka T, Nakashima H, Tomaka H: Direct activation of the ATP sensitive potassium channel by oxygen &ee radicals in guinea pig ventricular cells: its potentiation by MgADP. J MoZ Cell CardioZ 1996;28:1867-1877 378. Marber MS, Latchman DS, Walker JM,Yellon DM: Cardiac stress protein elevation 24 hours after brief ischemia or heat stress is associated with resistance to myocardial infarction- Circulation 1993;88 :1264-1 272 379. Knowlton 4 Brecher P, Apstein CS: Rapid expression of heat shock protein in the rabbit after brief cardiac ischemia. J CZin Invest 1991;s 7: 139- 147 380. Thornton J, Striplin S, Liu GS, Swafford A, Stanley AWH, VanWinkle DM, Downey JM: Inhibition of protein synthesis does not block myocardial protection by preconditioning. Am J Physiol l99O;Zg:H 1822-H1825 38 1. Das DK, Engelman RM, Kimura Y: Molecular adaptation of cellular defences following preconditioning of the heart by repeated ischemia. Cmdiovasc Res 1993;27:578-584 382. Taggart DP, Bakkenist CJ, Biddolph SC, Graham AK, McGee JO: Induction of myocardial heat shock protein 70 during cardiac surgery. J Pathol1997; 182:3 62-3 66 383. Neely CF, Haile DM, Cahill BE, Kadowitz PI: Adenosine and ATP produce vasoconstriction in the feline pulmonary vascular bed by different mechanisms. J Pharmacolq Ther 1991;258:753-761 3 84. Vegh A, Szekeres L, Parratt JR: Protective effects of preconditioning of the ischemic myocardium involve cyclo-oxygenase products. Cardovasc Res 1990;24:1020- 1023 385. Li Y, Kloner RA: Cardioprotective effects of ischemic preconditioning are not mediated by prostanoids. Cardiovasc Res 2992;26:226-231 386. Tosaki A, Cordis GA, Szerdahelyi P, Engelman RM, Das DK: Effects of preconditioning on repefision arrhythmias, myocardial function, formation of fiee radicals, and ion shifts in isolated ischemidrepexfked rat hearts. J Cardovasc Pharmucol * l994;23 13 65-3 73 387. Osada Ad, Takeda S, Sato T, Komori S, Tamura K: The protective effect of preconditioning on reperfusion-induced arrhythmia is lost by treatment with superoxide dismutase. Jap Circ J 1994;58:259-263 388. Steenbergen C, FralLv T& Murphy E: Role of increased cytosolic fiee calcium concentration in myocardial ischemic injury. Basic Res Cardiol 1993 ;8 8 :456-470 389. Steenbergen CJ, Perlrnan ME, London RE, Murphy E: Mechanism of preconditioning: ionic alterations. Cnrdiovasc Res 1993 ;72:1 12-125 390. Allen DG, Xiao )i?I: Activity of the Na+/H+ exchanger contributes to cardiac damage following ischemia and reperfusion. CZin Ekp Phamacol Physiol 2OOO;27: 727- 73 3 3 9 1. Hayashida N, Weisel RD, Shirai T: Tepid antegrade and retrograde carioplegia. Ann Thorac Surg 1995;59:723-729 392. Shirai T, Rao V, Weisel RD: Antegrade and retrograde cardioplegia: alternate or simultaneous? J Thorac Cardiovasc Surg 1996;1 12787-796 393. Cheng DCH, Karski J, Peniston CE: Morbidity outcome of early versus conventional tracheal extubation following coronary artery bypass graft (CABG) surgery: A prospective randomized, controlled clinical study. J 27zorac Cardiovasc Surg 1996; 112:755-764 394. Rao V, Ivanov J, Weisel RD, Ikonomidis JS, Christakis GT, David TE: Predictors of low output syndrome following coronary bypass surgery. J Thornc Cardiovasc Surg 1996; 112:38-51 395. Burt JM, Copeland JG: Myocardial hnction after preservation for 24 hours. J 7horac cmdiavasc Surg 1986;92:238-246 396. See YP, Weisel RD, Mickie DAG, et al. Prolonged hypothermic cardiac storage for transplantation. The effects on myocardial metabolism and mitochondria1 function. J morac Cardiovasc Surg 1992; 1O4:8 1 7-824 397. Weisel RD, Mickle DAG, Finkle CD, et al. Delayed myocardial recovery after blood cardioplegia. Ann Thorac Surg 1989;48:503-507 398. Weisel RD, Lipton IE3, Lyall RN, Baird RJ: Cardiac metabolism and performance following cold potassium cardioplegia. CircuIatton 1978; 5 8 :I2 1 7-1226 399. Cohen G, Schwartz D, Feindel CM, Boylen P, Rao V, Borger Weisel RD: Donor blood perfhion improves hernodynamic performance following prolonged storage of cardiac allografts. Can J CardioZ 1 999;1 5 :37(Abstract) 400. Ferrera R, Larese A, Marcsek P, et al. Comparison of different techniques of hypothermic pig heart presenration. Ann XJzorac Surg 199437:123 3 - 123 9 401. Wicomb WN, Collins GM: Twenty four hour rabbit heart storage with UW solution. Effects of low flow perfusion, colloid and shelf storage. TranqIantation 1989;48:6-9 402. Rao V, Feindel CM, Weisel RD, Boylen P, Cohen G: Donor blood pefision improves myocardial recovery after heart transplantation. J Heart Lung Transplanr 1997; 16:667-673 403. Saldanha C, Hearse DJ: Coronary vascular responsiveness to 5-hydroxytryptamine before and after infusion of hyperkalemic crystalloid cardioplegic solution in the rat heart: possible efidence of endothelial damage. J Thorac Cmdiovasc Surg 1989;98:783-787 404. Follette Dm Buckberg GD, Mulder DG, Fonkalsrud EW: Deleterious effects of crystalloid hyperkalemic cardioplegic solutions on arterial endothelial cells. Surg Form l98O;3 1 :253-255 405. Feindel CM, Tait GA, Wilson GI, Klement P, MacGregor DC: Multidose blood versus crystalloid cardioplegia: comparison by quantitative assessment of irreversible myocardial injuiy. J Thorac Cardiovasc Surg 1984;87:585-595 406. Fremes SE, Christakis GT, Weisel RD, et al. A clinical trial of blood and crystalloid cardioplegia. J ishorac Cordovasc Surg l984;88:726-74 1 407. Christakis GT, Fremes SE, Weisel RD, et al. Reducing the risk of urgent revascularization for unstable angina: a randomized clinical trial. J Vasc Surg 1986;3:764- 772 408. Buckberg GD,Brazier JR, Nelson RL, et al. Studies of the effects of hypotheha on regional myocardial flow and metabolism during cardiopulmonary bypass. The adequately perked, beating, fibrillating and arrested heart. J Xhorac Cardovasc Surg 1977;73:87-94 409. Lichtenstein SV, Ashe KA, El Dalati II-, et al. Warm Heart Surgery. J norac Cardiovasc surg 199 1 ;10 1 :269-274 410. Hayashida N, Ikonomidis JS, Weisel RD,et d.The optimal cardioplegic temperature. Ann Thorac Surg 1994;58:961-971 41 1. Yau TM, Ikonomidis JS, Weisel RD, et al. Ventricular function after normothermic versus hypothermic cardioplegia. J Thorac Cardiovasc Surg 1993;I05 :833-844 412. Ikonomidis JS, Yau TM, Weisel RD, et al. Optimal flow rates for retrograde warm cardioplegia. J Kborac Cardovasc Surg 1994;107: 5 10-519 413. Hendry PJ, Walley VM, Koshal 4 et al. Are temperatures attained by donor hearts during transport too cold? J Thorac Cmdiovarc Surg 1989;98:517-522 414. Gott VL, GodezJL, Zhudi MN, et al. Retrograde perfision of the coronary sinus for direct vision aortic surgery. Surg Gynecol Obstet 1957;1 OM19 415. Feindel CM, Sandhu q Cruz J, Wdson JG: The effect in delay of retroperfbsion therapy on infarct size reduction. Ann liclorac Surg l992;54: 1120-1 1ZS 416- Carrier M, Leung TK, Solymoss BC, Cartier R, Leclerc Y,Pelletier LC: Clinical trial of retrograde warm blood pefision versus standard cold topical irrigation of transplanted hearts. Ann norac Surg 1W6;6 1 :13 10-13 15