The Role of Candidate G-protein Coupled Receptors

in Mediating Remote Myocardial Ischemic

Preconditioning.

by

Harinee Surendra

A thesis submitted in conformity with the requirements for the degree of

Master of Science

Graduate Department of Laboratory Medicine and Pathobiology

University of Toronto

© Copyright by Harinee Surendra (2009) i

ACKNOWLEDGEMENTS

I would like to extend my heartfelt gratitude to my family for supporting me through this rewarding and demanding experience. Their constant encouragement has made this thesis possible.

Also, my sincere thanks are given to the guidance and support of Dr. Gregory Wilson

(Division of Pathology, Department of Paediatric Laboratory Medicine, Hospital for Sick

Children) and Roberto Diaz (Dr. Wilson‘s Lab, Hospital for Sick Children). Their extensive knowledge in the field of ischemic preconditioning has provided excellent direction for this thesis and given me opportunities to progress in my career. Also, tremendous guidance was provided by members of my graduate committee through Dr. Herman Yeger (Research Institute,

Hospital for Sick Children) and Dr Aleksander Hinek (Research Institute, Hospital for Sick

Children). My genuine appreciation is given to Alina Hinek (Dr. Wilson‘s Lab, Hospital for Sick

Children) and Taneya Hossain (Dr. Wilson‘s Lab, Hospital for Sick Children) for their assistance in providing training.

I would like to thank Jing Li (Dr. Redington‘s Lab, Research Institute, Hospital for Sick

Children) for providing the dialysate used in this study and Michael Tropak (Dr. Callahan‘s Lab,

Research Institute, Hospital for Sick Children) for measuring substances in the dialysate with mass spectrometry. Finally, I would like to thank Foundation Leducq, the Heart and Stroke

Foundation of Ontario, and the University of Toronto for providing funding for my research. ii

ABSTRACT

The Role of Candidate G-protein Coupled Receptors in Mediating Remote Myocardial

Ischemic Preconditioning.

By Harinee Surendra

Degree of Master of Science (2009)

Graduate Department of Laboratory Medicine and Pathobiology

University of Toronto

This study investigated the role of opioid, adenosine, bradykinin, and -gene related peptide (CGRP) receptors, and potential ‗cross-talk‘ among suspected G-protein coupled receptors in a humoral model of remote ischemic preconditioning (rIPC) cardioprotection.

Compared to Control dialysate (from non-preconditioned donor rabbit blood), rIPC dialysate

(from remotely preconditioned blood) reduced cell death in rabbit cardiomyocytes following simulated ischemia and reperfusion. Non-selective, δ-, or κ-opioid blockade and non- selective blockade abolished rIPC dialysate protection; whereas, bradykinin

B2 and CGRP receptor blockade had no effect. Non-selective adenosine receptor blockade fully and partially abolished protection by κ- and δ-opioid receptors, respectively. Multiple reaction monitoring mass spectrometry detected low levels of adenosine, and other preconditioning substances, in the dialysate. An increase in extracellular adenosine was not detected during opioid-induced preconditioning to explain this cross-talk. These results suggest that δ-opioid, κ- opioid, adenosine receptors, and opioid-adenosine cross-talk are involved in rIPC of freshly isolated cardiomyocytes. iii

TABLE OF CONTENTS

ABSTRACT ...... ii

GLOSSARY...... viii

1. INTRODUCTION...... 1

ISCHEMIC PRECONDITIONING (IPC) 1.1 ...... 1

Background in IPC 1.1.a ...... 1

Signalling Mechanisms in IPC 1.1.b ...... 2

REMOTE ISCHEMIC PRECONDITIONING (RIPC) 1.2 ...... 5

Background in rIPC 1.2.a ...... 5

Signalling Mechanisms in rIPC 1.2.b ...... 5

INVOLVEMENT OF OPIOID RECEPTORS 1.3 ...... 8

Opioids and Their Receptors 1.3.a ...... 8

Evidence of Opioid Involvement in IPC1.3.b ...... 9

Evidence of Opioid Involvement in rIPC1.3.c...... 10

INVOLVEMENT OF BRADYKININ B2 RECEPTORS 1.4 ...... 11

Bradykinin and Their Receptors 1.4.a ...... 11

Evidence of Bradykinin Involvement in IPC 1.4.b ...... 12

Evidence of Bradykinin Involvement in rIPC 1.4.c ...... 13

INVOLVEMENT OF CGRP RECEPTORS 1.5 ...... 14

CGRP and Their Receptors 1.5.a ...... 14

Evidence of CGRP Involvement in IPC 1.5.b...... 15

Evidence of CGRP Involvement in rIPC 1.5.c ...... 16

INVOLVEMENT OF ADENOSINE RECEPTORS 1.6 ...... 17 iv

Adenosine and Their Receptors 1.6.a...... 17

Evidence of Adenosine Involvement in IPC 1.6.c...... 18

Evidence of Adenosine Involvement in rIPC 1.6.b ...... 19

OTHER POTENTIAL PRECONDITIONING SUBSTANCES 1.7 ...... 20

OPIOID-ADENOSINE RECEPTOR CROSS-TALK 1.8 ...... 22

Background 1.8.a ...... 22

Adenosine Levels in the Blood 1.8.b ...... 23

Potential Mechanisms of Cross-talk 1.8.c ...... 23

2. RATIONALE ...... 26

3. OBJECTIVES ...... 27

HYPOTHESIS 3.1 ...... 27

SPECIFIC AIMS 3.2 ...... 27

4. RESEARCH DESIGN & METHODS ...... 28

ANIMALS AND HUMAN SUBJECTS 4.1 ...... 28

ISOLATION OF ADULT CARDIOMYOCYTES 4.2 ...... 28

Operative Procedure 4.2.a ...... 28

Digestion Protocol 4.2.b ...... 29

Comparison of Digestion Protocols 4.2.c ...... 32

DIALYSATE PREPARATION 4.3 ...... 34

FRESH-CELL EXPERIMENTAL MODEL 4.4 ...... 37

General Protocol 4.4.a ...... 37

AIM (1) Protocol 4.4.b ...... 39 v

AIM (2) Protocol 4.4.c ...... 40

AIM (3) Protocol 4.4.d ...... 41

TRYPAN BLUE EXCLUSION ASSAY 4.5 ...... 42

Inter-Observer Error Data 4.5.a ...... 44

DRUGS 4.6 ...... 46

OTHER METHODS 4.7 ...... 47

Western Blotting 4.7.a ...... 47

Multiple Reaction Monitoring Mass Spectrometry 4.7.b ...... 48

Statistics 4.7.c ...... 50

5. CHARACTERIZATION OF THE PROTECTION INDUCED BY RIPC ...... 51

RABBIT AND HUMAN PRECONDITIONED DIALYSATE INDUCES PROTECTION 5.1 ...... 51

Dialysate Characterization 5.1.a ...... 53

Discussion 5.1.b ...... 53

6. THE ROLE OF CELL MEMBRANE RECEPTORS IN RIPC ...... 56

THE ROLE OF OPIOID RECEPTORS 6.1 ...... 56

Survey of Opioid Receptors in Rabbit Cardiomyocytes 6.1.a ...... 56

Non-selective Blockade of Protection 6.1.b ...... 58

δ-Opioid Receptor Blockade of Protection 6.1.c ...... 60

κ-Opioid Receptor Blockade of Protection 6.1.d ...... 62

Discussion 6.1.e...... 64

THE ROLE OF BRADYKININ B2 RECEPTORS 6.2 ...... 66

Bradykinin B2 Receptor Blockage Conserves Protection 6.2.a ...... 66

Discussion 6.2.b ...... 68 vi

THE ROLE OF CGRP RECEPTORS 6.3 ...... 70

Existence of Calcitonin-like Receptors in Rabbit Cardiomyocytes 6.3.a ...... 70

CGRP Receptor Blockage Conserves Protection 6.3.b...... 71

Discussion 6.3.c...... 73

THE ROLE OF ADENOSINE RECEPTORS 6.4 ...... 74

Non-selective Adenosine Receptor Blockade of Protection 6.4.a ...... 74

Discussion 6.4.b ...... 76

7. RIPC MEDIATES OPIOID-ADENOSINE CROSS-TALK ...... 78

OPIOID-ADENOSINE CROSS-TALK 7.1 ...... 78

Adenosine Deamination of the Dialysate Conserves Protection 7.1.a ...... 78

Partial Adenosine Blockade of δ-Opioid Receptor-Induced Protection 7.1.b ...... 80

Compete Adenosine Blockade of κ-Opioid Receptor-Induced Protection 7.1.c ...... 82

Discussion 7.1.d ...... 84

THE INHIBITION OF ADENOSINE KINASE HYPOTHESIS 7.2 ...... 86

Exposure to Dynorphin B Does Not Accumulate Extracellular Adenosine 7.2.a ...... 86

Discussion 7.2.b ...... 89

8. SUMMARY OF FINDINGS ...... 92

9. GENERAL DISCUSSION ...... 93

OVERALL PERSPECTIVE 9.1 ...... 93

OTHER MECHANISMS OF OPIOID-ADENOSINE CROSS-TALK 9.2 ...... 95

Dimerization of G-protein Coupled Receptors 9.2.a ...... 95

FUTURE DIRECTIONS 9.3 ...... 96 vii

Specific Adenosine Receptor Subtypes in Cross-talk & rIPC 9.3.a ...... 96

Heterodimerization of Opioid and Adenosine Receptors 9.3.b ...... 98

Cell Signalling in rIPC 9.3.c ...... 98

LIMITATIONS 9.4 ...... 103

CONCLUSIONS 9.5 ...... 104

APPENDIX ...... 105

APPENDIX I: LIST OF FIGURES ...... 105

APPENDIX II: LIST OF TABLES ...... 107

APPENDIX III: RIPC SUMMARY: MYOCARDIUM AS THE TARGET ORGAN ...... 108

APPENDIX IV: RIPC SUMMARY: SKELETAL MUSCLE AS THE PRECONDITIONING ORGAN ...... 109

APPENDIX V: MRM MASS SPECTROMETRY PLOTS OF STANDARD CONCENTRATIONS ...... 110

REFERENCE LIST ...... 113 viii

GLOSSARY

TERM DEFINITION

8-SPT 8-(p-sulfophenyl)theophylline

ADA Adenosine deaminase

ADO Adenosine

BNTX 7-benzylidenealtrexone cAMP cyclic adenosine monophosphate

CAO Coronary artery occlusion

CCPA 2-chloro-N6-cyclopentyladenosine

CGRP (8-37) Human fragment of the calcitonin-gene related peptide

DynB Dynorphin B

FAO Femoral artery occlusion

GNTI 5‘-guanidinyl-17-(cyclopropylmethyl)-6,7-dehyrdo-4,5α-epoxy-3,14-

dihydroxy-6,7-2‘,3‘-indolomorphinan

HOE140 Hoechst 140

IPC Ischemic preconditioning

IR Ischemia-reperfusion

KH Krebs-Henseleit

MAO Mesenteric artery occlusion

ME Met-enkephalin mKATP Mitochondrial ATP sensitive potassium channels ix

MPG N-2-mercaptopropionyl glycine mPTP Mitochondrial permeability transition pore

MRM MS Multiple reaction monitoring mass spectrometry

Nal Naloxone

NO Nitric oxide

NOS nitric oxide synthase

NTI Naltrindole

PGI2 Prostacyclin

PI3 Kinase Phosphatidylinositol 3-kinase

PKC Protein kinase C

RAO Renal artery occlusion rIPC Remote ischemic preconditioning

ROS Reactive oxygen species

SI Simulated ischemia sKATP Sarcolemmal ATP sensitive potassium channels

SR Simulated reperfusion

TNF-α Tumour necrosis factor –α

WB Western blotting

Wort Wortmannin 1

- Chapter 1 -

INTRODUCTION

1.1 Ischemic Preconditioning (IPC)

1.1.a Background in IPC:

Ischemic preconditioning (IPC) is one of the most potent strategies, under experimental conditions, to reduce myocardial infarction to date. One or more periods of brief ischemia (~5 min) and reperfusion (~5 min) render the myocardium protected from a much longer damaging ischemic stress. This phenomenon, first discovered by Murry et al.1 in 1986, exhibits two distinct windows of protection. This thesis will focus on the first window of protection (called acute preconditioning), which occurs within 2 hours of the IPC stimulus, whereas the second window

(called delayed preconditioning) occurs 24-72 hours later and is thought to be gene transcription- dependent.

Since the discovery of IPC to induce protection in a wide range of tissues and species, the triggers involved in this phenomenon have been of great interest as a therapeutic intervention for myocardial infarction. Thornton et al.2 determined that G-protein coupled receptors were vital to preconditioning when pertussis toxin (an inhibiter of G-protein coupled receptors) administered in vivo to rabbits blocked myocardial preconditioning. Since then, the major extracellular molecules involved in IPC at the trigger phase were found to be adenosine, bradykinin and opioid peptides. Goto et al.3 postulated that receptors for these three peptides exert an additive effect to reach a hypothetical threshold that is required to induce cardioprotection. Thus, blockade of any one of these G-protein coupled receptors is enough to abolish protection by IPC. 2

1.1.b Signalling Mechanisms in IPC:

The downstream signalling pathways with respect to classical IPC have been well established. According to Downey et al. 4, cell signalling exhibits three distinct phases: the

‗trigger‘ phase, the ‗mediator‘ and the ―end effector‖ phase.

Mocanu et al.5 demonstrated that cardioprotection was abolished by the phosphatidylinositol 3-kinase (PI3 kinase) inhibitor, wortmannin and LY 294002. The activation of PI3 kinase results in translocation of Akt to the plasma membrane6 and its subsequent phosphorylation7. Akt then stimulates nitric oxide synthase (NOS) to generate nitric oxide (NO)8, an important molecule that also acts as a trigger in IPC9 as suggested by Lochner et al.10.

Oldenburg et al.11 also proposed the involvement of guanylyl cyclase (GC) in IPC by abrogating protection with the GC inhibitor, ODQ. GC then produces cGMP and activates protein kinase G

(PKG).

PKG then induces the opening of mitochondrial potassium channels (mKATP) which results in swelling of the mitochondria due to potassium influx. This leads to reactive oxygen

12 species (ROS) generation as shown by Forbes et al. when protection from diazoxide (an mKATP channel opener) was abolished by ROS scavengers. Finally, the target of this downstream signalling pathway converges on protein kinase C (PKC)13.

To summarize, the trigger phase involves the release of endogenous substances, such as opioid peptides and bradykinin, that activate a complex pathway which includes, in the following 3 order: phosphatidylinositol 3-kinase (PI3-kinase), Akt, nitric oxide synthase (NOS), nitric oxide

(NO), guanylyl cyclase, protein kinase G, opening of mitochondrial KATP channels, which in turn generates reactive oxygen species (ROS) and actives protein kinase C (PKC). In addition, opioid peptides undergo an additional step of activating an epidermal growth factor receptor (EGFR) before activating PI3-kinase and Akt. Adenosine, another important trigger in IPC, is thought to

4 activate PKC directly through adenosine A1 and A3 receptors.

The ‗mediator‘ phase occurs either during the long ischemia (called the index ischemia) or early during reperfusion and is characterized by adenosine-dependent activation of adenosine

A2b receptors. PKC can also modulate the activity of this adenosine receptor directly. Adenosine

A2b receptors, in turn, lead to ERK and PI3-kinase/Akt activation. These kinases phosphorylate

GSK-3β, which is thought to inhibit formation of mitochondrial permeability transition pores

(mPTP) (see Figure 1). The mPTP has been proposed to be an end effector of IPC but this a subject of intense debate. 4 (See Figure 1 for an overall summary) 4

14

Figure 1. IPC Signalling Mechanisms in the Target Organ. Preconditioning exhibits three distinct phases: the ‗trigger‘ phase (prior to index ischemia), the ‗mediator‘ phase (during index ischemia and reperfusion) and the ―end effector‖ phase.

During the trigger phase, endogenously released substances, such as opioid peptides and bradykinin, activate in the following order: phosphatidylinositol 3-kinase (PI3 kinase), Akt, nitric oxide synthase (NOS), produce nitric oxide (NO), guanylyl cyclase, protein kinase G, opening of mitochondrial K channels, generation of oxygen radicals (ROS), finally activating protein ATP kinase C (PKC). Opioids undergo an additional step of activating an epidermal growth factor receptor (EGFR, or HB-EGF in this figure) before activating PI3 kinase and Akt. Adenosine activates PKC directly through adenosine A1 and A3 receptors.

The ‗mediator‘ phase is characterized by adenosine-dependent activation of adenosine A2b receptors. PKC can also modulate the activity of this adenosine receptor directly. Adenosine A 2b receptors activate in the following order: ERK and PI3-kinase/Akt, phosphorylation of GSK-3β, which inhibits formation of mitochondrial permeability transition pores (mPTP). The mPTP has been proposed to be an end effector of IPC.

This schematic diagram is identical to Figure 1 from the review by Cohen & Downey in 2008.14 5

1.2 Remote Preconditioning (rIPC):

1.2.a Background in rIPC:

In 1993, Przyklenk et al.15 first conceived the idea of remote ischemic preconditioning

(rIPC) wherein repeated brief episodes of ischemia and reperfusion in a non-local tissue/distant organ has the ability to render the myocardium protected against ischemia/reperfusion injury.

Remote preconditioning of the myocardium has can be induced by other organs such as the liver, small intestine, kidneys, and also from skeletal muscle ischemia (see Appendix III & IV).

Similarly, two windows of protection have been confirmed for rIPC16. However, the focus of my thesis will be on the first window of protection (called acute preconditioning/rIPC) which occurs within 2 hours of the preconditioning stimulus and not the second window (called delayed preconditioning/rIPC).

1.2.b Signalling Mechanisms in rIPC:

There is much controversy over the involvement of either a humoral (via blood) or neurogenic (via nerves) pathway, or both, as a mechanism of transferring protection from the preconditioned tissue/organ to the myocardium16. Coronary effluent from preconditioned donor rabbit hearts elicited protection in untreated hearts in the study by Dickson et al.17, suggesting the involvement of humoral protective factors. However, Gho et al.18 demonstrated that protection by mesenteric artery occlusion (MAO) was abolished by ganglion blockade, providing support for a neurogenic pathway in rIPC.

The actual trigger mechanisms involved to induce protection in the myocardium are unclear. Multiple triggers have been suggested such as opioid peptides, adenosine, bradykinin, 6 and calcitonin-gene related peptide (CGRP). Once G-protein coupled receptors are triggered on the cardiomyocyte cell surface, there is translocation of PKCε from the cytosol to the mitochondrial membrane fraction, an event thought to be dependent on mitochondrial ROS generation. However, it is yet to be determined whether activation of survival kinases during reperfusion or inhibition of mPTP occurs. 19 (See Figure 2 for an overall summary) 7

Figure 2. rIPC Signalling Mechanisms of the Myocardium. Either humoral or neurogenic pathways, or both, act as a mechanism of transferring protection from the preconditioned tissue/organ to the myocardium. Once this signal triggers G-protein coupled receptors on the cardiomyocyte cell surface, kinases such as ERK or Akt may be activated, and there is translocation of protein kinase C-epsilon (PKCε) from the cytosol to the mitochondrial membrane fraction, an event thought to be dependent on mitochondrial reactive oxygen species (ROS) generation. Finally, inhibition of mitochondrial permeability transition pore (mPTP) may occur, resulting in preconditioning of the myocardium.

This schematic diagram is identical to Figure 2 from the review by Hausenloy & Yellon in 2008.19 8

1.3 Involvement of Opioid Receptors:

1.3.a Opioids and Their Receptors:

Opioids exhibit various functions in the heart such as cardiac arrhythmogenesis and modulation of vasculature20. Opioid peptides consist of enkephalins, endorphins, dynorphins, and endomorphins as well as other non-opioid substances, such as nociceptin, that exhibit similar pharmacological properties20. Enkephalins are most abundant in cardiac ventricles compared to any other organ in the body aside from the central nervous system21, suggesting the important role of opioids in cardiomyocyte regulation and maintenance during stress. Each opioid class is derived from a corresponding gene which produces a distinct hormone precursor. Enkephalins are produced from pro-enkephalin, endorphins from pro-opiomelanocortin (POMC), dynorphins from pro-dynorphin, and finally the endomorphin precursor has yet to be identified20. In particular, opioids in the myocardium exhibit unique features such as larger molecular weighted peptides (e.g. Met5-enkephalin-Arg6-Phe7 or MERF and dynorphin A)22. Regardless, the myocardium contains all necessary machinery to generate all peptide forms and receptor types. 20

Minimum concentrations reported to protect rabbit cardiomyocytes from ischemia/reperfusion damage by common endogenous opioids such as dynorphin B is 10µM152 and 1µM148 for Met-enkephalin. However, this does not include all known and unknown opioids which may induce protection at lower concentrations.

The above mentioned peptide classes bind with varying affinities to three subtypes of Gi- protein coupled opioid receptors, which are: delta (δ), kappa (κ), and mu (μ). Endorphins and enkephalins are known to associate mostly with δ receptors (and to some extent μ), while 9 dynorphins are highly selective for κ opioid receptors. Endomorphins are most potent at μ receptors. The κ receptor can be divided into κ1 and κ2, and the δ receptor into δ1 and δ2, and all of these have been identified on rat and human cardiomyocytes20. However, there is evidence

23 that the μ subtype (which exists as three isoforms, µ1, µ2, µ3 ) is not expressed in adult cardiomyocytes24,25. The μ receptor is highly abundant in the central nervous system, suggesting this subtype may be present at nerve ends in the myocardium; however, this requires further investigation. Nonetheless, the tissue specificity of these opioids and their receptors as well as their pharmacological functions within the heart is still a subject of study.

1.3.b Evidence of Opioid Involvement in IPC:

During episodes of myocardial ischemic stress, opioid levels were found to be elevated in humans26, suggesting an involvement of opioid peptides in preconditioning. A number of studies have investigated the role of opioids in IPC. Mesenteric artery occlusion (MAO) in rats has shown that morphine (a non-selective opioid agonist) could mimic IPC and this protection was abolished by naloxone (a non-selective opioid antagonist)27. In addition, coronary effluent that was collected following IPC measured increased levels of endogenous opioid peptides.

There is controversy regarding which opioid receptor subtype is involved in ischemic preconditioning. The involvement of δ receptors was proposed by Schultz et al.28 when µ and κ receptor antagonists failed to block protection from 3 cycles of 5 min ischemia-5min reperfusion in in vivo rat hearts. In addition, the detrimental effects of κ receptors to aggravate ischemia- reperfusion injury have been reported in κ-opioid perfused isolated rat hearts29 and found to be proarrhythmic for in vivo κ opioid-induced preconditioning in swine30. Interestingly, κ receptors 10 have also been reportedly involved in the beneficial effects of IPC in rats by Wang et al. 31.

Though δ and κ agonists reduced infarct size in isolated perfused hearts, Wang and colleagues suggested that only the κ opioid agonist mimicked the antiarrhythmic effects of IPC in this study.

Very few studies have investigated the role of opioids during reperfusion. Gross et al.32 in

2004 determined that activation of opioid receptors during the reperfusion phase by morphine

(non-selective opioid agonist) and BW373U86 (selective δ agonist) reduced infarct size in rats.

However, this is an aspect of IPC that requires further exploration but is not the focus of my thesis.

1.3.c Evidence of Opioid Involvement in rIPC:

The involvement of opioids in rIPC was proposed in a study by Dickson et al.33 in which pre-treatment with naloxone (a non-selective opioid antagonist) blocked protection from transferred coronary effluent from one isolated rabbit heart to another. The coronary effluent was analyzed and found to contain Met- and Leu-enkephalin (endogenous agonists of δ and µ opioid receptors)33,34. Patel et al.35 had found that MAO in rats could reduce myocardial infarction, and this effect was blocked by naloxone. Preconditioning by femoral artery occlusion (FAO) in rats also reduced infarct size and lactate dehydrogenase levels (a marker of oxidative stress)36. In a separate study in isolated rat hearts, Weinbrenner et al.37 found that protection was abolished by pre-treatment with naloxone and the free radical scavenger N-2-mercaptopropionyl glycine

(MPG), thus suggesting a relationship between opioid signalling in rIPC and ROS generation.

The same study demonstrated the involvement of the δ1 opioid receptor subtype in protection 11 induced by limb ischemia. However, a recent study by Zhang et al.36 has proposed the idea that it is κ receptors, not δ, that mediate rIPC through femoral artery occlusion in rats.

The exact mechanism of opioid involvement is unclear. Skeletal muscle ischemia with ganglion blockade by hexamethonium did not affect preconditioning in pigs38. Thus, it is thought that opioid peptides enter the blood stream through a humoral pathway and mediate their effects through receptor binding at the target organ20. In addition, similarities in signalling pathways between rIPC and IPC are even less clear. However, there are implications of ROS generation and inhibition of the mPTP with regards to opioids and rIPC37.

1.4 Involvement of Bradykinin B2 Receptors:

1.4.a Bradykinin and Their Receptors:

Bradykinin (BK), a major player of the kinin family, is a potent vasodilatory protein that is released into the blood stream to lower blood pressure following stimuli such as ischemia and tissue damage. BK is produced when high-molecular-weight kininogen is released into the blood stream following proteolytic cleavage by serine proteases called kallikreins39,40. Bradykinin is also degraded in the blood by angiotensin converting enzyme (ACE) (in this role, ACE is called kininase II), carboxypeptidase N, and neutral endopeptidase 1.Kinins generally reduce vascular resistance and increase vascular permeability41 through the release of nitric oxide (NO) and

42 prostacyclin (PGI2) from the endothelium .

43,44 The kinin receptors, B1 and B2 were first cloned in the 1990‘s from humans . The constitutively active rabbit bradykinin B2 receptor (a Gi- and Gq- protein coupled receptor) was 12 cloned in 199545 and is the target of endogenous kinins such as bradykinin (BK) and Lys- bradykinin (aka: kallidin). This receptor is also highly expressed in rabbit kidney and duodenum

45 but mRNA is present in rabbit heart . Bradykinin B2 receptors were reported on rat

46 cardiomyocytes in 1994 . The cloned rabbit B2 receptor is 92% homologous with humans, indicating the homogeneity of this receptor across species. The inducible B1 receptor is thought

47 to activate as a result of tissue injury . Also, bradykinin is highly selective for the B2 receptor

48 over B1 in rabbit smooth muscle cells . The potent antagonist, Hoechst 140 (HOE140) is highly selective for B2 receptors such that it is considered a non-competitive antagonist for this receptor49,50.

There is also some controversy over the existence of other kinin receptor subtypes such

51 as B3, B4, and B5 which are species specific . In addition, these receptor subtypes may be expressed as varying homologs in different species52.

The source of bradykinin in the heart is cardiac endothelium53. BK levels are ten-fold higher in tissues, such as the kidneys, heart, and brain, compared to plasma in rats53 and levels of circulating BK in the blood are low in humans54 at basal conditions. However during ischemia, kinin levels dramatically increase five-fold in plasma55,56.

1.4.b Evidence of Bradykinin Involvement in IPC:

The first studies of bradykinin in IPC illustrated the beneficial effects of exogenous BK to recover cardiac function through increased coronary flow and an improved metabolic profile

57 following ischemia . However, this protection in rats was abolished by the B2 receptor 13 antagonist, HOE140. This finding was recapitulated in dogs58 and rabbits59 when investigating cardiac necrosis following ischemia. In vivo canine studies in which BK was injected into the coronary artery showed a decrease in infarct size following coronary artery occlusion (CAO)60, which was subsequently abolished by HOE140. With respect to rabbits, ischemic preconditioning was blocked by the administration of HOE140 and exogenous bradykinin has been shown to reduce infarct size59. Studies have also revealed increased kinin release following local and global ischemia in rats, dogs, and humans61,62,63.

Ebrahim et al.64 determined that exogenous bradykinin limits infarct size through a concentration-dependent manner. Male rats were preconditioned on a Langendorff apparatus with increasing concentrations of bradykinin. Concentrations >0.1µM were able to induce protection from ischemia-reperfusion injury in rat hearts.

Kinin activation is thought to increase intracellular endothelial cyclic guanine monophosphate (cGMP) through NO and increase cyclic adenosine monophosphate (cAMP)

65 through activation of prostacyclin (PGI2) . Also, B2 receptors from neonatal rats show G-protein coupled activation of PI3 kinase66.

1.4.c Evidence of Bradykinin Involvement in rIPC:

Bradykinin has been implicated in a combined neuronal and humoral pathway in rIPC

67 through the G-protein coupled B2 receptor. Schoemaker et al. abolished rIPC protection with mesenteric artery occlusion (MAO) in early reperfusion through administration of the bradykinin

B2 receptor antagonist, HOE140. The authors suggested that bradykinin exerts its effects in rIPC 14 by activating the neuronal pathway via stimulatory sensory nerves. In a later finding by Wolfrum et al.68, the involvement of PKCε downstream of bradykinin was also implicated.

1.5 Involvement of CGRP Receptors:

1.5.a CGRP and Their Receptors:

Calcitonin gene-related peptide (CGRP) is a vasodilatory released by vagal and capsaicin-sensitive sensory nerves. CGRP is widely recognized as the most potent vasodilator in the cardiovascular system69 through its actions on vascular smooth muscle70. In addition to these roles, CGRP is thought to be involved in neurogenic inflammation, nociception71,72, and vascular hypertrophy73. Recently, this peptide has been implicated in protection from myocardial infarction70,74.

CGRP is a 37 amino acid peptide present in cardiac C-fibres (unmyelinated free nerve fibres)75 and cardiomyocytes76, and is highly abundant in central and peripheral neurons77. Of the two isoforms, α- and β-CGRP, α-CGRP is known to bind to the CGRP receptor. α-CGRP is a neuropeptide derived from the calcitonin gene in neural tissues78. β-CGRP is highly homologous to the α isoform but is derived from another gene located near the calcitonin gene on 1179. Since the cloning of human CGRP in the early 1980‘s, CGRP is found to be highly homologous among mammalian species80.

The binding of CGRP to its receptor depends on the association of the calcitonin-like receptor with two accessory proteins. Calcitonin-like receptor (CLR) is a -like Gs- protein couple receptor first discovered in the 1990s81,82 exhibiting an unknown function83. The 15 association of the CLR to specific receptor activity-modifying proteins (RAMPs) determine which ligands can bind to the receptor84,85. RAMPs are single-pass transmembrane proteins which are 148 amino acids in length. CLR coupled to RAMP1 allows binding of CGRP to the receptor (now a CGRP receptor). CLR coupled to RAMP2 allows binding of another vasodilator, adrenomedullin (a peptide associated with the adrenal medulla tumour, pheochromocytoma).

Finally, CLR coupled with RAMP3 produces a dual CGRP and adrenomedullin receptor. When comparing the different accessory proteins, RAMP2 and RAMP3 are 30% homologous to

RAMP185. The second accessory protein associated with CLR is the receptor component protein

(RCP) which forms a component of the CLR-RAMP1 complex86. Both rat and human CLRs have been cloned and expression of CLR in rat aortic smooth muscle cells has been reported87,88, however, no such investigations have been conducted in rabbits.

1.5.b Evidence of CGRP Involvement in IPC

CGRP, adrenomedullin89, and intermedin90 (another protein that binds to CLR in association to all three RAMPs) are considered cardioprotective 91,92,93. With respect to cardioprotection, coronary effluent from isolated rat hearts displayed elevated CGRP levels during preconditioning94. Several clinical studies have also shown elevated CGRP during early reperfusion following acute myocardial infarction in human patients74 suggesting that CGRP is an endogenous substance produced by the myocardium95. In isolated rat hearts, preconditioning by global ischemia was abolished by CGRP (8-37), a CGRP receptor antagonist91,94. In vivo studies with a CGRP antibody also abolished ischemic preconditioning in rats96 (only the abstract is available, since the article is in Chinese). CGRP has also been implicated in delayed preconditioning and has been extensively studied in gastrointestinal preconditioning97,98,99. 16

Chai et al.92 examined the role of CGRP in isolated rat hearts to induce IPC. The authors measured CGRP release in coronary effluent and determined that CGRP improved left ventricular pressure and coronary flow in a heart subjected to ischemia and reperfusion. A concentration of 1µM improved coronary flow and left ventricular pressure, suggesting that concentrations >1µM will protect isolated rat hearts from ischemia-reperfusion injury.

CGRP downstream of receptors is known to activate protein kinase C, but not ATP-

+ 100,101 sensitive K (KATP) channels, and inhibits tumour necrosis factor –α (TNF-α) . Also, suggestions have been made that CGRP-induced cardioprotection in rats is activated by nitric oxide release102.

1.5.c Evidence of CGRP Involvement in rIPC:

A number of remote intestinal preconditioning studies of the myocardium suggest that

CGRP is released by capsaicin sensory nerves into the blood stream97,98 and activates PKCε in the myocardium103. Wolfrum et al.103 found that administration of the CGRP receptor antagonist,

CGRP (8-37) abolished this protection. The experiments by Wolfrum et al. demonstrated increased CGRP plasma levels following preconditioning, thus suggesting a humoral pathway in rIPC. However, this CGRP protective effect was abolished by ganglion blockade, yet CGRP levels in the plasma remained unaffected. Also, a clinical study by Li et al.104 has shown that cardiac ischemic preconditioning improved lung preservation during value replacement operations through CGRP receptor activation.

17

1.6 Involvement of Adenosine Receptors:

1.6.a Adenosine and Their Receptors:

Adenosine plays several important biological roles, such as energy transfer, an anti- inflammatory agent, and can act as an inhibitory . This purine nucleoside is formed both intracellularly and extracellularly in cardiomyocytes, endothelial cells, and vascular cells by dephosphorylation of AMP by 5‘-nucleotidase or by hydrolysis of S-adenosyl- homocysteine105,106. Degradation of adenosine occurs when extracellular concentrations of adenosine increase, facilitating diffusion of adenosine into the cell. Adenosine is then broken down to AMP by adenosine kinase or into inosine by adenosine deaminase (ADA) which is found in all mammalian tissues107. Though extracellular adenosine is mostly broken down by

ADA via erythrocytes in the blood stream105, ventricular cardiomyocytes do not posses extracellular adenosine deaminase108.

There are four known G-protein coupled adenosine receptors: A1, A3 (both inhibitory; coupled to Gi, thus resulting in a decrease in cAMP), A2a and A2b (both stimulatory, coupled to

Gs which increases cAMP; A2b is also coupled to Gq, which mediates phosphoinositide

109 metabolism) . A1 and A3 receptors are involved in the trigger phase of classical IPC, whereas

14 the A2b subtype has been implicated in the mediator phase . The binding affinity of adenosine to

110 rat A1 receptors is 3-30nM and is >1µM for A3 receptors . A2b is considered a low affinity receptor as adenosine concentrations required to stimulate cAMP levels in the brain are >10µM

111 compared to A2a (requires 0.1-1µM of adenosine) . Only A1, A2b and A3 receptors have been

112 shown to be definitively expressed in cardiomyocytes , while A2a has been found in coronary 18

113 arterioles . With respect to cell signalling, A1 receptors are known to activate ,

109 while A3 receptors activate phospholipase D .

1.6.b Evidence of Adenosine Involvement in IPC:

Studies have shown that in vivo infusion of adenosine deaminase through coronary arteries in swine throughout the preconditioning event and index ischemia abolished IPC

114 115 protection . Also, in vivo rabbit studies by Thornton et al. demonstrate that A1 adenosine agonists, PIA and 2-chloro-N6-cyclopentyladenosine (CCPA), triggered protection before the index ischemia (i.e. longer, damaging ischemia), but not activation via A2a receptors by

CGS21680. This finding was later recapitulated in other species such as dogs116, swine114, rabbits117, and humans118. In vivo studies with rabbits indicated that 8-(p- sulfophenyl)theophylline (8-SPT) and PD115,199, non-selective and A2 adenosine blockers respectively, blocked protection from ischemia by coronary artery occlusion (CAO)119.

6 In addition, rabbit hearts perfused with adenosine and the A1 agonist, N -1-(phenyl-2R- isopropyl) adenosine (abbreviated: PIA), conferred protection on a level similar to IPC 119. The above study determined that saturation of adenosine receptors was required to induce cardioprotection and short pulses or low concentrations of receptor activation was insufficient to confer protection. Armstrong et al.120 states that the minimum adenosine concentration required to protect rabbit cardiomyocyte is 10µM. This is also supported by Peart et al.121 in which 10µM of adenosine reduced cell necrosis but did not limit contractile dysfunction in isolated perfused rat hearts. However, 50µM of adenosine was able to reduced cell death and improve contractile dysfunction. 19

122 6 Lui et al. demonstrated the involvement of A3 receptors by agonism with N -2-(4- aminophenyl)ethyl-adenosine (APNEA) and A3 antagonism with BW-A1433 in isolated rabbit

123 6 hearts. Later in 1997, Auchampach et al. illustrated that activation of A3 receptors by N -(3- iodobenzyl) adenosine-5‘-N-methyluronamide (IB-MECA) mimicked IPC in rabbits as well.

Murry et al.1 discovered that IPC protection could be abolished when 8-SPT, a non- selective adenosine blocker was administered during reperfusion following the index ischemia.

In addition, the A1/A2 receptor agonist 5‘-(N-ethylcarboxamido) adenosine (NECA) given at

124 reperfusion induced protection, but was blocked by the A2b blocker MRS1754 in rabbits .

MRS1754 administered at reperfusion in classical IPC also abolished protection125. Recently in

2007, the novel agonist BAY60-6583, that is highly selective for A2b receptors, induced protection at reperfusion126. However, this protection was abolished by MRS1754. Thus, the above studies demonstrate the role of A2b receptors in the mediator phase (i.e. during reperfusion) of IPC.

1.6.c Evidence of Adenosine Involvement in rIPC:

The mechanism of adenosine activation in rIPC was proposed by Liem et al.127 to act via a non-humoral pathway that differed from classical IPC. In this study, 4 cycles of 15 min CAO followed by 2 cycles of 15 min adenosine-dependent CAO induced preconditioning tolerance in rats. Also, interstitial adenosine initially increased, but rapidly decreased to basal levels.

However, 4 cycles of 15 min CAO followed by 3 cycles of adenosine-independent 3 min mesenteric artery occlusion (MAO) maintained cardioprotection. Even though tolerance of 20 adenosine-dependent preconditioning can occur in IPC (i.e. repeated cycles of ischemia- reperfusion can no longer induce protection), rIPC may employ alternate pathways to maintain this protection. To further support the role of adenosine in rIPC, Pell et al.128 in 1998 demonstrated that protection from renal artery occlusion (RAO) in rabbits was abolished by treatment with 8-(p-sulfophenyl)theophylline (8-SPT). The same study determined that rIPC blockade could also be achieved when 8-SPT was administered during reperfusion, suggesting the role of adenosine in both the trigger and mediator phase.

A study by Pang et al.129 demonstrated that plasma adenosine concentrations increased with skeletal muscle rIPC, and this effect was partially abolished by reserpine, an inhibitor of the vesicular monoamine transporter (VMT, a transporter of catecholamines at synaptic nerve endings). In addition, Addison et al.38 has shown that blockade by 8-SPT and the free radical scavenger, mercaptopropionyl glycine (MPG) completely abolished skeletal muscle rIPC cardioprotection in pigs.

1.7 Other Potential Preconditioning Substances:

A number of G-protein coupled and non-G-protein coupled receptors have been confirmed to play a role in classical IPC and their involvement in rIPC has been suggested.

However, the focus of my thesis will be on G-protein coupled receptors, and in particular, the major receptors that have been implicated: δ and κ opioid receptors, adenosine receptors, and bradykinin B2 receptors; as well as the recently proposed CGRP receptor. Unfortunately, investigating the claims of other receptors (both G-protein and non- coupled) is beyond 21 the scope of this thesis. Nonetheless, this section will bring to light other receptors that may be involved in rIPC.

With respect to other GPCRs, α1 adrenergic receptors which bind both norepinephrine and epinephrine have been implicated in myocardial rIPC; however, the results in this area are conflicting130,131. Losartin (an angiotensin I receptor blocker) was found to block renal artery

132 occlusion (RAO) preconditioning in rat myocardium . Prostaglandin E2 (PGE2) has also been implicated in rIPC gastric preconditioning by Brzozowski et al.133. However, there is yet to be a study on the role of PGE2 in myocardial preconditioning. The endocannabinoid receptor CB2 has also been implicated in a humoral role in rIPC. Intestinal ischemia studies have shown that blockage of the CB2 receptor abolished myocardial protection in mice134 and rats135. Finally, studies have demonstrated that carbachol (a muscarinic M2 receptor agonist) mimicked preconditioning in isolated rabbit whole hearts, suggesting the role of acetylcholine in IPC2. To date, no studies have been conducted in rIPC involving muscarinic M2 receptors.

A number of substances have been implicated to trigger rIPC. These include nitric oxide

(NO) and the following cytokines: tumour necrosis factor-α (TNF-α), nuclear factor- κB (NF-

κB), interleukin-6 (IL-6), and interleukin-1β (IL-1β). Also, free radicals and heat shock proteins such as hemoxygenase-1 (HO-1) have been suggested. However, these substances do not activate

G-protein coupled receptors and are beyond the scope of this thesis. That being said, the findings presented in this section are not conclusive and requires exploration of the mechanisms involved in rIPC. Consequently, many of the above studies are preliminary and require further testing.19

22

1.8 Opioid-Adenosine Receptor Cross-talk:

1.8.a Background:

A number of non-preconditioning studies have implicated opioid-adenosine cross-talk in the central nervous system. Interestingly, adenosine A1 and A2 blockers antagonize the

136 synergistic signalling actions between opioid and dopamine D2 receptors in the rat CNS . In this study, adenosine deaminase and the adenosine blocker, BW A1434U, prevented increased activation of cAMP downstream of colocalized δ opioid and dopamine D2 receptors in transfected NG108-15/D2 cells. Also, Eisenach et al.137 conducted a clinical trial in which patients were administered intrathecal opioid (morphine and fentanyl), which resulted in increased adenosine release in cerebrospinal fluid. This study provided support for an opioid- adenosine role in analgesia in humans.

The novel idea of ‗cross-talk‘ between opioid and adenosine receptors in ischemic preconditioning was first proposed by Peart et al.138 in 2003. Peart et al. found that myocardial protection induced by either morphine (non-selective opioid receptor agonist) or CCPA (A1 adenosine receptor agonist) was blocked, for both morphine and CCPA, by either δ opioid or A1 adenosine specific blockers. This study was conducted in an in vivo coronary artery occlusion

(CAO) model in rats. Later, Peart et al.139 in 2005, they claimed that adenosine kinase inhibition reduced infarct size in the same in vivo CAO model in rats. A1 adenosine, A3 adenosine, and δ opioid receptor antagonists abolished this protection, suggesting that stimulation of these receptors resulted in adenosine kinase inhibition. Currently, there are no studies of opioid- adenosine receptor cross-talk in rIPC.

23

1.8.b Adenosine Levels in the Blood:

Concentrations of extracellular adenosine are 30-300nM in tissues and can increase during ischemia to 10µM when ATP is converted to adenosine111. An increase in adenosine during ischemia suggests that adenosine could enter the blood stream to reach the target organ.

However, the half-life of adenosine in the blood stream is 0.6-1.5 seconds due to constant deamination by adenosine deaminase from erythrocytes, pericytes, and endothelial cells140. Since there are no studies demonstrating an inhibition of nucleoside transportation that prevents adenosine degradation in the blood, it is postulated that adenosine either works entirely through the neurogenic pathway, or partially activates this pathway to induce the release of humoral mediators4. Liem et al.141 provided evidence of a neurogenic pathway in which exogenous adenosine mimicked MAO rIPC, however this protection was abolished by prior ganglion blockade.

1.8.c Potential Mechanisms of Cross-talk:

Two mechanisms have been proposed by Peart et al.139 to mediate ‗cross-talk‘ between opioid and adenosine receptors. One possibility for cross-talk is via adenosine kinase inhibition in cardiomyocytes (see Figure 3). Adenosine kinase continually converts adenosine to AMP, effectively maintaining very low concentrations of adenosine within cardiomyocytes. In turn, adenosine continually diffuses into cardiomyocytes to replenish adenosine levels. However,

Peart et al. postulates that opioid receptor stimulation inactivates adenosine kinase, resulting in a build-up of adenosine within cardiomyocytes. Since the uptake of adenosine by cells is dependent on a concentration gradient, there is an accumulation of extracellular adenosine. The amassing of adenosine around cardiomyocytes activates adenosine receptors on the cell 24 membrane and initiates downstream signalling that ultimately results in ischemic preconditioning. In support of this mechanism, Deussen et al.142 provides evidence that inhibition of adenosines deaminase doubles cardiac adenosine release, whereas inhibition of adenosine kinase causes a 30-fold increase in adenosine. In addition, the role of adenosine kinase is thought to be important in IPC since a low pH due to hypoxia inhibits adenosine kinase, which may contribute to increases in adenosine during ischemia142.

The second mechanism of cross-talk proposed by Peart et al.139 is heterodimerization of opioid and adenosine receptors. This hypothesis is addressed in detail in the General Discussion chapter of this thesis (see section 9.2.a Dimerization of G-protein Coupled Receptors). 25

A

B

Figure 3. The Adenosine Kinase Inhibition Hypothesis.

(A) Adenosine kinase continually converts adenosine to AMP, effectively maintaining very low concentrations of adenosine within cardiomyocytes. In turn, adenosine continually diffuses into cardiomyocytes to replenish adenosine levels.

(B) Opioid receptor stimulation inactivates adenosine kinase, resulting in a build-up of adenosine within cardiomyocytes. Since the uptake of adenosine by cells is dependent on a concentration gradient, there is an accumulation of extracellular adenosine. The amassing of adenosine around cardiomyocytes activates adenosine receptors on the cell membrane and initiates downstream signalling that ultimately results in ischemic preconditioning. (Peart et al., 2005)139

26

- Chapter 2 -

RATIONALE

The mechanisms involved in IPC have been studied extensively; however, the exact triggering molecules involved in rIPC are a subject of controversy. Possible candidates for rIPC include opioid peptides, bradykinin, adenosine, and CGRP. Even less is known about possible cross-talk between the G-protein coupled receptors for these molecules in IPC or rIPC. Thus, a comprehensive study is needed to evaluate the similarities between classical IPC and rIPC in regard to receptor activation and explore the possibility of receptor cross-talk, a novel concept in myocardial preconditioning.

The most appealing aspect of rIPC is its clinical applicability. Unlike classical IPC and the recent phenomenon of post-conditioning (i.e. short periods of re-occlusion during long reperfusion), which is restricted to specific settings such as cardiac surgery, rIPC does not require invasive treatment that can initiate harmful side effects. Cheung et al.143 have already demonstrated the use of rIPC in a clinical setting wherein four cycles of 5 min limb ischemia and reperfusion was able to reduce myocardial injury in children undergoing cardiac surgery. The clinical use of rIPC in acute myocardial infarction may prove to be a major medical advance.

Given the practical value of rIPC alone, the elucidation of mechanisms involved in rIPC is of significant importance. 27

- Chapter 3 -

OBJECTIVES

3.1 Hypothesis

I hypothesize that one or more humoral protective factor(s) released by hind limb rIPC activate two G-protein coupled receptors on the cardiomyocyte cell surface, adenosine and opioid receptors, and involves a cross-talk mechanism between these two receptors to induce protection against cardiomyocyte cell death from ischemia-reperfusion injury.

3.2 Specific Aims

1. To characterize the protection induced by rIPC in freshly isolated rabbit ventricular

cardiomyocytes.

2. To determine the role of G-protein coupled cell membrane receptors in rIPC in

cardiomyocytes.

3. To determine whether rIPC involves cross-talk between adenosine and opioid receptors. 28

- Chapter 4 -

RESEARCH DESIGN & METHODS

4.1 Animals and Human Subjects

Male New Zealand rabbits were used to obtain both preconditioned and control dialysate.

Isolated cardiomyocytes for fresh cell experiments were obtained from either male or female

New Zealand adult rabbits. All rabbits were ~12-14 weeks old, weighed ~3.2-3.5 kg, and had been designated specific pathogen free. All animals were maintained and used in accordance with recommendations from the Department of Laboratory Animal Services at the Hospital for

Sick Children.

Human dialysate was obtained from male human volunteers. Prior to obtaining blood, subjects were instructed not to exercise or consume caffeine and remain calm during the procedure.

4.2 Isolation of Adult Cardiomyocytes

4.2.a Operative Procedure:

Male or female New Zealand White rabbits were treated with a mild topical anaesthetic

(xylocaine) prior to being anesthetised via the ear vein with pentobarbital (30mg/kg) and heparin

(100U/kg) to prevent coagulation. Once rabbits reached a surgical level of anaesthesia, hearts were rapidly excised and hung by the aortic root onto a Langendorff apparatus.

29

4.2.b Digestion Protocol:

Buffers were made in 1L of distilled water from a Millipore filtration system according to specifications in Table 1and Figure 4. This Krebs-Henseleit buffer (contains the following in mM: 0.5 MgSO4, 4.7KCl, 10.0 NaHCO3, 1.2 KH2PO4, 10.0 Dextrose, 20.0 HEPES, and 128.3

NaCl) was filtered and adjusted to pH 3.7. All buffers were oxygenated at 37°C with 95% O2 and

5% CO2. Rabbit hearts were mounted on a pressure-controlled Langendorff apparatus via the

+ aorta and immediately perfused with Ca Buffer (with 0.99mM CaCl2) for 2 min in order to expel blood from the heart. Hearts were then perfused with Ca- Buffer containing 9.99mM ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) for 7 min to chelate calcium and suppress heart contraction. Enzymatic digestion was conducted via recirculation of the Collagenase Buffer (with 200U/ml crude collagenase) until the heart was soft. Samples were taken throughout the digestion process to ensure sufficient perfusion of the heart. Once digestion was complete, the heart was agitated in the Mincing Buffer to separate cardiomyocytes from connective tissue.

The isolate then underwent subsequent filtration and wash steps in Wash Buffer (2% albumin). Dead cells were removed by low centrifugation (at 500g for 2 min, all subsequent spins occur at 500g for 1min) and removal of the supernatant. Cardiomyocytes in Wash Buffer were gradually re-introduced to calcium in a step-wise manner until concentrations reached

0.9mM CaCl2. Finally, calcium tolerant cardiomyocytes were re-suspended in Reactive Buffer

(0.1% albumin, 0.99mM CaCl2) to remove excess calcium from the re-introduction mentioned earlier. Only calcium tolerant cardiomyocyte isolates with <30% cell necrosis were used in fresh cell experiments. 30

Table 1. Krebs-Henseleit Buffer Composition. Reagent Concentration (mM) Osmolarity (mOsm) MgSO4 0.5 1.0 KCl 4.7 9.4 NaHCO3 10.0 20.0 KH2PO4 1.2 4.8 Dextrose 10.0 10.0 HEPES 20.0 20.0 NaCl 128.3 256.6 31

Krebs- Henseleit Buffer

1L

400ml 400ml 100ml

Ca- Buffer Ca+ Buffer Wash Buffer

0.1% albumin 0.99 mM CaCl2 2% albumin 9.99mM EGTA

100ml 150ml

Reactive Buffer

Collagenase Buffer 0.1% albumin

200U/ml collagenase

40ml

Mincing Buffer

Figure 4. Preparation of Buffers Derived from Krebs-Henseleit Buffer. Krebs-Henseleit (KH) buffer was filtered and adjusted to pH 3.7. Rabbit hearts were mounted on + - a Langendorff apparatus and perfused with Ca Buffer (with 0.99mM CaCl2) then with Ca Buffer (9.99mM EGTA, 0.1% albumin). Enzymatic digestion was conducted via recirculation of the Collagenase Buffer (with 200U/ml crude collagenase, 0.1% albumin, 9.99mM EGTA). Once digestion was complete, the heart was agitated in the Mincing Buffer (from the Collagenase Buffer). The isolate then underwent subsequent filtration and wash steps in Wash Buffer (2% albumin) to remove dead cells. Cardiomyocytes in Wash Buffer were gradually re-introduced to calcium in a step-wise manner until concentrations reached 0.9mM CaCl2. Finally, calcium tolerant cardiomyocytes were re-suspended in Reactive Buffer (0.1% albumin, 0.99mM CaCl2). All buffers were oxygenated at 37°C with 95% O2 and 5% CO2. 32

4.2.c Comparison of Digestion Protocols:

A number of modifications to the digestion protocol were studied in an effort to improve isolate yields. Digestions were modified in two protocols, each supplemented with DNase I

(degrades DNA, 0.02mg/ml; Worthington)144, hyaluronidase (degrades hyaluronic acid, chondroitin, and chondroitin sulphates, 0.6mg/ml; Sigma)145 , ovamucoid trypsin inhibitor

(inhibits trypsin, derived from ovamucoid extracts, 0.1mg/ml; Worthington)145, or soybean trypsin inhibitor (derived from soybean extracts, 1mg/ml; Worthington)144 and compared to digestions with only collagenase (200U/ml crude collagenase; Worthington).

In the first protocol, hearts were initially digested with collagenase for 10 min, removed from the Langendorff system, minced, and re-suspended in collagenase buffer supplemented with a reagent for a further 10 min (n=2). This protocol was designed to ensure that cardiomyocytes from the same heart were used in order to avoid heart-to-heart variability. Also, these were preliminary experiments to confirm the supplemented reagents were non-toxic. The second protocol involved re-circulation of collagenase with the supplemented reagent in a

Langendorff until digestion was complete (n=1-2). All isolates underwent similar wash and filtration steps as in a non-supplemented digestion. The results of these modifications indicate that re-circulation with only collagenase is the most optimal method of digestion. (See Figure 5)

33

Figure 5. Comparison of Digestion Protocols. Digestions with collagenase were modified in two protocols that were supplemented with DNase I (degrades DNA, 0.02mg/ml), hyaluronidase (degrades hyaluronic acid, chondroitin, and chondroitin sulphates, 0.6mg/ml), ovamucoid trypsin inhibitor (inhibits trypsin, derived from ovamucoid extracts, 0.1mg/ml), or soybean trypsin inhibitor (derived from soybean extracts, 1mg/ml) and compared to digestions with only collagenase (200U/ml crude collagenase).

In the first protocol (Minced Heart), hearts were initially digested with collagenase for 10 min, removed from the Langendorff system, minced, and re-suspended in collagenase buffer supplemented with a reagent for a further 10 min. The second protocol (Perfusion) involved re- circulation of collagenase with the supplemented reagent in a Langendorff until digestion was complete. All isolates underwent similar wash and filtration steps for a non-supplemented digestion. The results of these modifications indicate that re-circulation with only collagenase is the most optimal method of digestion.

34

4.3 Dialysate Preparation

Male New Zealand White rabbits were anaesthetized with akmezine (0.25mg/kg) (a pre- anaesthetic containing ketamine, acepromazine, and atropine) followed by pentobarbital

(30mg/kg) with heparin (100U/kg) via the ear vein then placed on a ventilator. rIPC rabbits were preconditioned by 4 cycles of 5 min hind limb ischemia and 5 min reperfusion using a blood pressure cuff. Control rabbits were only anaesthetized and ventilated for the same time period as rIPC rabbits. About 100-150ml of blood was quickly drawn from the left carotid artery immediately after the preconditioning or control protocol. Rabbits were monitored throughout the procedure to detect any hemodynamic variability. The whole blood obtained was centrifuged

(3000g for 20 min) to obtain 50 or 100ml of plasma which was then dialysed using a 12-14kDa cut-off membrane against a 10 fold greater volume of water. Thus, the dialysate contents were limited to <14 kDa. Dialysate was divided into 900µl aliquots and immediately stored at -80°C.

Prior to use, the frozen aliquots were thawed only once and reconstituted with 10µl of 10 fold concentrated Krebs-Henseleit buffer. Since dialysate was created against water, a concentrated salt solution was supplemented in order to achieve the same physiological salt levels as in 1x

Krebs-Henseleit buffer. Osmolarity and pH was measured in the dialysate before exposing the solution to cardiomyocytes.

For human subjects, 100 ml of blood was withdrawn before (Control Human Dialysate) and after (rIPC Human Dialysate) 4 cycles of 5 min ischemia (>20mmHg above systolic pressure) and 5 min reperfusion to the upper arm by a blood pressure cuff. Dialysate was obtained using the identical procedure as described above for rabbits. The protocol for preparing 35 both rabbit and human dialysate is shown on Figure 6 and was previously described by Shimizu et al.146. All dialysate used in these studies were prepared by an external laboratory*.

* Dialysate prepared by Jing Li (Dr. Andrew Redington‘s Lab; Research Institute, Hospital for Sick Children). 36

Figure 6. Rabbit and Human Dialysate Preparation. rIPC rabbits were preconditioned by 4 cycles of 5 min hind limb ischemia and 5 min reperfusion using a blood pressure cuff. Control rabbits were only anaesthetized and ventilated for the same time period as rIPC rabbits. About 100-150ml of blood was quickly drawn from the left carotid artery immediately after the preconditioning or control protocol.

For human subjects, 100 ml of blood was withdrawn before (Control Human Dialysate) and after (rIPC Human Dialysate) 4 cycles of 5 min ischemia (>20mmHg above systolic pressure) and 5 min reperfusion to the upper arm by a blood pressure cuff. Dialysate was obtained using the identical procedure as described above for rabbits.

The whole blood obtained was centrifuged (3000g for 20 min) to obtain 50 or 100ml of plasma which was then dialysed using a 12-14kDa cut-off membrane against a 10 fold greater volume of water. Thus, the dialysate contents were limited to <14 kDa. Dialysate was divided into 900µl aliquots and immediately stored at -80°C. Prior to use, the frozen aliquots were thawed only once and reconstituted with 10µl of 10 fold concentrated Krebs-Henseleit buffer. Osmolarity and pH was measured in dialysate before exposing the solution to cardiomyocytes.

(Shimizu et al., 2009)146 37

4.4 Fresh-Cell Experimental Model

4.4.a General Protocol:

Cardiomyocyte isolates were evenly divided into Eppendorff tubes such that each group contained >200µl per group. Cardiomyocytes were re-suspended in 1ml Reactive Buffer and placed in 12-well plates (2ml total volume) at 37°C and 100% O2. Samples from the Baseline group were taken after stabilization and at the end of the experiment to ensure that cardiomyocyte viability remained stable. All cardiomyocytes underwent a 20 min stabilization period in suspension. Cardiomyocytes were subjected to 20 min of a particular treatment, depending on the protocol. This was followed by another 20 min wash period in suspension with fresh Reactive Buffer such that any residual drug(s) in the supernatant was/were removed. All treatment groups subsequently underwent 45 min damaging simulated ischemia (SI) and 60 min simulated reperfusion (SR). SI was conducted by low speed centrifugation (500g for 1 min) of cardiomyocytes into a pellet and leaving only a thin layer of supernatant with an oil layer on top of cells. Cardiomyocytes then underwent SR through suspension in fresh Reactive Buffer.

Samples from the treatment groups were taken before SI and after SR. The purpose of these fresh cell protocols was to observe the effect of a treatment on cell survival following the simulation of normally damaging ischemia and reperfusion.147 (See Figure 7) 38

Figure 7. General Protocol: Isolated Rabbit Cardiomyocytes. All cardiomyocytes underwent a 20 min stabilization period in suspension. Cardiomyocytes were subjected to 20 min of a particular treatment, depending on the protocol. This was followed by another 20 min wash period in suspension with fresh Reactive Buffer such that any residual drug(s) in the supernatant was/were removed. All treatment groups subsequently underwent 45 min damaging simulated ischemia (SI) and 60 min simulated reperfusion (SR). SI was conducted by low speed centrifugation (500g for 1 min) of cardiomyocytes into a pellet, leaving only a thin layer of supernatant with an oil layer on top of cells. Cardiomyocytes then underwent SR through suspension in Reactive Buffer. Samples from the treatment groups were taken before SI and after SR (see arrows).

39

4.4.b AIM (1) Protocol:

AIM (1): To characterize the protection induced by rIPC in freshly isolated rabbit ventricular cardiomyocytes.

Cardiomyocytes freshly isolated from non-preconditioned donor rabbit hearts were preconditioned by exposing them to10 min ischemic pelleting (IPC treatment group) or rIPC dialysate, human or rabbit derived (rIPC Dialysate group). Cardiomyocyte groups that were not preconditioned were subjected to 10 min re-suspension in buffer (IR group) or control human or rabbit dialysate (Control Dialysate group). Treatment groups underwent a wash period in fresh buffer for 20 min. All groups were then subjecting to a long period of 45 min simulated ischemia

(SI) and 60 min simulated reperfusion (SR). Samples were taken before and after SI/SR. (See

Figure 8)

Figure 8. AIM (1) Protocol: To Characterize Dialysate Protection. Cardiomyocytes freshly isolated from non-preconditioned donor rabbit hearts were preconditioned by exposing them to10 min ischemic pelleting (IPC treatment group) or rIPC dialysate, human or rabbit derived (rIPC Dialysate group). Cardiomyocyte groups that were not preconditioned were subjected to 10 min re-suspension in buffer (IR group) or control human or rabbit dialysate (Control Dialysate group). Treatment groups underwent a wash period in fresh buffer for 20 min. All groups were then subjecting to a long period of 45 min simulated ischemia (SI) and 60 min simulated reperfusion (SR). Samples were taken before and after SI/SR. 40

4.4.c AIM (2) Protocol:

AIM (2): To determine the role of G-protein coupled cell membrane receptors in rIPC in cardiomyocytes.

Freshly isolated cardiomyocytes were either subjected to 10 min IR or rIPC (using dialysate) in the presence of an antagonist (or its vehicle) for 20 min to prevent specific activation of each receptor being studied (adenosine, bradykinin B2,  and κ opioid receptors, and CGRP receptors). Treatment groups underwent a wash period in fresh buffer for 20 min. All groups were then subjecting to a long period of 45 min simulated ischemia (SI) and 60 min simulated reperfusion (SR). Samples were taken before and after SI/SR. (See Figure 9)

Figure 9. AIM (2) Protocol: The Role of Cell Membrane Receptors. Freshly isolated cardiomyocytes were either subjected to 10 min IR or rIPC (using dialysate) in the presence of an antagonist (or its vehicle) for 20 min to prevent specific activation of each receptor being studied (adenosine, bradykinin B2,  and κ opioid receptors, and CGRP receptors). Treatment groups underwent a wash period in fresh buffer for 20 min. All groups were then subjecting to a long period of 45 min simulated ischemia (SI) and 60 min simulated reperfusion (SR). Samples were taken before and after SI/SR. 41

4.4.d AIM (3) Protocol:

AIM (3): To determine whether rIPC involves cross-talk between adenosine and opioid receptors.

Cardiomyocytes were subjected to 10 min classic IPC, specific opioid receptor agonists

(Ag), such as Met-enkephalin (δ-specific agonist) or dynorphin B (κ-specific agonist) in the presence of adenosine receptor blockers, such as 8-SPT (non-selective adenosine blocker) for 20 min. Treatment groups underwent a wash period in fresh buffer for 20 min. All groups were then subjecting to a long period of 45 min simulated ischemia (SI) and 60 min simulated reperfusion

(SR). Samples were taken before and after SI/SR. (See Figure 10)

Figure 10. AIM (3) Protocol: Investigate Receptor Cross-talk. Cardiomyocytes were subjected to 10 min classic IPC, specific opioid receptor agonists, such as Met-enkephalin (δ-specific agonist) or dynorphin B (κ-specific agonist) in the presence of adenosine receptor blockers, such as 8-SPT (non-selective adenosine blocker) for 20 min. Treatment groups underwent a wash period in fresh buffer for 20 min. All groups were then subjecting to a long period of 45 min simulated ischemia (SI) and 60 min simulated reperfusion (SR). Samples were taken before and after SI/SR.

42

4.5 Trypan Blue Exclusion Assay

Cardiomyocyte samples were taken before and after simulated ischemia (SI) and reperfusion (SR) to assess cell death by a trypan blue exclusion assay. 10µL of cardiomyocytes were suspended in 13µl of 85mOsm hypotonic Tyrode Buffer (contains the following in mM:

10.7 KCl, 1.5 NaH2PO4, 14.7 NaHCO3, 5.6 glucose, 1.6 MgSO4, 7.2 CaCl2, 3.0 amylobarbitone,

0.5% glutaraldehyde, 0.5% trypan blue; see Table 2 for buffer composition) and 13µl of Reactive

Buffer for 1 min in order to allow dead cardiomyocytes to take up the trypan dye. Samples were place on a hemocytometer and images were taken with a light microscope at 20x magnification.

Cardiomyocytes unable to exclude the trypan blue dye due to the loss of membrane integrity were counted as dead cells, whereas live cells that maintained membrane integrity were able to exclude the dye and were seen as clear (see Figure 11). The percent cell death was counted from

>300 cells per sample. Images were taken by the Micro-Cap software (Spectrum) and ImageJ software (NIH) was used to count sample images.

Table 2. Tyrode Buffer Composition. Reagent Concentration (mM) Osmolarity (mOsm) KCl 10.7 21.4 NaH2PO4 1.5 3.0 NaHCO3 14.7 29.4 Glucose 5.6 5.6 MgSO4 1.6 3.2 CaCl2 7.2 21.6 Total Osmolarity ~85 mOsm Amylobarbitone 3.0 Glutaraldehyde 0.5% Trypan Blue 0.5%

43

Figure 11. Trypan Blue Exclusion Assay. Cardiomyocyte samples were taken before and after ischemia (SI) and reperfusion (SR) to assess cell death by a trypan blue exclusion assay. Cardiomyocytes unable to exclude the trypan blue dye due to the loss of membrane integrity were counted as dead cells (Nonviable Cells), whereas live cells (Viable Cells) that maintained membrane integrity were able to exclude the dye and were seen as clear

44

4.5.a Inter-Observer Error Data:

Inter-observer measurements were collected to ensure consistency in cell counting from various observers and experiments. Three experiments (Sept. 17, 2007; Sept 19, 2007; Sept 25,

2007) were conducted using the rabbit dialysate protocol described in Figure 8. Treatment groups included a Baseline, ischemia-reperfusion (IR), ischemic preconditioning (IPC), control rabbit dialysate (Control Dialysate), and preconditioned rabbit dialysate (rIPC Dialysate).

Sample pictures were taken before and after simulated ischemia and simulated reperfusion.

Cardiomyocytes were exposed to hypotonic trypan blue and images were counted. Four different observers (Harinee Surendra, Elena Kuzmin, Sue Omar, and Mohammad Escandarian) counted cell necrosis from the same images in all three experiments (a total of 45 images). The mean was calculated for the following parameters: live, dead, and total cardiomyocytes counted and the percent necrosis for reach observer. Statistical analysis was conducted via correlation coefficients and correlation p-values. The results indicated that all counts correlated among observers for a given parameter (coefficients approached 1) and the probability of incorrectly concluding this correlation was very small among the observers (p<0.0001). Thus, the method of counting cell necrosis through trypan blue staining showed little variation among observers. (See

Table 3 for results) 45

Table 3. Inter-Observer Error Data.

Elena Harinee Sue Mohammad Live 175.3 228.3 253.3 160.1 Mean of Dead 83.58 88.53 87.84 59.53 Counts Total 258.9 316.8 341.1 219.6 % Necrosis 32.51 28.84 26.60 27.37

Elena Harinee Sue Mohammad Live 0.82 0.71 0.82 Dead 0.97 0.93 0.91 Elena Total 0.86 0.74 0.87 % Necrosis 0.93 0.91 0.88 Correlation Coefficient Live <0.0001 0.90 0.90 Dead <0.0001 0.93 0.87 Harinee Total <0.0001 0.90 0.88 % Necrosis <0.0001 0.94 0.91 Live <0.0001 <0.0001 0.85 Dead <0.0001 <0.0001 0.93 Sue Total <0.0001 <0.0001 0.85 % Necrosis <0.0001 <0.0001 0.92 Live <0.0001 <0.0001 <0.0001 Dead <0.0001 <0.0001 <0.0001 Mohammad Total <0.0001 <0.0001 <0.0001 % Necrosis <0.0001 <0.0001 <0.0001

Correlation p-value

46

4.6 Drugs

All drugs used as agonists or antagonists for these studies competitively bound to cell- membrane receptors and the concentrations used have previously been published in peer- reviewed preconditioning literature to pharmacologically precondition (agonist) or inhibit the protection of classical or remote IPC (antagonists).

Antagonists used for opioid receptors were naloxone (Nal), naltrindole (NTI), and GNTI.

Naloxone (chemical name: (5a)- 4,5-Epoxy-3,14-dihydro-17-(2-propenyl)morphinan-6-one hydrochloride; from Sigma) is a highly potent, non-selective, competitive inhibitor of opioid receptors used at 100µM148,149,150,151. Naltrindole (chemical name: 17-(cyclopropylmethyl)-6,7- dehydro-4,5a-epoxy-3,14-dihyd roxy-6,7-2',3'-indolomorphinan hydrochloride; Sigma) is a δ opioid blocker used at 10nM152 with a 223- and 346-fold greater selectivity for δ over µ and κ opioid receptors, respectively153,154,155. GNTI (chemical name: 5'-Guanidinyl-17-

(cyclopropylmethyl)-6,7-dehydro-4,5a-ep oxy-3,14-dihydroxy-6,7-2',3'-indolomorphinan dihydrochloride; Tocris Biosciences) is a κ-opioid blocker used at 1nM152 with a 208- and 799- fold selectively for κ receptors over µ and δ opioid receptors, respectively156,157,158.

Agonists used for opioid receptors were Met-enkephalin (ME) and dynorphin B (DynB).

Met-enkephalin is an endogenous ligand of δ opioid receptors used at 100µM152 that is highly selective for δ receptors, and to a lesser extent µ, over κ receptors20. The minimum concentration of Met-enkephalin required to protect rabbit cardiomyocytes from ischemia/reperfusion damage is 1µM148. Dynorphin B (aka: rimorphin; Phoenix Pharmaceuticals) is an endogenous peptide that is highly selective for κ opioid receptors over µ and δ receptors20. DynB was used at 100µM 47 and the minimum concentration reported to protect rabbit cardiomyocytes from ischemia/reperfusion damage is 10µM152.

This study used Hoechst 140 (HOE140) as a blocker of bradykinin B2 receptors and calcitonin gene-related peptide 8-37 (CGRP (8-37)) as a blocker for CGRP receptors. Hoechst

140 (aka: icatibant; Sigma) is a highly selective inhibitor of bradykinin B2 receptors and is

49,49 ,159 160 negligibly active against B1 receptors when used at 5µM . CGRP (8-37) (calcitonin gene-related peptide, fragment 8-37; Sigma) is a fragment of the endogenous α-CGRP human peptide and inhibits CGRP receptors and not calcitonin receptors at 5nM103,161,162.

Finally, the antagonist used for adenosine receptors was 8-SPT. 8-SPT (chemical name:

8-(p-sulfophenyl)theophylline; Sigma) is non-selective blocker of adenosine receptors used at

100µM120,163.

4.7 Other Methods

4.7.a Western Blotting:

Western blots were performed to determine the presence of cell-membrane receptors on rabbit cardiomyocytes. For µ opioid receptors, a synthetic rabbit monoclonal antibody corresponding to amino acid residues 220–250 of the human µ opioid receptor sequence was used (Santa Cruz Biotechnology). This human epitope is 100% homologous with both the cloned rat and mouse µ-opioid receptor sequence over residues 220-250. The δ opioid receptor was detected with a rabbit polyclonal synthetic antibody which corresponded to amino acids 3-17 of both the mouse and rat δ opioid receptor (Abcam). The antibody used for κ opioid receptors was 48 a goat polyclonal antibody corresponding to amino acids 330 – 380 of the human κ-opioid receptor (Santa Cruz Biotechnology). This fragment is 88% homologous to the rat and mouse κ- opioid receptor sequence over these residues.

The calcitonin-like receptor in rabbits was detected using a mouse polyclonal antibody of human origin, corresponding to amino acids 23-133 (Abnova). This immunogen has a 92% homology to the mouse calcitonin-like receptor over these residues.

4.7.b Multiple Reaction Monitoring Mass Spectrometry:

Multiple reaction monitoring (MRM) mass spectrometry was used to identify molecules in the rabbit dialysate. MRM mass spectrometry is a form of tandem mass spectrometry which ionizes chemical compounds into multiple fragment ions. Once the particular fragments are detected by a magnetic and/or electric field, the mass-to-charge ratio is calculated and data analysis allows identification of the unknown compound. The purpose of utilizing MRM mass spectrometry, compared to other methods such as chromatography, is the high degree of sensitivity (can detect substances at concentrations as low as 1-3nM) and selectivity (is able to distinguish adenosine from other purines, such as inosine which differs by 1Da). In order to measure accurate concentrations, all substances were compared to known standard concentrations prepared in water (see Figure 28 in Appendix V for a plot of the standard concentrations).

In order to investigate the role of adenosine kinase inhibition in cross-talk, MRM mass spectrometry was also used to measure levels of adenosine in supernatant samples. Supernatant 49 was obtained from freshly isolated rabbit cardiomyocytes after 20 min stabilization, after 10 min of exposure to dynorphin B (κ-opioid receptor agonist) in fresh Reactive Buffer, and after a 20 min wash period in which cardiomyocytes were suspended in fresh Reactive Buffer (see Figure

12). Measured adenosine concentrations were compared to known standard concentrations prepared in Krebs-Henseleit buffer (see Figure 29 in Appendix V for a plot of the standard concentrations).

All mass spectrometry measurements were conducted on an API 4000 Triple Quadrupole with Agilent LC by an external laboratory†.

Figure 12. Adenosine Kinase Inhibition Protocol. Supernatant was obtained from freshly isolated rabbit cardiomyocytes after 20 min stabilization, after 10 min of exposure to dynorphin B (κ-opioid receptor agonist) in fresh Reactive Buffer, and after a 20 min wash period in which cardiomyocytes were suspended in fresh Reactive Buffer. MRM mass spectrometry was used to measure levels of adenosine in supernatant samples in order to investigate the role of adenosine kinase inhibition in cross-talk.

† MRM mass spectrometry was conducted by Michelle Young (AIMS Lab; Department of Chemistry, University of Toronto) and Michael Tropak (Dr. Callahan‘s Lab, Hospital for Sick Children). 50

4.7.c Statistics:

Averages and standard error of the mean were reported for all results. For all experiment sets, the N (number of different rabbit dialysates used) and n (number of times experiments were replicated for a particular dialysate) were stated. All treatment groups were statistically assessed by an Analysis of Variance (ANOVA) Scheffé post hoc test for multiple comparisons. For groups that were not distributed normally, an ANOVA Kruskal-Wallis post hoc test was conducted. Statistics was conducted with Statview (Abacus Corporation). 51

- Chapter 5 -

CHARACTERIZATION OF THE PROTECTION INDUCED BY RIPC

5.1 Rabbit and Human Preconditioned Dialysate Induces Protection

Cardiomyocytes were subjected to 10 min. exposure to either preconditioned rIPC dialysate or non-preconditioned control dialysate. Results indicate that remotely preconditioned dialysate, whether rabbit (31.9% ±3.7 vs. 48.0%±2.7 IR, p=0.0002; N=6, n=1 each) or human derived (32.4%±2.9 vs. 48.0%±2.7 IR, p=0.0001; N=1, n=6), reduces SI/SR induced cell death similar to classical IPC (30.5%±3.1 vs. 48.0%±2.7 IR, p=0.0006; n=6). (See Figure 13 and Table

4).

52

Figure 13. Rabbit and Human Dialysate Administered Prior to Ischemia-Reperfusion. Cardiomyocytes were subjected to 10 min. exposure to either preconditioned rIPC dialysate or non-preconditioned control dialysate. Results indicate that remotely preconditioned dialysate, whether rabbit (31.9% ±3.7 vs. 48.0%±2.7 IR, p=0.0002; N=6, n=1 each) or human derived (32.4%±2.9 vs. 48.0%±2.7 IR, p=0.0001; N=1, n=6), reduces SI/SR induced cell death similar to classical IPC (30.5%±3.1 vs. 48.0%±2.7 IR, p=0.0006; n=6).

Table 4. Rabbit and Human Dialysate Results (Mean ± SEM). Stabilization End Baseline 26.8 ± 1.0 32.3 ± 2.3 Before SI After SR IR 33.1 ± 2.9 48.0 ± 2.7 IPC 30.1 ± 1.6 30.5 ± 3.1 Control Rabbit Dialysate 29.5 ± 2.0 49.1 ± 4.8 rIPC Rabbit Dialysate 29.5 ± 1.4 31.9 ± 3.7 Control Human Dialysate 31.0 ± 1.7 47.4 ± 1.9 rIPC Human Dialysate 28.9 ± 1.5 32.4 ± 2.9 53

5.1.a Dialysate Characterization:

Substances in rabbit control and rIPC dialysate were identified using MRM mass spectrometry. Opioids such as Met-enkephalin and dynorphin B had no detectable peaks and

Met5-enkephalin-Arg6-Phe7 (MEAP) had a concentration of <0.033 µM in control dialysate and

<0.031 µM in rIPC dialysate. Similarly, acetylcholine and angiotensin II were undetectable in the dialysate. Adenosine in control dialysate was <0.035µM and <0.80 µM in rIPC dialysate.

Inosine was detected at maximum concentrations of 0.583µM in control dialysate and 0.456µM in rIPC. Bradykinin was <0.0034 µM in control dialysate and was at levels below measurement in rIPC dialysate. Norepinephrine was <0.021 µM in control dialysate and <0.019 µM in rIPC dialysate. The above substances were at either undetectable or at minuscule levels that remained unaffected in either the control or rIPC dialysate (with the exception of adenosine). (See Table 5 for results; see Figure 28 in Appendix V for a plot of the standard concentrations).

Table 5. Characterization of Rabbit Dialysate Using MRM Mass Spectrometry. Control Rabbit Dialysate (µM) rIPC Rabbit Dialysate (µM) Met-Enkephalin No peak No peak Dynorphin B No peak No peak MEAP No peak – 0.033 No peak – 0.031 Adenosine 0.001-0.035 0.003-0.80 Inosine 0.103-0.583 0.056-0.456 Bradykinin No peak-0.0034 <0 Norepinephrine 0.0087-0.021 0.0080-0.019 Acetylcholine No peak No peak Angiotensin II No peak No peak

5.1.b Discussion:

Remotely preconditioned (rIPC) rabbit dialysate conferred protection to rabbit cardiomyocytes on a level similar to IPC. Non-preconditioned (Control) dialysate and cardiomyocytes subjected to ischemia-reperfusion alone did not provide protection (see Figure 54

13). Thus, there is a protective factor in rIPC dialysate that travels to distant organs through a humoral route.

Literature on rIPC suggests two mechanisms of transferring protection from one organ to another. The first is a neuronal pathway, in which substances are released from the stimulus organ and activate local nerves to release protective factors at the target organ. As evidence for this pathway, Gho et al.18 demonstrated that ganglion blockade could abolish protection from mesenteric artery occlusion (MAO). Another method of transferring protection is the humoral pathway and is the focus of this thesis. Coronary effluent from preconditioned donor hearts provided protection to untreated virgin hearts in a study by Dickson et al17. For many substances implicated in rIPC, there is evidence for a dual neurogenic and humoral pathway which may work simultaneously in concert or independent of each other16. However, our model does not involve the role of the neurogenic pathway, since there is no neuronal involvement in isolated rabbit cardiomyocytes, and the dialysate used to elicit protection is derived from blood plasma.

Therefore, the protection from rabbit rIPC dialysate indicates that a humoral mechanism alone can confer protection to distant organs.

Human dialysate can also protect rabbit cardiomyocytes, indicating cross-species reactivity of the preconditioned dialysate. Though preconditioning has been shown in many species, very few papers have investigated if protection from one species can elicit protection in another, i.e. inter-species rIPC, except Shimizu et al. 146 (our data regarding human and rabbit dialysate to induced protection was presented in this paper) . Since rIPC mechanisms in rabbits may also apply to humans, cross-species reactivity of preconditioned dialysate is clinically 55 relevant for humans. However, human dialysate from different volunteers are needed to determine variability in cardioprotection, since control conditions are difficult to enforce in humans compared to rabbits.

MRM-MS analysis has shown various G-protein coupled receptor ligands are either undetectable (Met-enkephalin, dynorphin B, acetylcholine, and angiotensin II) or well below levels that protect in tissues (MEAP, adenosine, bradykinin, and norepinephrine) (see Table 5).

However, this list is a sample survey of molecules that have been suggested in rIPC. For example, there are a diverse number of opioids and opioid-like molecules that bind to opioid receptors. To further complicate the matter, Pugsley20 points out that many opioid peptides have yet to be discovered. Similarly, a number of molecules can bind to a single GPCR.

Unfortunately, an exhaustive survey of substances that bind to G-protein coupled receptors is beyond the scope of this thesis. However, we did investigate the most common agonist for a particular receptor through MRM-MS. Considering the above mentioned limitations in characterizing the dialysate, a better approach would be to investigate the particular receptors involved in rIPC by blocking their activation.

56

- Chapter 6 -

THE ROLE OF CELL MEMBRANE RECEPTORS IN RIPC

6.1 The Role of Opioid Receptors

6.1.a Survey of Opioid Receptors in Rabbit Cardiomyocytes:

In order to determine the existence of the δ, κ, and µ opioid receptors in cardiomyocytes, western blotting (n=2) revealed the presence of all three receptors in rabbit whole heart tissues.

The δ and κ subtype were found in rabbit cardiomyocytes, δ was also located in rabbit and rat brain. The µ subtype was in rabbit brain tissue but absent in rabbit cardiomyocytes, suggesting the µ subtype is present in neuronal tissue within rabbit whole hearts. (See Figure 14).

57

Survey of Opioid Receptors in Rabbit Cardiomyocytes

Figure 14. Western Blot Analysis of Opioid Receptors in Rabbit Tissues. In order to determine the existence of the δ, κ, and µ opioid receptors in cardiomyocytes, western blotting (n=2) revealed the presence of all three receptors in rabbit whole heart tissues. The δ and κ subtype were found in rabbit cardiomyocytes, δ was also located in rabbit and rat brain. The µ subtype was in rabbit brain tissue but absent in rabbit cardiomyocytes, suggesting the µ subtype is present in neuronal tissue within rabbit whole hearts. 58

6.1.b Non-selective Opioid Receptor Blockade of Protection:

The effect of naloxone (a non-selective opioid blocker; 100µM concentration was used;

Takasaki, 1999148) in conjunction with rabbit dialysate was studied. Blockers of opioid receptor activity abolished the protection induced by the preconditioned dialysate (43.5%±1.5 vs. 31.3%

± 2.0, p=0.001; N=2, n=4,1). (See Figure 15 and Table 6).

59

Figure 15. Naloxone Administered Prior to Rabbit Dialysate. The effect of naloxone (a non-selective opioid blocker; 100µM concentration was used) in conjunction with rabbit dialysate was studied. Blockers of opioid receptor activity abolished the protection induced by the preconditioned dialysate (43.5%±1.5 vs. 31.3% ± 2.0, p=0.001; N=2, n=4,1).

Table 6. Naloxone Results (Mean ± SEM). Group Stabilization End Baseline 27.4 ± 1.4 28.2 ± 1.5 Baseline + Nal 26.8 ± 0.9 31.0 ± 1.7 Before SI After SR IR 27.9 ± 2.3 43.0 ± 1.1 IR + Nal 26.7 ± 2.8 45.0 ± 1.6 rIPC Rabbit Dialysate 28.1 ± 1.9 31.3 ± 2.0 rIPC Rabbit Dialysate + Nal 28.7 ± 1.6 43.5 ± 1.5 60

6.1.c δ-Opioid Receptor Blockade of Protection:

The effect of naltrindole (NTI; a δ opioid blocker with a 350 fold selectivity for δ receptors over κ receptors; 10nM concentration was used on cardiomyocytes; Cao, 2003152) in conjunction with rabbit dialysate was studied. Blockers of δ opioid receptor activity completely abolished the protection induced by the preconditioned dialysate (43.2%±2.0 vs. 29.6%±1.6, p=0.0003; N=2, n=3,2). (See Figure 16 and Table 7).

61

Figure 16. Naltrindole Administered Prior to Rabbit Dialysate. The effect of naltrindole (NTI; a δ opioid blocker with a 350 fold selectivity for δ receptors over κ receptors; 10nM concentration was used on cardiomyocytes) in conjunction with rabbit dialysate was studied. Blockers of δ opioid receptor activity completely abolished the protection induced by the preconditioned dialysate (43.2%±2.0 vs. 29.6%±1.6, p=0.0003; N=2, n=3,2).

Table 7. Naltrindole Results (Mean ± SEM). Group Stabilization End Baseline 27.4 ± 1.4 29.2 ± 2.4 Baseline + NTI 28.1 ± 1.6 29.3 ± 1.9 Before SI After SR IR 28.0 ± 1.5 42.4 ± 0.8 IR + NTI 27.6 ± 1.2 45.2 ± 2.1 rIPC Rabbit Dialysate 28.5 ± 1.4 29.6 ± 1.6 rIPC Rabbit Dialysate + NTI 28.8 ± 1.5 43.2 ± 2.0 62

6.1.d κ-Opioid Receptor Blockade of Protection:

The effect of GNTI (a κ selective opioid blocker with an 800 fold selectivity for κ receptors over δ receptors; 1nM concentration was used; Cao, 2003152) in conjunction with rabbit dialysate was studied. Blockers of κ-opioid receptor activity completely abolished the protection induced by the preconditioned dialysate (46.8%±2.7 vs. 29.6%±1.6, p=0. 0002; N=2, n=3, 2).

(See Figure 17 and Table 8).

63

Figure 17. GNTI Administered Prior to Rabbit Dialysate. The effect of GNTI (a κ selective opioid blocker with an 800 fold selectivity for κ receptors over δ receptors; 1nM concentration was used) in conjunction with rabbit dialysate was studied. Blockers of κ-opioid receptor activity completely abolished the protection induced by the preconditioned dialysate (46.8%±2.7 vs. 29.6%±1.6, p=0.0002; N=2, n=3, 2).

Table 8. GNTI Results (Mean ± SEM). Group Stabilization End Baseline 27.4 ± 1.4 29.2 ± 2.4 Baseline + GNTI 30.9 ± 0.4 32.0 ± 1.3 Before SI After SR IR 28.0 ± 1.5 42.4 ± 0.8 IR + GNTI 26.5 ± 0.9 45.0 ± 2.3 rIPC Rabbit Dialysate 28.0 ± 1.5 29.6 ± 1.6 rIPC Rabbit Dialysate + GNTI 29.3 ± 1.3 46.8 ± 2.7 64

6.1.e Discussion:

Literature regarding µ opioid receptors confirms that this receptor subtype is not present in rabbit cardiomyocytes24,25. Also, there is indirect evidence that denies the involvement of µ receptors in preconditioning. Fentanyl (a selective µ agonist) administered during preconditioning was unable to abolish protection in Langendorff perfused rabbit hearts164.

There is little surprise regarding the involvement of opioids in rIPC. Multiple studies have suggested that naloxone (a non-selective opioid antagonist) can abolish protection across species and in different organs. Dickson et al. 33 used naloxone to blocked protection from preconditioned coronary effluent in untreated rabbit hearts. Patel et al.35 determined that MAO- induced cardioprotection was blocked by naloxone in rats. Weinbrenner et al.37 attests to a similar findings when failing to precondition isolated rat hearts in the presence of naloxone.

However, controversy lies in which receptor subtype is involved in cardioprotection. In our isolated rabbit cardiomyocyte model, both δ and κ opioid receptors are involved in a humoral mechanism of rIPC (see Figure 16 and Figure 17). Interestingly, Cao et al.152 has also shown that both Met-enkephalin (δ opioid agonist) and dynorphin B (κ opioid agonist) administered before the index ischemia can protect isolated rabbit cardiomyocytes. Naltrindole (δ receptor blocker) and GNTI (κ receptor blocker) administered during classical IPC abolished this protection. Also,

37 Weinbrenner et al. has observed the involvement of δ1 in isolated rat hearts. More recently,

Zhang et al.36 has reported the involvement of only κ receptors in rIPC when δ receptor blockers failed to abolish protection from femoral artery occlusion (FAO). However, the authors 65 attributed the controversy of their findings to variations with the experimental models and species used when compared to previous studies.

Another reason for the discrepancies regarding opioid receptor subtype involvement is promiscuity of opioid peptides and heterodimerization of receptors. Opioids have the ability to bind to multiple receptor subtypes20. Also, many endogenous peptides that are subtype-specific can operate as non-selective agonists at higher concentrations. The δ-specific Met-enkephalin and the µ-specific morphine are such examples. The versatility of endogenous opioid peptides to bind to multiple receptors can be explained by the high degree of homology among receptor subtypes. The µ subtype is 58% and 67% homologous to δ and κ receptively, whereas δ and κ share 61% homology165. Another explanation for the involvement of both δ and κ opioid receptors is the evidence of dimerization across and within different subtypes. There is evidence of hybrid δ and µ receptors that have unique structures, pharmacology, and functions upon activation by either δ or µ-specific agonists166.

The results so far suggest the involvement of δ and κ opioid receptors in mediating a humoral mechanism of rIPC protection. However, dialysate characterization using MRM-MS was unable to detect Met-enkephalin, dynorphin B, or significant levels of the more common endogenous opioid peptide in coronary effluent, MEAP. Therefore, at least one of the trigger molecule(s) in dialysate is/are opioid-like in nature. However, since the factor(s) is/are not these common endogenous peptides, this provides room for discovering a novel/unfamiliar opioid involved in both δ and κ receptor activation in rIPC.

66

6.2 The Role of Bradykinin B2 Receptors

6.2.a Bradykinin B2 Receptor Blockage Conserves Protection:

The effect of Hoechst (HOE140) (a bradykinin B2 receptor blocker; 5µM concentration was used; Schoemaker, 200067) in conjunction with rabbit dialysate was studied. Blockers of bradykinin B2 receptor activity conserved the protection induced by preconditioned rIPC dialysate (31.2%±3.0 vs. 26.9%±2.2, p=0.60; N=2, n=4, 1). (See Figure 18 and Table 9).

67

Figure 18. HOE140 Administered Prior to Rabbit Dialysate. The effect of Hoechst (HOE140) (a bradykinin B2 receptor blocker; 5µM concentration was used) in conjunction with rabbit dialysate was studied. Blockers of bradykinin B2 receptor activity conserved the protection induced by preconditioned rIPC dialysate (31.2%±3.0 vs. 26.9%±2.2, p=0.60; N=2, n=4, 1).

Table 9. HOE140 Results (Mean ± SEM). Group Stabilization End Baseline 28.9 ± 3.1 26.5 ± 1.7 Baseline + HOE140 26.9 ± 2.0 28.3 ± 2.1 Before SI After SR Control Rabbit Dialysate 24.5 ± 1.3 41.3 ± 1.1 Control Rabbit Dialysate + HOE140 26.1 ± 2.2 44.6 ± 1.9 rIPC Rabbit Dialysate 25.1 ± 1.5 26.9 ± 2.2 rIPC Rabbit Dialysate + HOE140 26.2 ± 1.6 31.2 ± 3.0 68

6.2.b Discussion:

Blockage with HOE140 did not abolish rIPC dialysate protection, suggesting that bradykinin B2 receptors are not involved (see Figure 18). To reiterate this finding, levels of bradykinin were detected at minuscule levels using MRM-MS (see Table 5).

According to Schoemaker et al.67, MAO-induced rIPC of the myocardium was abolished when HOE140 (bradykinin B2 receptor blocker) was administered during early reperfusion in in vivo rats. Also, infusion of bradykinin into the mesenteric artery resulted in cardioprotection, but was subsequently abolished by the ganglion blocker, hexamethonium. Thus, bradykinin is thought to exert its effects on distant organs through a dual neurogenic and humoral (since plasma bradykinin levels increase following ischemia55) pathway. Once the neurogenic pathway is blocked, there may be insufficient bradykinin from the humoral pathway to evoke protection at the target organ. Knocking out one pathway may be enough to abolish protection entirely, as protection from ischemia-reperfusion injury is commonly an all-or-none phenomenon that requires a threshold to be met in order to induce protection according to Goto et al.3. To confirm that dialyzing blood plasma did not dilute bradykinin concentrations (dialysate is 10x diluted compared to plasma), dialysate was lyophilized into a powder and reconstituted with 1x Krebs-

Henseleit buffer such that dialysate concentrations were comparable to rabbit plasma. This reconstituted dialysate still protected cardiomyocytes from ischemia-reperfusion damage and

HOE140 did not abolish this protection (N=1, n=1; see Table 10). Thus, in isolated rabbit cardiomyocytes, the bradykinin B2 receptor and bradykinin molecule do not play a role in humorally-mediated rIPC nor cross-talk with other receptors.

69

Table 10. 1x Dialysate with HOE140 Results (% Cardiomyocyte Death). Group Stabilization End Baseline 24.3 26.4 Before SI After SR 1x Control Rabbit Dialysate 24.9 40.8 1x Control Rabbit Dialysate + HOE140 25.3 34.5 1x rIPC Rabbit Dialysate 25.5 24.4 1x rIPC Rabbit Dialysate + HOE140 24.7 27.3

70

6.3 The Role of CGRP Receptors

6.3.a Existence of Calcitonin-like Receptors in Rabbit Cardiomyocytes:

The presence of calcitonin-like receptors (CLRs) in rabbit cardiomyocytes was investigated using western blotting (n=1). CLRs were found in rabbit whole hearts, isolated cardiomyocytes, and cardiomyocyte particulate fractions (i.e. the mitochondrial membrane was excluded). (See Figure 19).

Existence of Calcitonin-Like Receptors in Rabbit Cardiomyocytes

Figure 19. Western Blot Analysis of Calcitonin-Like Receptors in Rabbit Tissues. The presence of calcitonin-like receptors (CLRs) in rabbit cardiomyocytes was investigated using western blotting (n=1). CLRs were found in rabbit whole hearts, isolated cardiomyocytes, and cardiomyocyte particulate fractions (i.e. the mitochondrial membrane was excluded).

71

6.3.b CGRP Receptor Blockage Conserves Protection:

The effect of CGRP (8-37) (a CGRP receptor blocker; 5nM concentration was used;

Wolfrum, 2005103) in conjunction with rabbit dialysate was studied. Blockers of CGRP receptor activity conserved the protection induced by the preconditioned dialysate (24.8%±1.9 vs.

26.0%±3.3, p=0.96; N=2, n=2, 2). (See Figure 20 and Table 11).

72

Figure 20. CGRP (8-37) Administered Prior to Rabbit Dialysate. The effect of CGRP (8-37) (a CGRP receptor blocker; 5nM concentration was used) in conjunction with rabbit dialysate was studied. Blockers of CGRP receptor activity conserved the protection induced by the preconditioned dialysate (24.8%±1.9 vs. 26.0%±3.3, p=0.96; N=2, n=2, 2).

Table 11. CGRP (8-37) Results (Mean ± SEM). Group Stabilization End Baseline 23.4 ± 2.2 23.3 ± 2.6 Baseline + CGRP (8-37) 22.6 ± 7.7 21.8 ± 4.0 Before SI After SR IR 21.7 ± 2.3 41.6 ± 2.0 rIPC Rabbit Dialysate 22.4 ± 3.3 26.0 ± 3.3 rIPC Rabbit Dialysate + CGRP (8-37) 23.4 ± 2.7 24.8 ± 1.9 73

6.3.c Discussion:

Calcitonin-like receptors (CLRs) have been identified in humans and rat smooth muscle cells87, 88. However, calcitonin-like receptors in rabbit tissues and in particular, rabbit cardiomyocytes have not been studied. Western blotting indicates that CLRs are present in rabbit whole heart, cardiomyocyte lysate, and the particulate fraction (with mitochondria excluded) (see

Figure 19). Even though this is a significant finding, there are limitations to concluding that

CGRP receptors exist on rabbit cardiomyocytes. The CGRP receptor is a receptor complex that consists of the CLR and associated receptor activity-modifying proteins (RAMPs). CLR association with RAMP1 and RAMP3 allows binding of CGRP to the receptor, resulting in CLR becoming a CGRP receptor85. CLR association with RAMP2 permits the binding of another cardioprotective factor, adrenomedullin84. Thus, in order to conclusively determine the existence of CGRP receptors on rabbit cardiomyocytes, it is important to establish that RAMP1 and

RAMP3 associate with this CLR.

CGRP (8-37) blocked CGRP receptors and did not affect rIPC dialysate protection, suggesting that the CGRP receptor is not involved in humoral rIPC (see Figure 20). In addition,

CGRP (8-37) has been used to abolish the protective effects of adrenomedullin167 in rat cardiomyocytes. Therefore, the results presented in Figure 20 used CGRP (8-37) to block all

CLRs on rabbit cardiomyocytes. CGRP (8-37) was also administered to isolated rabbit cardiomyocytes under basal conditions (i.e. the Baseline + CGRP (8-37) treatment group). Since the CGRP blocker did not increase cell necrosis, this antagonist was deemed as non-toxic in rabbit cardiomyocytes.

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The claim by Wolfrum et al.103 regarding CGRP and rIPC cardioprotection suggests the involvement of a dual neurogenic and humoral pathway. The authors determined that infusion of

CGRP could mimic MAO-preconditioned cardioprotection in rats. MAO preconditioning with

CGRP (8-37) abolished protection. Also, CGRP-induced protection was abolished by hexamethonium (a ganglion blocker).Throughout MAO preconditioning, the level of CGRP was found to be elevated by radioimmunoassay regardless of ganglion blockade. However, Wolfrum et al. did not investigate the role of CGRP in a humoral model alone. Since CGRP is released by caspasin sensitive sensory nerves, most likely this peptide‘s mechanism of action occurs through a neurogenic pathway. Thus, there may be insufficient CGRP circulating in the blood following preconditioning to induce cardioprotection. In addition, the work regarding CGRP is in remote preconditioning of the intestine97,103 in rats to induced cardioprotection, suggesting that CGRP may play a unique role when the intestine as the preconditioning organ but not in skeletal muscle ischemia.

6.4 The Role of Adenosine Receptors

6.4.a Non-selective Adenosine Receptor Blockade of Protection:

The effect of 8-SPT (a non-selective adenosine receptor blocker; 100µM concentration was used; Armstrong, 1995120) in conjunction with rabbit dialysate was studied. Blockers of adenosine receptor activity abolished the protection induced by the preconditioned dialysate

(41.6%±2.2 vs. 28.1%±1.7, p=0 0002; N=2, n=5,1). (See Figure 21 and Table 12).

75

Figure 21. 8-SPT Administered Prior to Rabbit Dialysate. The effect of 8-SPT (a non-selective adenosine receptor blocker; 100µM concentration was used) in conjunction with rabbit dialysate was studied. Blockers of adenosine receptor activity abolished the protection induced by the preconditioned dialysate (41.6%±2.2 vs. 28.1%±1.7, p=0.0002; N=2, n=5,1).

Table 12. 8-SPT Results (Mean ± SEM). Group Stabilization End Baseline 26.9 ± 2.3 27.3 ± 0.9 Baseline + SPT 25.2 ± 1.3 26.7 ± 2.2 Before SI After SR Control Rabbit Dialysate 25.6 ± 1.2 43.7 ± 1.4 Control Rabbit Dialysate + SPT 24.5 ± 1.4 42.3 ± 0.8 rIPC Rabbit Dialysate 25.3 ± 1.4 28.1 ± 1.7 rIPC Rabbit Dialysate + SPT 26.5 ± 1.5 41.6 ± 2.2 76

6.4.b Discussion:

My results indicate the involvement of adenosine in rIPC since 8-SPT (a non-selective adenosine blocker) was able to abolish protection from rIPC dialysate (see Figure 21). To support this finding, Pell et al.128 suggested the involvement of adenosine in rabbit renal artery occlusion (RAO). Also, Liem et al.127 confirmed this in rat mesenteric artery occlusion (MAO) to induce cardioprotection. However, these studies have suggested a neurogenic model for rIPC in renal and mesenteric preconditioning. Pang et al.129 demonstrated that skeletal muscle ischemia increased plasma adenosine concentrations and inhibition of vesicular monoamine transport (inhibition of catecholamines at synaptic nerve endings) partially abolished preconditioning. Thus, it is possible that skeletal muscle ischemia releases adenosine that activates local nerves, which in turn affect distant organs like the myocardium through a dual neurogenic/humoral pathway. However, the protective factor in rIPC dialysate is humoral in nature and this alone can protect isolated cardiomyocytes without the need of neuronal intervention (see Figure 13).

According to Pang et al.129, levels of adenosine in the plasma were elevated during skeletal muscle ischemia, hinting at this purine‘s involvement in a humoral pathway. However, the half-life of adenosine is very short (0.6-1.5 sec in plasma) due to continual breakdown by circulating adenosine deaminase140. To support this finding, the level of adenosine was determined to be <0.80µM by MRM-MS in rIPC dialysate (see Table 5). However, adenosine concentrations are required to be >10uM to protect isolated rabbit cardiomyocytes according to

Armstrong et al.120. Based on the above evidence, the involvement of the adenosine molecule in 77 a humoral route is unlikely. Therefore, the blockage of dialysate protection with 8-SPT (Figure

21) suggests the involvement of the adenosine receptor and not the adenosine molecule.

A number of controversies exist with the role of adenosine in IPC and rIPC. Auchampach

168 et al. demonstrated that administration of three A1 antagonists (DPCPX, BG9719, BG9928) in dogs throughout preconditioning until before index ischemia did not block protection. Mice with knockouts for adenosine receptors also show contradictory results. Lankford et al.169 could not

170 precondition A1 knockouts, however Guo et al. demonstrated that A3 knockout mice are still capable of preconditioning, thus illustrating the importance of A1 receptors in IPC. However, a

171 study conducted by Eckle et al. demonstrated that A1, A3, and A2a knockouts had preserved protection following ischemia. It was A2b receptors alone that could not be preconditioned through IPC. To explain these controversies, a review by Cohen and Downey14 suggests the involvement of multiple receptor agonists acting in parallel during IPC. This also suggests the involvement of other receptors in the form of cross-talk.

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- Chapter 7 -

RIPC MEDIATES OPIOID-ADENOSINE CROSS-TALK

7.1 Opioid-Adenosine Cross-talk

7.1.a Adenosine Deamination of the Dialysate Conserves Protection:

The effect of adenosine deamination of dialysate was studied. Rabbit dialysate was passed through a column containing adenosine deaminase (ADA) coupled to beads‡. Levels of adenosine and inosine before and after deamination were measured using MRM mass spectrometry to ensure complete elimination of adenosine from the dialysate. The adenosine deamination of the dialysate conserved the protection induced by rIPC dialysate§ (34.9% vs.

33.3% and 30.1% vs. 31.8%, n=2). (See Table 13).

NOTE: These experiments were originally conducted with an n=3.However, adenosine in rIPC dialysate from one experiment was not deaminated, thus this experiment was excluded.

‡ Dialysate deamination was conducted by Amy Yeung (Dr. John Callahan‘s Lab; Department of Paediatric Laboratory Medicine, Hospital for Sick Children). § These experiments were conducted by Alina Hinek (Dr. Wilson‘s Lab, Hospital for Sick Children). 79

Table 13. ADA Results (% Cardiomyocyte Death). Experiment A Stabilization End Baseline 24.1 35.1 Before SI After SR Control Rabbit Dialysate 24.3 42.6 rIPC Rabbit Dialysate 28.4 34.9 rIPC Rabbit Dialysate + ADA 31.1 33.3 Pre-ADA (µM) Post-ADA (µM) Adenosine in rIPC Rabbit Dialysate 0.01 No peak Inosine in rIPC Rabbit Dialysate 0.22 0.39

Experiment B Stabilization End Baseline 26.6 33.6 Before SI After SR Control Rabbit Dialysate 24.0 45.0 rIPC Rabbit Dialysate 29.5 30.1 rIPC Rabbit Dialysate + ADA 30.1 31.8 Pre-ADA (µM) Post-ADA (µM) Adenosine in rIPC Rabbit Dialysate 0.01 No peak Inosine in rIPC Rabbit Dialysate 0.26 0.20

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7.1.b Partial Adenosine Blockade of δ-Opioid Receptor-Induced Protection:

The effect of 8-SPT (a non-selective adenosine receptor blocker; 100µM concentration was used; Armstrong, 1995120) in conjunction with Met-enkephalin (a δ-opioid receptor agonist,

100µM; Cao, 2003152) was studied. The adenosine receptor blocker partially abolished the protection induced by δ -opioid receptor activation (41.8%±1.5 vs. 32.8%±1.0 compared to ME, p=0.0007, and vs. 50.7%±1.1 compared to IR, p=0.0008; n=5). (See Figure 22 and Table 14).

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Figure 22. 8-SPT Administered Prior to Met-Enkephalin. The effect of 8-SPT (a non-selective adenosine receptor blocker; 100µM concentration was used) in conjunction with Met-enkephalin (a δ-opioid receptor agonist, 100µM) was studied. The adenosine receptor blocker partially abolished the protection induced by δ -opioid receptor activation (41.8%±1.5 vs. 32.8%±1.0 compared to ME, p=0.0007, and vs. 50.7%±1.1 compared to IR, p=0.0008; n=5).

Table 14. Met-Enkephalin with 8-SPT Results (Mean ± SEM). Group Stabilization End Baseline 29.3 ± 0.8 31.2 ± 0.7 Baseline + ME 23.9 ± 0.0 21.7 ± 0.0 Before SI After SR IR 28.6 ± 1.3 50.7 ± 1.1 ME 30.6 ± 1.9 32.8 ± 1.0 ME + 8-SPT 29.4 ± 0.8 41.8 ± 1.5 ME + NTI 33.4 ± 0.0 51.7 ± 0.0

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7.1.c Complete Adenosine Blockade of κ -Opioid Receptor-Induced Protection:

The effect of 8-SPT (a non-selective adenosine receptor blocker; 100µM concentration was used; Armstrong, 1995120) in conjunction with dynorphin B (a κ-opioid receptor agonist,

100µM; Cao, 2003152) was studied. The adenosine receptor blocker abolished the protection induced by κ-opioid receptor activation (44.3%±2.6 vs. 31.8%±2.1 compared to DynB, p=0.02, and vs. 51.3%±5.0 compared to IR, p=0.17; n=5). (See Figure 23 and Table 15).

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Figure 23. 8-SPT Administered Prior to Dynorphin B. The effect of 8-SPT (a non-selective adenosine receptor blocker; 100µM concentration was used) in conjunction with dynorphin B (a κ-opioid receptor agonist, 100µM) was studied. The adenosine receptor blocker abolished the protection induced by κ-opioid receptor activation (44.3%±2.6 vs. 31.8%±2.1 compared to DynB, p=0.02, and vs. 51.3%±5.0 compared to IR, p=0.17; n=5).

Table 15. Dynorphin B with 8-SPT Results (Mean ± SEM). Group Stabilization End Baseline 26.4 ± 1.1 27.2 ± 2.4 Baseline + DynB 19.2 ± 0.0 22.4 ± 0.0 Before SI After SR IR 29.1 ± 2.1 51.3 ± 5.0 DynB 28.7 ± 1.8 31.8 ± 2.1 DynB + 8-SPT 30.5 ± 1.9 44.3 ± 2.6 DynB + GNTI 29.1 ± 1.6 48.3 ± 3.1 84

7.1.d Discussion:

Preconditioned dialysate was exposed to adenosine deaminase (ADA) conjugated beads in order to remove any trace of adenosine. The results of our study confirm that adenosine removal from the rIPC dialysate maintains protection, indicating with certainty that the protective factor(s) is/are not adenosine (see Table 13). ADA-conjugated beads were utilized in order to remove ADA prior to cardiomyocyte exposure, thus avoiding deamination of vital adenosine released during the mediator phase of preconditioning. To summarize the results so far: the half-life of adenosine in plasma is 0.6-1.5sec140, MRM-MS detected low levels of adenosine in the dialysate (see Table 5), and rIPC protection was conserved even in the absence of adenosine through adenosine deamination. However, rIPC dialysate protection was abolished by the adenosine receptor blocker, 8-SPT (see Figure 21). Based on this accumulating evidence, there is involvement of adenosine receptors in rIPC but not the adenosine molecule itself.

ADA also increases inosine concentrations during the breakdown of adenosine. Jin et al.172 has reported that inosine increases mast cell degranulation during ischemia or inflammation. The same study demonstrated that inosine can bind only to adenosine A3 receptors

173 and not A1 or A2a. Recently, Shen et al. suggested that inosine preconditions rats from cerebral

174 brain injury via adenosine A3 receptors. Litsky et al. found inosine protects mouse neuronal and glial cell cultures from hypoxia. The authors constructed a concentration response curve for inosine and determined the minimum concentration to protect neuronal cells is 500µM in rats.

However, the level of inosine in rIPC dialysate is <0.26µM and upon deamination of adenosine, inosine levels elevated to <0.39µM (see Table 13). Therefore, the protection seen after adenosine deamination is not due to inosine. 85

The non-selective adenosine blocker 8-SPT partially abolished protection from Met- enkephalin (δ-specific opioid agonist) (see Figure 22). The activation of κ receptors by dynorphin B (κ-specific opioid agonist) was fully abolished by 8-SPT since there was no statistical significance between ischemia-reperfusion alone and administration of dynorphin B with 8-SPT (see Figure 23). However, increased number of experiments may result in dynorphin

B exhibiting partial protection similar to Met-enkephalin. Regardless, these studies have confirmed cross-talk between δ and κ opioid receptors with adenosine receptors during the trigger phase of classical IPC.

Interestingly, Peart et al.138 suggests that cross-talk between opioid and adenosine receptors work both ways. However, our rIPC dialysate model does not involve adenosine due to its deamination in the blood. Also, preliminary data from our lab indicates that cross-talk is unidirectional (n=2, see Table 16). This may be due to species differences or due to experimental models since Peart et al. utilized an in vivo rat model.

86

Table 16. Adenosine with Naloxone Results (% Dead Cardiomyocytes). Experiment A Stabilization End Baseline 19.2 22.1 Baseline + Adenosine 19.3 22.1 Before SI After SR IR 26.8 52.9 Adenosine 25.1 29.0 Adenosine + Nal 23.3 31.0 Adenosine + SPT 25.3 45.1

Experiment B Stabilization End Baseline 26.9 29.0 Before SI After SR IR 26.2 36.2 Adenosine 28.4 36.2 Adenosine + Nal 26.9 37.6

One explanation, suggested by Peart et al.139, of opioid-adenosine cross-talk is the adenosine kinase inhibition hypothesis. Adenosine kinase degrades adenosine into AMP within cardiomyocytes. According to the authors, activation of opioid receptors inhibits this enzyme, which leads to an accumulation of intracellular adenosine. Since diffusion of adenosine across the cell membrane is dependent on a concentration gradient, extracellular adenosine remains outside cardiomyocytes. The build-up of adenosine activates adenosine receptors and results in ischemic preconditioning. Therefore, a vital component of the adenosine kinase inhibition hypothesis is the amassing of extracellular adenosine.

7.2 The Inhibition of Adenosine Kinase Hypothesis

7.2.a Exposure to Dynorphin B Does Not Accumulate Extracellular Adenosine:

The inhibition of adenosine kinase to increase levels of extracellular adenosine was measured using MRM mass spectrometry. Supernatants from freshly isolated rabbit cardiomyocytes were obtained after stabilization, dynorphin B (a κ-opioid receptor agonist, 87

100µM; Cao, 2003152) exposure, and after a wash period. Levels of adenosine did not change significantly (contained the following in µM: 1.54±0.5 after stabilization, 0.96±0.1 after DynB exposure, 1.14±0.1 after wash; n=5) and were well below 10µM (the minimum concentration of adenosine required to protect cardiomyocytes; Armstrong, 1995120). (See Figure 24 and Table 17 for results; see Figure 29 in Appendix V for a plot of the standard concentrations).

88

Figure 24. Adenosine Concentrations After Exposure to Dynorphin B. The inhibition of adenosine kinase to increase levels of extracellular adenosine was measured using MRM mass spectrometry. Supernatants from freshly isolated rabbit cardiomyocytes were obtained after stabilization, dynorphin B (a κ-opioid receptor agonist, 100µM) exposure, and after a wash period. Levels of adenosine did not change significantly (contained the following in µM: 1.54±0.5, 0.96±0.1, 1.14±0.1; n=5) and were below 10µM (the minimum concentration of adenosine required to protect cardiomyocytes).

Table 17. Adenosine Concentrations After Exposure to Dynorphin B Results (Mean ± SEM). Group Concentration (µM) After Stabilization 1.54 ± 0.5 After DynB 0.96 ± 0.1 After Wash 1.14 ± 0.1

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7.2.b Discussion:

Supernatant was collected before, during and after cardiomyocytes were exposed to dynorphin B (DynB, κ-selective agonist) and immediately placed on ice to prevent deamination of adenosine. The same MRM-MS method used to detect adenosine in the dialysate (see Table 5) was used to measure adenosine concentrations in supernatant samples (see Figure 24). The results indicate that adenosine concentrations do not change after DynB exposure and are well below protective levels (<10µM). Therefore, there is no increase in extracellular adenosine as a result of opioid receptor stimulation during the trigger phase of IPC.

Another method of increasing interstitial adenosine concentrations is through inhibition of adenosine deaminase which is normally situated outside cardiomyocytes. Silva et al.175 examined the effects of an adenosine deaminase inhibitor (pentostatin) on myocardial infarction in dogs. Pentostatin increased plasma adenosine levels 3.5-fold above basal conditions prior to ischemia and sustained these levels until early reperfusion. However, this attenuation of adenosine had no effect on infarct size and did not precondition myocardium.

Peart et al.138 in 2003 first noted a relationship between opioid and adenosine receptors in in vivo cardioprotection in rats. Rats were pre-treated with morphine (a non-selective opioid agonist) and 2-chloro-cyclopentyladenosine (CCPA, adenosine A1 agonist) throughout the experiment and this mimicked preconditioning by coronary artery occlusion. This protection was abolished by the δ1 opioid blocker, 7-benzylidenealtrexone (BNTX). Based on these findings,

Peart et al. concluded receptor cross-talk and/or parallel signalling pathways downstream of opioid and adenosine receptors. Later in 2005, Peart et al.139 postulated that the opioid-adenosine 90 cross-talk seen in 2003 was attributed to inhibition of adenosine kinase in cardiomyocytes (see

Figure 3). In this study, rats were treated in vivo with the adenosine kinase inhibitor, 5- iodotubercidin which protected myocardium from damaging CAO. 8-Cyclopentyl-1,3- dipropylxanthine (DPCPX, adenosine A1 blocker) and MRS1523 (adenosine A3 blocker) abolished this protection. In addition, the δ1 opioid blocker, BNTX, also eliminated protection.

However, my results measuring adenosine in cardiomyocyte supernatant samples do not support this hypothesis. This may be due to differences in experimental protocols. For example, our isolated rabbit cardiomyocyte model was not in vivo and therefore, independent of many factors such as neuronal tissue in the whole heart. Also, Peart et al. administered adenosine kinase inhibitors throughout the experimental protocol which may have allowed time for adenosine kinase inhibition and accumulation of extracellular adenosine, whereas my cardiomyocyte model only treated cells with an opioid agonist during the trigger phase of IPC.

139 Most importantly, Peart et al. determined that δ1 opioid receptor inhibitors abolished protection induced by adenosine kinase inhibition. However, this finding contradicts their proposed hypothesis. The model undergoes the following steps: (1) activation of opioid receptors, (2) which inhibits adenosine kinase, (3) leading to extracellular adenosine accumulation, (4) resulting in cardioprotection. Inhibition of opioid receptors eliminates step (1), however, since an adenosine kinase inhibitor is still present throughout the procedure, steps (2) –

(4) are still possible and should result in cardioprotection. Nonetheless, Peart et al. states that this is only a tentative hypothesis that requires further investigation and proposes other possibilities 91 such as heterodimerization of opioid and adenosine receptor or parallel signalling pathways downstream of these receptors.

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- Chapter 8 -

SUMMARY OF FINDINGS

The data presented confirms these major findings:

1. Remotely preconditioned dialysate (both human and rabbit derived) protect

cardiomyocytes against ischemia-reperfusion injury to the same extent as classical IPC.

2. Compounds that precondition in IPC are either undetectable (Met-enkephalin, dynorphin

B, bradykinin, acetylcholine, angiotensin II) or at orders of magnitude below the

threshold to confer protection (MEAP, adenosine, inosine, norepinephrine) in rIPC

dialysate.

3. Cardioprotection induced by rIPC dialysate requires activation of δ or κ opioid receptors

or adenosine receptors.

4. Cardioprotection induced by rIPC dialysate does not require the activation of bradykinin

B2 or CGRP receptors.

5. The adenosine molecule does not play a role in rIPC dialysate protection, but the

adenosine receptor does.

6. Protection induced by κ or δ opioid receptors is fully or partially abolished by non-

selective adenosine blockade. This indicates cross-talk between these receptors.

7. The cross-talk between opioid and adenosine receptors is not due to accumulation of

extracellular endogenous adenosine.

These findings support the hypothesis that δ or κ opioid receptors, through cross-talk with adenosine receptors, act as possible triggers in remote ischemic preconditioning. 93

- Chapter 9 -

GENERAL DISCUSSION

9.1 Overall Perspective

The investigations conducted in this thesis have determined that rIPC can be mediated from rabbit or human skeletal muscle to rabbit cardiomyocytes through a humoral pathway (see

Figure 13). Also, there is involvement of δ and κ opioid receptors33 (see Figures 15, 16, & 17) in the target organ, but this is not mediated by molecules (Met-enkephalin, dynorphin B, MEAP, adenosine, inosine, bradykinin, acetylcholine, norepinephrine, and angiotensin II) that have been widely involved in classical IPC (see Table 5). In addition, claims have been made regarding

67 103 bradykinin B2 (see Figure 18) and CGRP (see Figure 20) receptors, but these do not play a role in our isolated cardiomyocyte model. Interestingly, adenosine receptors128 have also been implicated in rIPC (see Figure 21). However, the level of adenosine in rIPC dialysate and subsequent deamination (Table 13), excludes the involvement of the adenosine molecule to explain these results. However, I determined there is cross-talk between δ and κ opioid receptors with adenosine receptors (see Figures 22 & 23) to explain these results. However, this relationship is not due to the inhibition of adenosine kinase139 increasing extracellular adenosine concentrations (see Figure 24).Thus, this cross-talk may occur through heterodimerization of opioid and adenosine receptors (See Figure 25)

94

Opioid Adenosine Opioid

Opioid Adenosine Opioid- Receptor Receptor Adenosine Receptor

Cross-talk

Activate cell signalling pathways

Figure 25. General Diagram of Opioid-Adenosine Cross-talk. The involvement of δ and κ opioid and adenosine receptors has been determined to play a role in humorally-mediated rIPC. However, the level of adenosine in rIPC dialysate and subsequent deamination excludes the involvement of the adenosine molecule to explain these results. This can be explained through cross-talk between δ and κ opioid and adenosine receptors. However, this relationship is not due to the inhibition of adenosine kinase increasing extracellular adenosine concentrations. Thus, this cross-talk may occur through heterodimerization of opioid and adenosine receptors.

95

9.2 Other Mechanisms of Opioid-Adenosine Cross-talk

9.2.a Dimerization of G-protein Coupled Receptors:

Heterodimerization among G-protein coupled receptors has been implicated in opioid- adenosine ‗cross-talk‘138. Heterodimerization between G-protein coupled receptors is a well documented phenomenon that can lead to unique pharmacological properties such as altered receptor sensitivity, endogenous adenosine release, ligand binding and signalling. Also, these uniquely dimerized receptors may bind to orphan peptides that have yet to be identified.166,176,177

In addition, there is evidence that opioid receptors dimerize with β2-adrenergic and somatostatin receptors. Jordan et al.178 studied oligomerization between these distant GPCR family members: δ and κ opioid receptors with β2-adrenergic receptors. Human embryonic kidney (HEK)-293 cells were cotransfected with anti-myc tagged opioid receptors and anti-Flag tagged β2-adrenergic receptors. Immunoprecipitation determined that these receptors associate with each other. Though these receptors did not display altered ligand binding, there were changes in protein trafficking such as endocytosis. Coimmunoprecipitation of sst(2A) somatostatin and µ1 opioid receptors were found to heterodimerize in HEK-293 cells by Pfeiffer et al. 179. Binding of either sst(2A) or µ ligands altered receptor phosphorylation and desensitization of this hybrid receptor.

Adenosine A1 receptors can form dimers with P2Y1 and dopamine D1 receptors. Gines et al. 180 cotransfected cultured mouse Ltk- fibroblasts and rat embryonic cortical neurons with adenosine A1, dopamine D1, and dopamine D2 human cDNA. Coimmunoprecipitation and colocalization with immunofluorescence determined that A1 heterodimerized with D1 receptors 96

but not D2. The authors determined that heterodimerization of these receptors resulted in receptor

181 desensitization. Yoshioka et al. extensively investigated in vivo heterodimerized P2Y1 and adenosine A1 receptors in rat brain tissues and cortical neurons using immunofluorescence and coimmunoprecipitation. In addition, adenosine A2 receptors exist as naturally occurring

182 oligomers with dopamine D2 receptors . Though GPCR heterodimerization has been described for both adenosine receptors and opioid receptors individually, there is currently no study that investigates heterodimerization of opioid and adenosine receptors with each other.

9.3 Future Directions

The further directions of these studies will be to further investigate the mechanism of

‗cross-talk‘ between opioid and adenosine receptors. Studies will involve investigating another mechanism of opioid-adenosine cross-talk, i.e. heterodimerization of G-protein protein receptors.

In addition, cell signalling downstream of G-protein coupled cardiomyocyte cell membrane receptors in rIPC and opioid-adenosine cross-talk is another logical avenue for further investigation.

9.3.a Specific Adenosine Receptor Subtypes in Cross-talk & rIPC:

Before determining if opioid and adenosine receptors heterodimerize, it is important to elucidate which adenosine receptor subtype(s) cross-talk(s) with δ and κ opioid receptors. To investigate this, cardiomyocytes will be subjected to 10 min classic IPC, specific opioid receptor agonists, such as Met-enkephalin (δ-specific agonist) or dynorphin B (κ-specific agonist) in the presence of adenosine receptor subtype blockers, such as DPCPX (A1 adenosine blocker),

MRS1523 (A3 adenosine blocker), or MRS1754 (A2b adenosine blocker) for 20 min. Treatment 97

groups will undergo a wash period in fresh buffer for 20 min. All groups will then be subjected

to a long period of 45 min simulated ischemia (SI) and 60 min simulated reperfusion (SR).

Samples will be taken before and after SI/SR. (See Figure 26).

Similar experiments will then be conducted with rIPC dialysate and the above mentioned

adenosine blockers in order to confirm which adenosine receptor subtype is involved in rIPC.

Figure 26. Protocol of Adenosine Receptor Subtypes in Opioid-Adenosine Cross-talk. Cardiomyocytes will be subjected to 10 min classic IPC, specific opioid receptor agonists, such as Met-enkephalin (δ-specific agonist) or dynorphin B (κ-specific agonist) in the presence of adenosine receptor subtype blockers, such as DPCPX (A1 adenosine blocker), MRS1523 (A3 adenosine blocker), or MRS1754 (A2b adenosine blocker) for 20 min. Treatment groups will undergo a wash period in fresh buffer for 20 min. All groups will then be subjected to a long period of 45 min simulated ischemia (SI) and 60 min simulated reperfusion (SR). Samples will be taken before and after SI/SR. 98

9.3.b Heterodimerization of Opioid and Adenosine Receptors:

Heterodimerization of GPCRs in cardiomyocytes can be investigated using coimmunoprecipitation of the opioid and adenosine receptors. In addition, immunofluorescence of the two receptors may also be examined. While these techniques provide information regarding receptor association, they cannot prove functionally significant heterodimerization.

A technique that can conclusively state the existence of dimerization is fluorescence energy transfer (FRET) and bioluminescence energy transfer (BRET) to study protein-protein interactions in living cells. Hebert et al.183 provides a highly detailed overview of these techniques. FRET involves fluorescence energy transfer between two adjacent proteins, one being the donor and the other the acceptor. When these proteins are dissociated, only the donor emission of fluorescence is detected. However, if they are in close proximity, there is significant energy transfer between the two proteins and the acceptor‘s emission is detected. BRET is similar to FRET, yet utilizes bioluminescent luciferase. This technique does not require external illumination to initiate fluorescence transfer like in FRET, but instead uses luciferase which has less background noise. Currently, these techniques study interactions between receptors and downstream signalling molecules, however, Gandia et al.184 describes the potential of the FRET and BRET techniques to study GPCR oligomerization.

9.3.c Cell Signalling in rIPC:

Signalling downstream of receptors has been studied in rIPC. However, the mechanisms involved are unclear compared to IPC. Similar to IPC, activation of PKC68, nitric oxide185,

186 128 reactive oxygen species , mitochondrial KATP channels , and the reperfusion injury salvage 99 kinase (RISK) pathway146 have been implicated in rIPC. However, inhibition of the mitochondrial permeability transition pore (mPTP) in rIPC is still unclear.

As a reminder, signalling in IPC involves ligand binging to cell membrane receptors that result in activation of kinases (e.g. PI3 kinase, Akt, and ERK) and nitric oxide (NO) synthesis, causing mitochondrial (mKATP) channel opening that produces reactive oxygen species (ROS), which leads to PKC translocation. During the mediator phase, pro-survival kinases187 of the reperfusion injury salvage kinase (RISK) pathway are activated and ultimately lead to inhibition of the mitochondrial permeability transition pore (mPTP) from opening188.

Nitric oxide (NO) is very important in early and delayed classical IPC, however, the role of NO in rIPC is still unclear. Tokuno et al.185 proposed that cerebral preconditioning could induce cardioprotection, however, this protection was absent in nitric oxide synthase (NOS) knockout mice. In addition, NFκβ and NOS have been implicated in delayed limb rIPC in rat hearts. Li et al.189 showed that rIPC increased translocation of NFκβ to the nucleus and increased

NOS mRNA. Inactivation of the NFκβ protein or the NOS gene abolished rIPC protection.

Phosphatidylinositol 3-kinase (PI3) is known to be involved in classical IPC. However, there are no current studies that have investigated the role of PI3 in rIPC. To address this question, I investigated the effect of wortmannin (a PI3 kinase inhibitor; 100nM concentration was used; Oldenburg, 2003160) in conjunction with rabbit dialysate. Blockers of PI3 kinase activity abolished the protection induced by the preconditioned dialysate (41.0%±4.9 vs.

24.3%±4.0, p=0.01; N=2, n=2,2). (See Figure 27 and Table 18). 100

Figure 27. Wortmannin Administered Prior to Rabbit Dialysate. The effect of wortmannin (a PI3 kinase inhibitor; 100nM concentration was used) in conjunction with rabbit dialysate was studied. Blockers of PI3 kinase activity abolished the protection induced by the preconditioned dialysate (40.2%±3.6 vs. 24.7%±3.0, p=0.01; N=2, n=2,2).

Table 18. Wortmannin Results (Mean ± SEM). Stabilization End Baseline 22.1 ± 1.7 22.9 ± 2.2 Baseline + Wort 23.1 ± 2.0 23.7 ± 1.9 Before SI After SR IR 21.0 ± 1.8 41.0 ± 1.7 rIPC Rabbit Dialysate 21.3 ± 2.5 24.7 ± 3.0 rIPC Rabbit Dialysate + Wort 20.3 ± 0.3 40.2 ± 3.6 101

The activation of mitochondrial KATP has been proposed in early and delayed rIPC. KATP channel opening is thought to reduce calcium overload and result in inhibition of the mPTP190,191,192. As evidence, rat cardioprotection by limb ischemia was blocked by glibenclamide (non-selective KATP blocker) and 5-HD (a mitochondrial KATP blocker) but not by

193,194,195 HMR 1098 (a sarcolemmal KATP blocker) . However, studies in pigs show that both

196,197 sKATP and mKATP are involved in early and delayed limb ischemia . In these studies, BMS

191095, a mKATP channel opener, mimicked rIPC by limb ischemia. However, 5-HD removed this protection.

Reactive oxygen species (ROS) generation plays a role in both detrimental lethal reperfusion injury and in the protective effects of preconditioning187 and post-conditioning198. As evidence, Weinbrenner et al.37 determined that a free radical scavenger (MPG) was able to abolish in vivo cardioprotection from renal artery occlusion (RAO) in rats.

In rIPC, the role of protein kinase C (PKC) has been confirmed in infrarenal aortic occlusion186 and MAO194, when chelerythrine (a PKC blocker) abolished cardioprotection in rats.

A number of studies investigating other triggers have suggested the role of PKC in rIPC, such as adenosine128, bradykinin68, and opioids16. In these studies, pharmacological preconditioning with

68 103 bradykinin B2 and CGRP receptor activation is thought to induce PKCε translocation in a manner similar to rIPC cardioprotection.

With respect to the RISK pathway, Heidbreder et al.199 suggest that mitogen-activated protein kinases (MAPKs), p38, and ERK1/2 are activated in the small intestine during 102 mesenteric rIPC to protect the myocardium. In addition, inhibition of these kinases reduced rIPC protection. Also, Shimizu et al.146 determined that these MAPK kinases from the RISK pathway were activated in the heart itself during in vivo skeletal muscle rIPC in rabbits.

To further complicate the role of adenosine in rIPC, cardiomyocytes not only utilize adenosine as a trigger, but also a mediator in IPC with its claimed involvement of adenosine A2b receptors during reperfusion14. Thus, it is important to investigate the role of adenosine as a mediator in rIPC, and potentially, a mediator in opioid-adenosine cross-talk. However, the role of adenosine receptors in the studies described by this dissertation has only been investigated in the trigger phase.

The role of mitochondrial permeability transition pore (mPTP) as an end effector in classical IPC is a subject of great interest, but to date, there are no published studies of the role of mPTP in rIPC. The mPTP is located on the inner mitochondrial membrane and opened during the beginning of reperfusion. Activation of the mPTP results in uncoupling of oxidative phosphorylation, ATP depletion, mitochondrial swelling, and ultimately results in cell death.

However, preconditioning is thought to inhibit mPTP opening200. Zhang et al.36 has shown that a

κ-opioid agonist (U-50,488H) induced mPTP opening, as seen by fluorescent calcein, in rat cardiomyocytes. In the same study, the mPTP activator (atractyloside) abolished femoral artery occlusion (FAO)-induced cardioprotection in rats.

Infiltration, adhesion and activation of neutrophils have also been proposed as an end effector in early and late rIPC201. Cardioprotection by limb ischemia in mice has shown down- 103 regulation of proinflammatory genes and up-regulation of genes involved in cytoprotection and protection from oxidative stress202. This suggests that rIPC suppresses neutrophils from releasing proinflammatory cytokines and from expressing adhesion markers. However, the role of neutrophils in rIPC requires an in vivo model and is not suitable to be studied by our isolated cardiomyocyte model.

9.4 Limitations of the Study:

There are some limitations to the model described in this dissertation. Unfortunately, utilizing an isolated cardiomyocyte model excludes the ability to study a neuronal pathway in protection. Though my results indicate that a humoral mediator is sufficient to induce rIPC cardioprotection (see Figure 13), this does not exclude a neurogenic involvement in vivo. An in vivo model is also useful in investigating the involvement of an inflammatory pathway via neutrophils in rIPC. Finally, my study did not measure apoptosis as an indicator of myocardial reperfusion injury. Such an investigation is more suitable for cultured cardiomyocytes since apoptosis takes several hours to develop. Regardless, it is important to note that an isolated cardiomyocyte model is more efficient (since many treatment groups can be derived from a single heart) and reduces heart-to-heart variability.

In addition, my studies only investigated rIPC during the trigger phase of acute preconditioning. A comprehensive study would involve the role of rIPC dialysate to induce protection during the reperfusion phase and in delayed rIPC. Fortunately, these investigations can be readily conducted either in isolated or cultured cardiomyocytes.

104

9.5 Conclusions:

This dissertation provides important insights into the role of particular G-protein coupled receptors in a humoral model of remote preconditioning. Also, there are benefits of using this model over others in investigating this area. For example, characterization of the dialysate (see

Table 5) using MRM-MS excluded the possibility of certain substances to mediate protection in the rIPC dialysate. However, this method does not encompass all endogenous ligands that can bind to a particular GPCR. Considering this, selective inhibition of receptors is a better method of determining which receptors are involved even if the stimulatory agent is not present.

Also, our model can be used to determine cross-talk between receptors on a functional level. MRM-MS also did not exclude the possibility of certain receptors involved in cross-talk with opioid receptors. However, our model is able to determine which receptors can participate in cross-talk (i.e. opioid with adenosine receptors) and which possibilities can be excluded (i.e. opioid cross-talk with bradykinin B2 and CGRP receptors).

In conclusion, remote preconditioning is a clinically relevant strategy to reduce myocardial infarction. However, the mechanistic pathways in remote precondition are still unclear, with some evidence resulting to conflicting results. Thus, it is important to compare the pathways in classical and rIPC in order to create beneficial therapeutic interventions. 105

APPENDIX

APPENDIX I: List of Figures

Figure 1. IPC Signalling Mechanisms in the Target Organ...... 4

Figure 2. rIPC Signalling Mechanisms of the Myocardium...... 7

Figure 3. The Adenosine Kinase Inhibition Hypothesis...... 25

Figure 4. Preparation of Buffers Derived from Krebs-Henseleit Buffer...... 31

Figure 5. Comparison of Digestion Protocols...... 33

Figure 6. Rabbit and Human Dialysate Preparation...... 36

Figure 7. General Protocol: Isolated Rabbit Cardiomyocytes...... 38

Figure 8. AIM (1) Protocol: To Characterize Dialysate Protection...... 39

Figure 9. AIM (2) Protocol: The Role of Cell Membrane Receptors...... 40

Figure 10. AIM (3) Protocol: Investigate Receptor Cross-talk...... 41

Figure 11. Trypan Blue Exclusion Assay...... 43

Figure 12. Adenosine Kinase Inhibition Protocol...... 49

Figure 13. Rabbit and Human Dialysate Administered Prior to Ischemia-Reperfusion...... 52

Figure 14. Western Blot Analysis of Opioid Receptors in Rabbit Tissues...... 57

Figure 15. Naloxone Administered Prior to Rabbit Dialysate...... 59

Figure 16. Naltrindole Administered Prior to Rabbit Dialysate...... 61

Figure 17. GNTI Administered Prior to Rabbit Dialysate...... 63

Figure 18. HOE140 Administered Prior to Rabbit Dialysate...... 67

Figure 19. Western Blot Analysis of Calcitonin-Like Receptors in Rabbit Tissues...... 70

Figure 20. CGRP (8-37) Administered Prior to Rabbit Dialysate...... 72 106

Figure 21. 8-SPT Administered Prior to Rabbit Dialysate...... 75

Figure 22. 8-SPT Administered Prior to Met-Enkephalin...... 81

Figure 23. 8-SPT Administered Prior to Dynorphin B...... 83

Figure 24. Adenosine Concentrations After Exposure to Dynorphin B...... 88

Figure 25. General Diagram of Opioid-Adenosine Cross-talk...... 94

Figure 26. Protocol of Adenosine Receptor Subtypes in Opioid-Adenosine Cross-talk...... 97

Figure 27. Wortmannin Administered Prior to Rabbit Dialysate...... 100

Figure 28. Standard Plot of Substances in Water...... 110

Figure 29. Standard Plot of Adenosine in Krebs-Henseleit Buffer...... 112

107

APPENDIX II: List of Tables

Table 1. Krebs-Henseleit Buffer Composition...... 30

Table 2. Tyrode Buffer Composition...... 42

Table 3. Inter-Observer Error Data...... 45

Table 4. Rabbit and Human Dialysate Results (Mean ± SEM)...... 52

Table 5. Characterization of Rabbit Dialysate Using MRM Mass Spectrometry...... 53

Table 6. Naloxone Results (Mean ± SEM)...... 59

Table 7. Naltrindole Results (Mean ± SEM)...... 61

Table 8. GNTI Results (Mean ± SEM)...... 63

Table 9. HOE140 Results (Mean ± SEM)...... 67

Table 10. 1x Dialysate with HOE140 Results (% Cardiomyocyte Death)...... 69

Table 11. CGRP (8-37) Results (Mean ± SEM)...... 72

Table 12. 8-SPT Results (Mean ± SEM)...... 75

Table 13. ADA Results (% Cardiomyocyte Death)...... 79

Table 14. Met-Enkephalin with 8-SPT Results (Mean ± SEM)...... 81

Table 15. Dynorphin B with 8-SPT Results (Mean ± SEM)...... 83

Table 16. Adenosine with Naloxone Results (% Dead Cardiomyocytes)...... 86

Table 17. Adenosine Concentrations After Exposure to Dynorphin B Results (Mean ± SEM). . 88

Table 18. Wortmannin Results (Mean ± SEM)...... 100

Table 19. Summary of Literature in which Myocardium was the Target Organ...... 108

Table 20. Summary of Literature in which Skeletal Muscle was the Preconditioning Organ.... 109

Table 21. Results of Standard Concentrations of Substances in Water...... 111

Table 22. Results of Standard Concentrations of Adenosine in Krebs-Henseleit Buffer...... 112 108

APPENDIX III: rIPC Summary: Myocardium as the Target Organ

Table 19. Summary of Literature in which Myocardium was the Target Organ. This table is identical to Table 1 from the review by Hausenloy & Yellon in 2008.19

109

APPENDIX IV: rIPC Summary: Skeletal Muscle as the Preconditioning Organ

Table203 20. Summary of Literature in which Skeletal Muscle was the Preconditioning Organ. This table is identical to Table 5 from the review by Tapuria et al. in 2008.203

110

APPENDIX V: MRM Mass Spectrometry Plots of Standard Concentrations

Figure 28. Standard Plot of Substances in Water. Known standard concentrations of all substances were prepared in water and measured by MRM mass spectrometry in order to measure accurate concentrations.

111

Table 21. Results of Standard Concentrations of Substances in Water. Standard Concentrations Measured Concentrations (nM)

(nM)

enkephalin

-

orepinephrine

DynorphinB MEAP Adenosine Inosine Bradykinin N Acetylcholine AngiotensinII Met Water ------2.5 2.64 ------2.45 0.068 3.4 3.57 3.5 3.77 5.0 4.46 ------5.08 4.94 5.9 7.20 6.9 6.79 10.0 9.9 ------10.5 10.3 14.2 12.4 14.3 14.4 15.0 17.1 17.0 15.8 25.0 24.8 27.0 27.6 24.7 25.1 50.0 50.7 --- 40.7 48.5 47.2 56.6 57.1 57.4 51.3 59.0 55.6 68.0 74.4 100 104 54.3 96.8 101 99.1 113 114 115 108 150 149 155 250 252 296 233 241 255 296 295 342 329 500 517 680 578 521 493 566 751 574 566 592 593 685 687 1000 981 823 959 NOTE: --- represents ‗No Peak‘. 112

Figure 29. Standard Plot of Adenosine in Krebs-Henseleit Buffer. Known standard concentrations of adenosine was prepared in Krebs-Henseleit buffer and measured by MRM mass spectrometry in order to measure accurate concentrations.

Table 22. Results of Standard Concentrations of Adenosine in Krebs-Henseleit Buffer. Standard Concentrations (nM) Measured Concentrations (nM) Water --- 100 96.3 1000 1070 10000 9940 10000 10800 NOTE: --- represents ‗No Peak‘. 113

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