NOTE TO USER Page(s) not included in the original manuscript are unavailable from the author or university. The manuscript was microfilmed as received. xiv-xv This is reproduction is the best copy available STUDIES on the ROLE of r-ATP CMELSin the PRECONDITIONING of CULTURED HI%U4N VENTRICULAR CARDIOIMYOCYTES Gideon Cohen, MD, BSc, MSc, PhD Institute for Medical Sciences, University of Toronto. Division of Cardiovascular Surgery, The Toronto Hospital. Toronto, Ontario, Canada. A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy. Graduate Department of the Institute of Medical Sciences, University of Toronto Copyright O 2001 National Library Bibliotheque nationale 1*1 of Canada du Canada Acquisitions and Acquisitions et Bibliographic Services services bibliographiques 395 Wellington Street 395, rue Wellington Ottawa ON KIA ON4 Ottawa ON KIA ON4 Canada Canada The author has granted a non- L'auteur a accorde une licence non exclusive licence allowing the exclusive pennettant a la National Library of Canada to Bibliotheque nationale du Canada de reproduce, low distribute or sell reproduke, preter, distribuer ou copies of this thesis in microform, vendre des copies de cette these sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format Bectronique. The author retains ownership of the L'autsur conserve la propriete du copyright ~JI this thesis. Neither the droit d'auteur qui protege cette these. thesis nor substantial extracts fiom it Ni la these ni des extraits substaatiels may be printed or otherwise de celle-ci ne doivent etre imprimes reproduced without the author's ou autrement reproduits sans son permission. autorisation. i ABSTRACT OBJECTIVES: Due to the increasing numbers of high risk patients presenting for CABG, improved methods of myocardial protection are necessary. Ischemic preconditioning (PC)is the most powetful endogenously mediated form of myocardial protection known. Udortunately, this phenomenon is quite ditticult to apply clinically. The following studies were designed to determine the sequence of events inherent in PC, with an attempt to harness its beneficial effects in the form of a simple pharmacologic additive. ME'IXODS: We have developed a mode1 of ischemia and repefision in human ventricular myocytes (HVh4s) obtained from the right ventricular outflow tract of patients undergoing corrective surgery for Tetralogy of Fallot. In this model, ischemia was reproduced by exposing the cells to low volume anoxic phosphate buffered saline (PBS) for a period of 90 minutes. Both stabilization and repefision were accomplished by exposing the cells to high volume normoxic PBS for a period of 30 minutes. Endogenous preconditioning was achieved by exposing the cells to a brief 20 minute episode of ischemia, followed by 20 rinutes of reperfhion. Exogenous preconditioning was achieved by exposing the cells to 50 uM Adenosine with or without SPT, 10 nM PMA with our without Calphostin-C, and 50 uM Pinacidil or 20 uM Diazoxide with or without Glybenclamide. Viability and metabolic assessments were undertaken in the cell model, and results were confirmed in both large animal and clinical studies. RESULTS: Ischemic preconditioning conferred significant protection to HVMs. This protective effect was reproduced with adenosine and PMA, and was abolished with SPT and calphostin-C. The second messenger system included PKC activation and translocation. The final effector in this process was K'-ATP channel opening, as glybenclamide abolished all preconditioning stimuli. Although preconditioning was reproduced with the non-specific R-ATP channel opener pinacidil, since HVMs are quiescent in nature, the final effector was believed to be mitochondria1 specific K--ATP channel opening. Indeed, mitochondrial- specific K'-ATP channel opening with diazoxide conferred protection, preserved cellular ATP .. 11 concentrations, and facilitated oxidative phosphorylation by increasing mitochondria1 matrix volume and upregulating respiratory chain enzymes. PKC was localized to HVM mitochondria1membranes using both Western blotting techniques and imrnunogold localization. Adenosine was found to preserve myocardial ATP concentrations throughout the crossclamp period in patients undergoing CABG, and diazoxide was found to preserve ATP and improve hnctional recovery of cardiac allografts in a porcine model of cardiac transplantation. CONCLUSIONS: In humans, preconditioning is mediated via adenosine release, PKC translocation and activation. The final effector in this process is the rnitochondrial KLATP channel, which when opened facilitates oxidative phosphoryIation and ATP preservation. iii THIS WORK IS DEDICATED TO MY PARENTS, MEfR AND M4Lh2 COHEN- NEHE~,AS ~LLAS TO msrsm, EFFRATAND DANYA. WlTHOUT =EM, 1 WOULD NOT BE MERE IAM TODAY. ACKNOWLEDGEMENTS First and foremost, I wish to thank my supervisor and mentor, Dr. Richard D. Weisel for his support and guidance, without which this work would have not been possible. His tireless efforts in pursuit of academic excellence and his dedication and commitment to his students are unsurpassed, and I am eternally gratell to have been granted the opportunity to work under his supervision I wish to thank the members of my thesis committee, Dr. Donald A. G. MickIe and Dr. Stephen E. Fremes, for their valuable input and guidance. Their expertise in their respective fields provided an invduable resource throughout my academic training and I am indebted to them for their time and effort on my behalf. As well, I wish to thank Drs. John Coles and Michael Johnston for agreeing to serve as thesis examiners. AIthough all experiments and assessments were undertaken by the author, this work would not have been possible without the guidance of the many laboratory technicians and post-doctoral fellows within the Centre for Cardiovascular Research at the Toronto Hospital. I would especially like to thank Frank Merank: PhD, Laura C. Turniati, MSc, Molly K. Mohabeer, BSc, and Ren-Ke Li, MD, PhD, I would also like to thank Cameron Ackerley, PhD, for his assistance in performing the eIectron microscopic assessments, and Charlene Weisel, RN, for her multi-media support. The continued commitment of the Division of Cardiovascular Surgery at the University of Toronto towards the training of surgical scientists remains a valuable asset and has provided an ideal backdrop for residents such as myself who are interested in pursuing an academic surgical career. I am indebted to the members of the division for their continued support and I hope to achieve a standard that is worthy of such cornmitment in the coming years. My family and friends have always provided the unending support necessary to achieve my persona1 and career goals. I am forever gratell to them for their love and commitment. Finally, I would like to thank the Heart and Stroke Foundation of Canada for their fellowship grants in support of this work. Portions of this work have been published in the follow in^ iournals: Cohen, et al. Circulation 1998; 9811-164-I96 Cohen, et al. J NY Acad Sci 1999; 874:30&319 Cohen, et al: Ann Thor Surg 1999; 68:1995-2OOI vi STUDIES on the ROLE ofK'-ATP CHANNELS in the PRECONDITIONING of CULTURED WUWKENTRICUZAR CARDIOIMYOCYTES TABLE OF CONTENTS Abstract ... I ... Dedication ...111 Acknowledgements ... iv List of Publications ...v Table of Contents .. .VI List of Abbreviations ...xii Figure Legends ...mi Introduction ... 1 Chapter One: KNOWLEDGE TO DATE ... 5 Myocardial Preconditioning 1.1.1 Historical Overview Adenosine 1-2.1 Historical Overview Adenosine Metabolism 1-3.1 Endogenous Adenosine Production 1-3 -2 Adenosine Transport 1-3 -3 Adenosine Catabolism 1-3 -4 Regulation of Interstitid Adenosine Concentrations 1-3.5 Adenosine Receptors Cardioprotective Properties of Adenosine 1.5 Signal Transduction I,5 - 1 Cyclic AMP System 1 -5-2 G proteins 16 Adenosine Effector Mechanisms 1-7 Protein Kinase C 1-7.1 Involvement of Alternate Protein Kinases 1-8 The Human Connection 1-8.1 Ischemic Preconditioning in Humans 1-8.2 Human Cardiomyocyte Studies of Endogenous Preconditioning 1-8.3 Clinical Studies of Ischemic Preconditioning 1.8 -4 Reproducing Ischemic Preconditioning 1.9 Exogenous Adenosine in Humans 1-9.1 Physiologic Effects 1-9 -2 ElectrophysioIogic Effects 1-9.3 Regulation of Coronary BIood Flow 1-9.4 Haernodynamic and Respiratory Effects 1-10 Adenosine Preconditioning in Humans 1.10.1 Human Cardiomyocyte Studies of Adenosine Preconditioning 1.10.2 Human Cardiomyocyte Studies of Protein Kinase C 1.10-3 Clinical Studies of Adenosine Preconditioning l.iO.3.1 Adenosine Pretreatment 1.20.3.2 Cardioplegic Adenosine Treatment 1.10-3-3 Adenosine post-treahnerzt 1.10.3.4 Contirtuozis Adenosine Treatment 1.1 1 Underlying Mechanisms of Preconditioning 2-12 Cardiac Potassium Channels 1.12.1 Knowledge to Date 1.13 ATP-Dependant Potassium Channels viii Overview The Search for a K-ATP Clone Physiologic Properties Distribution and Density Biophysical Properties Voltage Dependence ATP Modulation of K-ATP K-ATP Kinetics Other Intracellular Modulators of K-ATP Pharmacological Modulation of K-ATP 1.14 Therapeutic Implications in the Heart .,.55 1.14.1 Myocardial Infarction .,.57 1.14.2 Ischemia-Reperfhion Injury ...57 1- 14.3 Effects on Coronary Vascular Tone ...58 1.14.4 Myocardial Preconditioning ...58 1.14.5 Role of Sarcolemrnal KlAm Channels in Preconditioning ...60 1.14.6 Role of Mitochondria1 K+.xp Channels in Preconditioning ... 61 1-15 Summary of Study Rationale,