The heart of creatures is the foundation of life, the Prince of all, the sun of their microcosm, from where all vigor and strength does flow. -William Harvey, De Motu Cordis, 1628 University of Alberta The Role of TIMPs in Heart Disease by Vijay Saradhi Kandalam A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Physiology ©Vijay Saradhi Kandalam Fall 2012 Edmonton, Alberta Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission. DEDICATION To family, friends, and colleagues who have supported my pursuit of realizing my potential. I would like to share this accomplishment with all of you, as without you I could not have achieved this. ABSTRACT Heart disease is a leading cause of morbidity and mortality in the world with a growing prevalence in a variety of manifestations. Many of the events that occur in the heart during the progression of disease have been explored to identify a causative mechanism to develop effective treatments and possibly a cure for heart disease. There is a growing body of evidence demonstrating the necessity for adaptive remodeling of the extracellular matrix (ECM) for proper cardiac function in response to disease through balanced regulation of its structure. Cardiac ECM serves as a structural framework for the myocardium, and its integrity is maintained by the function of matrix metalloproteinases (MMPs) which are kept under control by their physiological inhibitors, Tissue Inhibitor of Metalloproteinases (TIMPs). A balance between the MMPs and TIMPs is important for optimal degradation and replacement of the ECM either in physiological ECM turnover or in adverse ECM remodeling in disease. The research presented in this thesis consists of our investigation of the role of two highly expressed TIMPs in the heart, TIMP2 and TIMP3, in response to two common models of human heart disease. Mice lacking TIMP2 or TIMP3 were subjected to myocardial infarction, while the outcome of TIMP2 deficiency was also studied in response to cardiac pressure overload. Myocardial infarction led to different outcomes in the TIMP2-/- and TIMP3-/- mice. While TIMP3-/- mice exhibited elevated rates of left ventricular rupture incidence and severely compromised survival, as well as significant cardiac dysfunction, lack of TIMP2 exacerbated cardiac function without compromising survival compared to parallel wildtype mice. Following pressure overload, TIMP2-/- mice showed left ventricular dilation and dysfunction, hypertrophy and fibrosis which we found to be linked to impaired ECM-myocyte connection due to excess degradation of integrin-1 via excess activity of membrane-type 1 MMP (MT1-MMP). We have therefore identified important and distinct roles for these TIMPs in heart disease. The results presented in this thesis are important contributions to the study of the role of ECM remodeling in the adaptive cardiac response to injury, as demonstrated through a wide variety of physiological, histological, and molecular analyses of the development of heart disease in each model. These findings provide novel evidence identifying distinct mechanisms or pattern of events associated with TIMP2 and TIMP3 that can alter the current understanding of the role of each TIMP in the development and progression of heart disease. ACKNOWLEDGEMENTS I would like to acknowledge the many people who have made contributions to the work presented in this thesis, to other projects directly influencing the work presented within, and those who have contributed to this important phase of my development into a research scientist. My supervisor Dr. Zamaneh Kassiri has been an ideal role model for me to learn from. From when we first started in the lab together to this day, we have shared many great accomplishments along the way. She has guided me through a variety of experiences, while always being available to provide me feedback to promote excellence in the work that I do. Her dedication and hardwork is inspirational and is why I strive to emulate her. I would like to thank my Supervisory Committee members Dr. Jason Dyck and Dr. Richard Schulz. They have been available, approachable, and supportive to give me a different perspective for me to incorporate into my training. I admire each of them for their respective accomplishments, and appreciate the insight that they have shared with me, either formally or informally. My laboratory has been the creative centre of great scientists with wonderful personalities and my professional and personal well-being in mind. We have accomplished quite a lot together and it has been very rewarding to have trained together with all of them, with each of them contributing to my life in some meaningful way. In particular I would like to acknowledge my fellow Ph.D. Candidate Ratnadeep Basu and Laboratory Technician Sue Wang. We have worked together since the beginning and they have been directly involved with all of the work you see in front of you. Thank you to the Department of Physiology in the Faculty of Medicine and Dentistry for their support and direction. They have assisted me through guidance, to provide me opportunities to appreciate the wealth of knowledge available and the value of the scientific community. The Cardiovascular Research Centre has provided an excellent environment to establish the skills I need to pursue my professional ambitions. I would like to give a special thank you to Alberta Innovates Health Solutions (AIHS) for rewarding my efforts and dedication with funding and opportunities to broaden my training. This exceptional training environment is what has made my time at the University of Alberta so influential for my future endeavors. My family has supported me in pursuing challenges in my life unconditionally, and with that freedom to become who I would like to be, I wish to share my joy and a thank you. TABLE OF CONTENTS CHAPTER 1 GENERAL INTRODUCTION ................................................................... 1 1.1. Introduction overview ...................................................................... 2 1.2. The Fundamentals of cardiovascular physiology ............................. 2 1.3. The complex machinery of the heart ................................................ 3 1.3.1. Structure of the heart muscle ................................................. 3 1.3.2. Cardiac cell types ................................................................... 3 1.4. Overview of heart disease ................................................................ 5 1.4.1. Myocardial infarction ............................................................. 6 Etiology of myocardial infarction ......................................... 6 Cellular & molecular processes of myocardial infarction ..... 7 Myocardial infarction: Ischemia-induced cellular necrosis ....................................................................... 7 Myocardial infarction: Oxidative stress ..................... 8 Myocardial infarction: Cardiac inflammation ............ 9 Myocardial infarction: Fibrosis ................................ 10 Myocardial infarction: Angiogenesis ....................... 11 Experimental models of myocardial infarction ................... 13 1.4.2. Pressure overload cardiomyopathy ..................................... 13 Etiology of pressure overload cardiomyopathy ................... 13 Cellular and molecular processes of pressure overload ...... 14 Pressure overload: Cardiomyocyte hypertrophy ...... 14 Pressure overload: Fibrosis ....................................... 15 Pressure overload: Calcium homeostasis ................. 16 Pressure overload: Angiogenesis .............................. 17 Experimental models of cardiac pressure overload .............. 18 1.5. The extracellular matrix of the heart .............................................. 19 1.5.1. Composition ......................................................................... 19 1.5.2. Synthesis .............................................................................. 20 1.5.3. ECM-cardiomyocyte adhesion and communication ............ 21 1.5.4. ECM remodeling .................................................................. 23 1.6. MMP and TIMP balance in heart disease ...................................... 25 1.6.1. Matrix metalloproteinases (MMPs) ..................................... 25 Classification of MMPs ....................................................... 25 Structure of MMPs .............................................................. 26 MMP pro-domain and catalytic domain interaction ........... 28 MMP activation ................................................................... 29 1.6.2. Tissue inhibitors of metalloproteinases (TIMPs) ................ 29 1.6.3. TIMP inhibition of MMPs .................................................. 30 1.6.4. MMPs in animal models of heart