Activation of Cannabinoid Receptors Prevents Endothelin-1-induced Cardiac Myocyte Hypertrophy and Mitochondrial Dysfunction By Yan Lu A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfillment of the requirements of the degree of Doctorate of Philosophy College of Pharmacy Faculty of Health Sciences University of Manitoba Winnipeg Copyright © 2016 by Yan Lu Abstract Objectives: Endocannabinoids are bioactive amides, esters, and ethers of long-chain polyunsaturated fatty acids that activate two cannabinoid receptors, CB1 and CB2. Evidence suggests that activation of the endocannabinoid pathway offers cardioprotection. Cardiac hypertrophy is a convergence point of risk factors for heart failure, and it is associated with aberrant mitochondrial function. We determined a role for endocannabinoids in attenuating endothelin-1 (ET1)-induced hypertrophy and mitochondrial dysfunction, as well as the signaling pathways involved. Design and Methods: Cardiac myocyte hypertrophy was provoked by ET1 in isolated neonatal rat ventricular myocytes. Effects of four cannabinoid receptor agonists (anandamide, R-methanandamide, JWH-133 and CB-13) on hypertrophic markers (myocyte enlargement and hypertrophic gene expression) were assessed in the presence or absence of selective antagonists of CB1 or CB2 receptors. Mitochondrial function was evaluated by assessing changes in membrane permeability transition (calcein-AM), membrane potential (JC-1 dye), and mitochondrial bioenergetics related to fatty acid and glucose oxidation (seahorse XF24 analyzer). Molecular pathway components were identified by western blot and real-time PCR. Results: Anandamide and its metabolically-inactive analogue, R-methanandamide, prevented ET1-induced increases in hypertrophic markers, and application of selective CB receptor antagonists revealed a distinct role of each receptor therein. Also, JWH-133, a selective CB2 agonist, and CB-13, a dual agonist of CB1/CB2 receptors with limited brain penetration, were investigated as strategies to theoretically avoid central CB1 i receptor-mediated psychoactive effects. CB-13 attenuated all indicators of hypertrophy, whereas JWH-133 did not. The anti-hypertrophic actions of CB-13 were mediated by AMPK-eNOS crosstalk. ET1 induced mitochondrial membrane depolarization in the presence of either palmitate or glucose as primary energy substrate, decreased mitochondrial bioenergetics and proteins related to fatty acid oxidation (i.e. PGC-1α, a driver of mitochondrial biogenesis, and CPT-1β, facilitator of fatty acid uptake), but did not affect glucose-related bioenergetics. CB-13 corrected all of these parameters, at least in part, via AMPK signaling. Conclusions: Activation of cannabinoid receptors by CB-13, a peripherally-restricted agonist of CB1/CB2 receptors, offers protective effects on cardiac myocyte hypertrophy and its related mitochondrial disorders. Therefore, a cannabinoid-based treatment for cardiac disease represents a potential therapeutic strategy that warrants further study. ii Acknowledgement I can’t believe I’m approaching the finish line, and how much I have gained throughout the Ph.D. program. First, I would like to express my deepest gratitude to my supervisor, Dr. Hope Anderson, who provided me this opportunity to step into the science world, guided and supported me through the journey. She helped and encouraged me during the difficult times of research, spent tons of times in helping me with my writing and presentation skills, gave me trust and freedom in planning and arranging my work. Dr. Anderson not only influenced me as a great scientist, her wisdom in life also made her my role model. It is impossible to finish this thesis without her, and I could not ask for a better supervisor. Second, I would like to thank my committee members Dr. Yuewen Gong, Dr. Karmin O, and Dr. Grant Pierce. Their valuable advice and feedback made my project better than I expected. Also, I want to thank Dr. Paul Fernyhough and Dr. Subir Chowdhury for their continual help in the mitochondria aspect of my thesis. Their expertise in this area helped me saved a lot of time and resource. Third, I would like to give my thanks to my colleagues and graduate peers: June, Kim, Basma, Haining, Bolanle, Caroline, Crystal, and Danielle. I’m lucky to have these wonderful people around me to discuss and share ideas. Special thanks go to June, who introduced me to Dr. Anderson, taught me so many techniques when I started, and helped me out with my project whenever I need. She is not just a colleague, but also my teacher and friend. In addition, thanks to R.O. Burrell lab and other support staff, who provided great service and ensured the progression of my project. Last but not least, I would like to thank my families and friends. My parents and sister have always been supportive and understanding for my decisions, and I know they iii always will. A big thank you goes to my aunt Yin, uncle Feng, and cousins (Lucy, Anna, and Rhea) in Winnipeg. They are like my parents and sisters in Canada and they made my life here much easier and happier. Also, thanks to my friends, without them I wouldn’t be able to come so far. Pengqi, my dearest roommate, we shared every moment in our life, ups and downs, during the past 5 years, and I know we are lifelong friends. Jiaqi, Yuhua, Zirui, and all of the friends in the College of Pharmacy have brought me a lot of special memories, they made my Ph.D program everything but boring. iv Table of Content Abstract ................................................................................................................................ i Acknowledgement ............................................................................................................ iii List of Tables ................................................................................................................... xii List of Figures ................................................................................................................. xiii List of Abbreviations ...................................................................................................... xvi List of Pharmacological Agents ..................................................................................... xxi Chapter I: Introduction ..................................................................................................... 1 Chapter II: Literature Review .......................................................................................... 5 1. Heart failure ............................................................................................................... 5 1.1. Definition .............................................................................................................. 5 1.2. Classifications ....................................................................................................... 5 1.3. Risk factors ......................................................................................................... 10 2. Cardiac remodeling.................................................................................................. 11 2.1. Categories ........................................................................................................... 11 2.1.1. Physiological cardiac remodeling ................................................................ 13 2.1.2. Pathological cardiac remodeling .................................................................. 14 2.2. Characteristics of cardiac remodeling ................................................................. 14 2.2.1. Loss of myocytes ......................................................................................... 14 2.2.2. Myocardial fibrosis ...................................................................................... 15 2.2.3. Cardiac hypertrophy ..................................................................................... 17 2.2.3.1. Categories of LVH ................................................................................ 17 v 2.2.3.2. Myocyte hypertrophy ............................................................................ 18 2.2.3.3. Hypertrophic signaling .......................................................................... 21 2.3. Hypertrophy Stimuli ........................................................................................... 24 2.3.1. Myocardial injury ......................................................................................... 24 2.3.2. Hemodynamic overload ............................................................................... 25 2.3.3. Neurohormonal activation ........................................................................... 25 2.3.3.1. Angiotensin II ....................................................................................... 25 2.3.3.2. ET1 ........................................................................................................ 26 2.3.3.3. Norepinephrine...................................................................................... 27 2.4. Cardiac hypertrophy - interventions ................................................................... 28 3. Mitochondria ............................................................................................................ 29 3.1. Mitochondrial physiology ................................................................................... 30 3.1.1. Structure ......................................................................................................