EXPLORATION OF THE MITOCHONDRIA AS A POTENTIAL THERAPEUTIC TARGET IN DUCHENNE MUSCULAR DYSTROPHY MEGHAN C HUGHES A DISSERTATION SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAM IN Kinesiology and Health Sciences YORK UNIVERSITY TORONTO, ONTARIO April 2019 Ó Meghan Hughes, 2019 Abstract Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease resulting from a mutation in the X-linked gene encoding the protein dystrophin. DMD is characterized by profound muscle weakness as degenerating muscle is replaced by fat and connective tissue. Early loss of ambulation followed by premature death due to cardiac and/or respiratory failure characterize the most debilitating aspects of DMD, a disease for which there is currently no cure. Limited success has been reported when treating DMD with gene based therapies. Current standard of care involves glucocorticoids, which target a secondary cellular myopathy; inflammation. While this line of treatment has provided promising benefits, these drugs present a variety of negative side effects for patients. As such, extensive research has been focused on identifying both therapeutic targets and corresponding novel therapies for the treatment of the DMD myopathy. The focus of this dissertation was to first determine the degree and precise mechanism of mitochondrial dysfunction in DMD followed by the evaluation of SBT-20, a mitochondrial- targeted peptide, as a therapeutic candidate for the treatment and prevention of DMD pathophysiology. In order to address these questions, we first comprehensively evaluated mitochondrial bioenergetics across a spectrum of oxidative and glycolytic muscles in the D2.B10- DMDmdx/2J mouse (D2.mdx) in early and late stages of disease progression. We then tested the efficacy of SBT-20 in improving both DMD myopathy in respiratory and skeletal muscles, as well as the underlying mechanism of mitochondrial dysfunction in dystrophic muscle. Our findings reveal that the mitochondrial H2O2 emission is elevated during impaired oxidative phosphorylation in cardiac, respiratory, oxidative and glycolytic muscle in young (4-week) and aged (52-week) D2.mdx mice and furthermore, that there is a specific defect in mtCK functionality in oxidative muscle. SBT-20 was effective in improving mitochondrial bioenergetics following ii short (4 weeks) and long (12 weeks) term treatment. This was associated with improved pathophysiology following short-term treatment. Taken together, this thesis identifies impairments in mitochondrial bioenergetics as a contributing factor to the pathophysiology in dystrophic muscle and further highlights SBT-20 as a promising therapy for the improvement of myopathy in DMD. iii Acknowledgements This thesis would not have been possible without the help and support from a variety of important individuals. First and foremost, I would like to thank my supervisor Dr. Chris Perry. May 2013 feels like a very long time ago at this point but it is enjoyable to look back and think about all that we have accomplished since that time. Thank you for your constant support, mentorship and guidance. Under your leadership, not only have I developed as an independent scientist, but my bad joke repertoire and fluid sense of time have been expanded immensely. While most of our meetings involved a series of eye rolls more often than not, I truly am grateful for everything you have helped me accomplish over the last 6 years. While a thank-you to express my gratitude doesn’t seem to suffice, a “Chris Sized” stein of beer and a framed copy of the quote board might start to cover it. Second, I would like to thank the Perry lab members, past and present. While things were certainly never dull (or quiet), I enjoyed coming to the lab each day and working with each of you. In particular, to Sofhia Ramos, we have spent enough hours together over the past 5 years to last a life time but I am incredibly grateful that it was you I spent those marathon experiment days with. While I can’t say I will miss spending holidays in the lab, our Easter feast following the Farquharson Flood of 2016 stands out as a favourite memory from the past 5 years with you. Looking forward to comparing our latest TV favourites out in the real world. To Patrick Turnbull (the Statler to my Waldorf?) thank you for always being my science sound board and my favourite person to debate sports with. While others may not have appreciated it as much as we did, our “hilarious” banter made day to day lab life much more enjoyable. My only regret from the past 5 years is how late in the game we discovered those $10 pitchers. I would also like to thank the amazing collaborators I have had the chance to work with during my time at York. Specifically, to Dr. Tom Hawke and the Hawke lab, working with and learning from you has been a true highlight of my PhD. To Tom in particular, thank you for your advice, support and contributions throughout my thesis. I really enjoyed hearing your perspectives, interpretations and insight on my data and your involvement in this thesis was pivotal, for which I am truly grateful. Finally, I would like to thank my parents, Bob and Louise Hughes. While you couldn’t really picture what day to day life was like in the Perry lab, you were always incredibly supportive and at least faked interest when I tried to talk science over dinner. Your unconditional love and support has meant the world during this long haul as a graduate student. I don’t think any of us will ever forget the Micky and Minnie Farewell Tour J. iv Table of Contents Abstract ii Acknowledgements iv Table of Contents v List of Tables vii List of Figures viii List of Supplemental Figures x List of Abbreviations xi 1 Introduction 1 2 Literature Review 4 2.1 Overview and Introduction to Duchenne muscular dystrophy 4 2.1.1 Overview of Duchenne muscular dystrophy 4 2.1.2 Genetic Nature of DMD 4 2.1.3 Current Therapeutic Strategies 6 2.1.4 Animal Models of DMD 8 2.1.4.1 Rodent Models of DMD 8 2.1.4.2 Canine Models of DMD 10 2.2 Primary Pathologic Outcomes in DMD 13 2.2.1 Skeletal Muscle Wasting 13 2.2.2 Respiratory Failure 15 2.2.3 Cardiomyopathy 17 2.3 Secondary Pathophysiologic Responses in DMD 18 2.3.1 Impaired Calcium Handling 18 2.3.2 Oxidative Stress 19 2.3.2.1 Oxidative Stress Overview 19 2.3.2.2 Evidence of ROS-induced Damage in DMD 21 2.3.2.3 Antioxidant Clinical Trials in DMD 22 2.3.2.4 Mechanisms of elevated ROS in DMD 24 2.3.3 Inflammation 26 2.4 Introduction to Mitochondrial Bioenergetics 29 2.4.1 Oxidative Phosphorylation 29 2.4.2 Mitochondrial Creatine Kinase 30 2.4.3 Mitochondrial Reactive Oxygen Species Production 33 2.4.4 Mitochondrial Induced Cell Death 37 2.4.4.1 Mitochondrial Ca2+ Handling 37 2.4.4.2 Mitochondrial Permeability Transition Pore 37 2.4.4.3 Mitochondrial-Derived Apoptosis 40 2.4.4.4 Mitochondrial-Derived Necrosis 41 2.5 Mitochondrial Function in Duchenne Muscular Dystrophy 42 2.5.1 Mitochondrial Oxidative phosphorylation 42 2.5.1.1 Creatine Impairments in DMD 43 2.5.2 Mitochondrial Reactive Oxygen Species Production 44 2.5.2.1 ADP as a Central Governor of Bioenergetics 45 2.5.2.2 Targeting Mitochondrial ROS in DMD 46 2.5.3 Mitochondrial Calcium Handling and Mitochondrial-Derived Cell Death 47 2.6 Mitochondrial Targeted Antioxidants 49 3 Objectives and Hypothesis 53 v 3.1 Overview of Thesis 53 3.2 Objective and Hypotheses for Study 1 (CHAPTER 4) 53 3.3 Objective and Hypotheses for Study 2 (CHAPTER 5) 53 3.4 Objective and Hypotheses for Study 3 (CHAPTER 6) 54 3.5 Objective and Hypotheses for Study 4 (CHAPTER 7) 54 3.6 Additional Contributions 56 Co-First Author - Published 56 Co-Author - Published 56 Co-First Author – In Progress 56 Co- Author – In Progress 57 4 Early myopathy in Duchenne muscular dystrophy is associated with elevated mitochondrial H2O2 emission during impaired oxidative phosphorylation 58 5 Impairments in left ventricular mitochondrial bioenergetics precede overt cardiac dysfunction and remodelling in Duchenne muscular dystrophy 108 6 The mitochondrial targeted peptide SBT-20 improves diaphragm and skeletal muscle pathophysiology in dystrophin-deficient mice 141 7 Mitochondrial creatine kinase deficits evident in Duchenne muscular dystrophy are ameliorated through treatment with cardiolipin-targeting peptide SBT-20 189 8 Summary of Findings 233 8.1 General Discussion and Future Directions 233 8.2 Limitations 241 8.3 Conclusions 243 References 245 Appendix A - Detailed Experimental Methods 272 A.1 - SkyScan 1278 – Scanning an Animal 272 A.2 - SkyScan 1278 µCT - Analyzing Hind-limb Muscle Volume 273 A.3 - SkyScan 1278 µCT - Analyzing % Body Fat 276 A.4 - In Vivo Hind-limb Plantarflexor Force Production 278 A.5 - In Situ Quadriceps Force Production 280 A.6 - In vitro Diaphragm Strip Force Production 281 A.7 - Serum Creatine Kinase 283 A.8 Measuring Mitochondrial Respiration 284 A.9 Measuring Mitochondrial H2O2 Emission 294 A.10 Measuring Calcium Retention Capacity 299 A.11 Caspase Activity Assay 301 A.12 Western Blotting 303 A.13 Evaluating Redox Status of Specific Protein 307 A.14 Evan’s Blue Dye 311 A.15 Glutathione 312 vi List of Tables Chapter 2 Table 2-1: Disease progression in DMD. .....................................................................................................................6 Table 2-2: Comparison of commonly used animal models of DMD. .....................................................................12 Table 2-3: Sites of Mitochondrial Superoxide Production. .....................................................................................34 Table 2-4: Comparison of Mitochondrial-Derived Apoptosis and Necrosis.