Identification and Characterization of a Cardiomyopathy Syndrome Resulting from Loss of the Melanocortin 4 Receptor By Michael Litt Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Molecular Biology and Biophysics August 31, 2017 Nashville, Tennessee Approved: David Wasserman Ph.D. Roger Cone Ph.D. Owen McGuinness Ph.D. Danny Winder Ph.D. David Weaver Ph.D. To Papa Jay and Papa Duck. ii ACKNOWLEDGEMENTS Any perception of my success in graduate school is a direct reflection of the tremendous guidance and support that I have received. First and foremost, I would like to thank Roger Cone for his tireless mentorship and unwavering support. It takes an extremely special mentor to train a graduate student working on an unfunded side project. His ‘sink or swim’ style has given me a confidence I will draw on for the rest of my career. For that, I am forever indebted. I will always be grateful to the guidance of Dr. Javid Moslehi. His ability to provide honest and critical feedback has been sine qua non. His willingness to find the time to help me with my project while simultaneously starting a lab and clinical department are a true testament to his dedication as a mentor. I would like to extend the deepest of thanks to the members of my thesis committee: Dave Wasserman, Owen McGuinness, Danny Winder and Dave Weaver. Their astute and timely insights into my project have provided a sense of clarity to both my science and professional development. I would also like to thank Josh, Masoud, Isin, Max, Luis, my fellow cone lab graduate students, Donald and Savannah for all of the spectacular advice, friendship, and technical acumen. I owe a debt of gratitude to Roger Colbran and Richard O’Brien for their wiliness to work with me under these unique circumstances. Furthermore, I want to thank the entirety of MPB for providing a wonderful environment in which to develop as a scientist. Lastly, I would like to thank the entirety of the MSTP leadership iii team, including Chris Williams, Terry Dermody, Danny Winder, Larry Swift, Sally York, and Melissa Krassnove for supporting me throughout this time. To My Parents: Thank you for giving me a loving home and always believing in my dreams. Despite my many flaws, your confidence in my abilities has never wavered – even when I got that D- in spelling class. While it may have seemed like I was not listening, the countless words of wisdom you have given to/yelled at me over the years have shaped me into who I am today. To Becca: The ups and downs of graduate school have been your daily source of laughter and concern. You may never believe in things that are microscopic, but your belief in me has given me the strength to push through the low points. You are my rock. To Alex: Your competitive drive and passion are something I strive towards every day. Thank you for being patient with me and always pointing me in the right direction. Maybe one day I will be as cu as you. To my grandparents: Thank you for the trips to Panera, rounds at Sawgrass, art lessons and unending love. It has been my greatest joy to have you all in my life. To my uncles, aunts, and cousins: Thank you for visiting me in Nashville, following me down ski runs, and demonstrating interest in what I am up to. I love you all very much. Lastly I would like to thank all of my friends. You all have helped make Nashville an amazing place to live, work and have fun. Without you guys, I would have lost my mind far before I did. iv TABLE OF CONTENTS Page DEDICATION ................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF TABLES ...........................................................................................................viii LIST OF FIGURES ...........................................................................................................ix Chapter I. Background and significance ................................................................................ 1 The central melanocortin system ..................................................................... 1 The cloning of MC4R .......................................................................... 2 The role of MC4R signaling in feeding behavior ................................. 4 AgRP – The putative MC4R antagonist .............................................. 6 POMC & AGRP neurons .................................................................... 8 MC4R Neurons ................................................................................. 15 MC4R Signal Transduction ............................................................... 18 MC4R and human disease ............................................................... 22 MC4R and energy expenditure ......................................................... 25 MC4R and cardiovascular function .................................................. 28 Cardiomyopathy pathophysiology .................................................................. 30 Dilated Cardiomyopathy ................................................................... 32 Hypertrophic Cardiomyopathy .......................................................... 33 Restrictive Cardiomyopathy ............................................................. 35 Arrhythmogenic right ventricular dysplasia ....................................... 37 Unclassified Cardiomyopathies ........................................................ 37 Treatment of cardiomyopathy ........................................................................ 38 II. Loss of the MC4R leads to dilated cardiomyopathy ............................................ 45 Introduction .................................................................................................... 45 Results ........................................................................................................... 48 Cardiac Function in Male MC4R-/- Mice ........................................... 48 Cardiovascular Function in female MC4R-/- mice ............................ 49 Cardiovascular function of MC4R-loxTB+/+ mice ............................. 50 Comparison of MC4R-/- heart function to weight matched controls . 51 Expression profile of MC4R .............................................................. 53 Effect of MC4R Deletion on Myocardial structure ............................. 54 Summary and conclusions ............................................................................. 56 III. Characterization of mitochondrial function in mc4r-/- hearts ............................... 70 v Introduction .................................................................................................... 70 Results ........................................................................................................... 70 Cardiac mitochondria DNA and protein content of MC4R-/- mice .... 72 High resolution respirometry of diseased MC4R-/- myocardium ...... 73 Pre-diseased MC4R-/- myocardial respirometry .............................. 75 Characterization of MC4R-/- red gastrocnemius fibers .................... 76 Respirometry of ARCPOMC-KO myocardium .................................. 77 Potential role for ROS in MC4R-/- cardiomyocyte dysfunction ......... 78 Summary and conclusions ............................................................................. 81 IV. Discussion and Future Directions ........................................................................ 95 Hyperinsulinemia and cardiomegaly .............................................................. 95 Role of mitochondrial dysfunction in MC4R associated cardiomyopathy ...... 97 Understanding the mechanism of MC4R-/- cardiomyopathy. ...................... 100 MC4R heterozygosity and personalized medicine ....................................... 103 V. Methods ............................................................................................................ 105 Mouse husbandry ........................................................................................ 105 Mouse lines .................................................................................................. 106 Echocardiography ........................................................................................ 107 Glucose tolerance testing ............................................................................ 108 Transmission electron microscopy .............................................................. 109 Histology ...................................................................................................... 110 Western blotting ........................................................................................... 111 High resolution respirometry ........................................................................ 112 ROS assays ................................................................................................. 113 RNA isolation ............................................................................................... 113 RNA-seq ...................................................................................................... 114 qRT-PCR ..................................................................................................... 115 ATP assay ..................................................................................................