Molecular physiology of ankyrin-G in the heart: Critical regulator of cardiac cellular excitability and architecture. DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Michael Anthony Makara Graduate Program in Integrated Biomedical Science The Ohio State University 2016 Dissertation Committee: Professor Peter J. Mohler, Ph. D, Advisor Professor Noah L. Weisleder, Ph. D Professor Thomas J. Hund, Ph. D Professor Philip F. Binkley, M.D. Copyright by Michael Anthony Makara 2016 Abstract Cardiovascular disease is the leading cause of death in the United States, claiming nearly 800,000 lives each year. Regardless of the underlying cardiovascular dysfunction, nearly 50% of these patients die of sudden cardiac arrest caused by arrhythmia. Development and sustainment of cardiac arrhythmia begins with dysfunction of excitability and structure at the cellular level. Therefore, in order to improve therapeutic options for these patients, a basic understanding of the molecular mechanisms regulating cardiac cellular excitability and structure is required. Decades of research have demonstrated that intracellular scaffolding polypeptides known as ankyrins are critical for the regulation of cellular excitability and structure in multiple cell types. Ankyrin-G (ANK3) is critical for regulation of action potentials in neurons and lateral membrane development in epithelial cells. Given its central importance for cellular physiology in excitable and non-excitable cell types, we hypothesized that functional ankyrin-G expression is critical for proper cardiac function. To test this hypothesis in vivo, we generated cardiac-specific ankyrin-G knockout (cKO) mice. In the absence of ankyrin-G, mice display significant reductions in membrane targeting of the voltage-gated sodium channel Nav1.5. This disruption in turn causes severely reduced whole cell sodium current, leading to significant conduction abnormalities, bradycardia, and ventricular arrhythmia and atrioventricular nodal block following infusion of NaV channel ii antagonists. In addition to regulating cardiac excitability, we also demonstrate a critical role for ankyrin-G in the regulation of the cardiomyocyte cytoarchitecture. Specifically, ankyrin-G cKO mice show disrupted cellular distribution of the desmosomal protein plakophilin-2 (PKP2) at baseline. In a setting of pressure overload-induced heart failure we observed severe disruptions to the cellular localization of PKP2. Further, as desmosomes mediate the integration of the intermediate filament protein desmin, we demonstrate the reduced expression of desmin at the intercalated disc (ID) in the setting of mislocalized PKP2. Mechanistically, we correlate these molecular changes with significant reductions in systolic function and increased propensity for bradyarrhythmia in ankyrin-G cKO mice following transverse aortic constriction (TAC). As ankyrin-G is significantly increased two weeks post TAC, we hypothesize that ankyrin-G expression is required for the early, compensatory phase of ventricular remodeling. Our hypothesis is further strengthened by the observation that functional ankyrin-G expression is severely reduced in multiple forms of human heart failure. We conclude that ankyrin-G is a critical regulator of both excitability and molecular architecture of the intercalated disc. We further hypothesize that remodeling of this ankyrin-G-dependent molecular environment is a critical step in the development of human arrhythmia and structural heart diseases. iii Dedication: To my parents, Ray and Trish, for their unending love, support, and wisdom throughout my life. Nothing I have accomplished would ever have been possible without you. From the bottom of my heart, I am so thankful that I have two such wonderful parents. Love you so much, Mike iv Acknowledgments After six months of late nights, extensive reading, and countless revisions, my dissertation is nearing completion. Throughout this whole process, my focus was locked on the daunting task of trying to condense an incredible expanse of information into a coherent story with specific hypotheses, results with significant scientific merit, and conclusions presented in the context of the current literature. And now, after adding the finishing touches and final references, I find myself sitting at my computer, reflecting on this whole process. The only thing that I can think at this point is how incredibly thankful I am to have such a wonderful group of family, friends, coworkers, and mentors in my life. Therefore, I would be remiss if I did not take this opportunity to sincerely thank all of you. To my parents, Ray and Trish. Words cannot possibly begin to describe the love and support that you have given me throughout my life. Mom and Dad, I dedicated this work to you because I truly would not be the person I am today without having such incredible people to call parents. For 26 years, you have been a constant source of love and understanding for which I am eternally grateful. I count myself blessed to be your son and I hope that I have made you proud to call yourselves my parents. To my brother David. Thank you for the love, the laughs, and all the good times. Without fail, you are able to brighten even my darkest day with your larger-than-life personality. I am so proud and thankful to have such an extraordinary person for a brother. v To my (at this point in time) fiancé Ting-Ting. With every day that passes, I realize how lucky I am to have fallen in love with such an amazing person. You have brought such happiness to my life, more happiness than one could ever hope to wish for. Thank you for being an untiring source of love, friendship, and compassion in my life and for being so supportive throughout this process. Although I do not know what life has in store for us, I do know that the only place I want to be is right by your side all the days of my life. I love you so much, Ting-Ting. To my family and friends. It is no exaggeration when I say that I have the best people I know looking out for me. I thank all of you for your unwavering love and friendship that has made my life so wonderful. I am forever grateful to have such an amazing group of people that I can call my friends and family. To my thesis committee, Noah Weisleder, Tom Hund, and Philip Binkley. I sincerely appreciate all the guidance and mentorship that you have provided me over these past four years. Your input has been invaluable to the advancement of my project, my scientific understanding, and my professional career. Sincerely, I could not wish for a better group of scientists or a better group of people to chair my thesis committee. Lastly, to my mentor Peter Mohler. Four years goes by pretty quick, doesn’t it? From the bottom of my heart, thank you so much for everything you have done for me on both a professional and personal level. Thank you for constantly demanding my best in lab and for challenging me to always ask and answer the tough questions in my research. I have learned so much from you over these past four years and I am incredibly thankful to have you as a mentor. vi Vita May 2007 .......................................................Canfield High School May 2011 .......................................................B.S. Biology, Youngstown State University July 2011 to Present ......................................Graduate Research Associate, Department of Physiology and Cell Biology, The Ohio State University Publications 1. Little SC, Curran J, Musa H, Kline CF, Makara MA, Higgins J, Hund TJ, Mohler PJ. Protein phosphatase 2A regulatory subunit B56α limits phosphatase activity in the heart. Sci Signal 2015; 386. 2. Curran J, Musa H, Kline CF, Makara MA, Little SC, Higgins JD, Hund TJ, Band H, Mohler PJ. Eps15 Homology Domain-containing Protein 3 Regulates Cardiac T-type Ca2+ Channel Targeting and Function in the Atria. J Biol Chem 2015. Epub ahead of print. 3. Curran J, Makara MA, Mohler PJ. Endosome-based protein trafficking and Ca(2+) homeostasis in the heart. Front Physiol. 2015; 6: 34. vii 4. Smith SA, Sturm, AC, Curran J, Kline CF, Little SC, Bonilla IM, Long VP, Makara M, Polina I, Hughes LD, Webb TR, Wei Z, Wright P, Voigt N, Bhakta D, Spoonamore KG, Zhang C, Weiss R, Binkley PF, Janssen PM, Kilic A, Higgins RS, Sun M, Ma J, Dobrev D, Zhang M, Carnes CA, Vatta M, Rasband MN, Hund TJ, Mohler PJ. Dysfunction in the βII spectrin-dependent Cytoskeleton Underlies Human Arrhythmia. Circulation 2015; 131: 695-708. 5. Radwański PB, Brunello L, Veeraraghavan R, Ho H, Lou Q, Makara MA, Belevych AE, Anghelescu M, Priori SG, Volpe P, Hund TJ, Janssen PM, Mohler PJ, Bridge JH, Poelzing S, Györke S. Neuronal Na+ Channel Blockade Suppresses Arrhythmogenic Diastolic Ca2+ Release. Cardiovasc Res. 2014; 262. 6. Chang KJ, Zollinger DR, Susuki K, Sherman DL, Makara MA, Brophy PJ, Cooper EC, Bennett V, Mohler PJ, Rasband MN. Glial ankyrins facilitate paranodal axoglial junction assembly. Nat. Neurosci. 2014; 17: 1673-168. 7. Makara MA, Curran J, Little SC, Musa H, Polina I, Smith SA, Wright PJ, Unudurthi SD, Snyder JS, Bennett V, Hund TJ, Mohler PJ. Ankyrin-G coordinates intercalated disc signaling platform to regulate cardiac excitability in vivo. Circulation Research. 2014; 115: 929-38. 8. Curran J, Makara MA, Little SC, Musa H, Liu B, Wu X, Polina I, Alecusan JS, Wright P, Li J, Billman GE, Boyden PA, Gyorke S, Band H, Hund TJ, Mohler PJ. Ehd3-dependent endosome pathway regulates cardiac