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Haploinsufficiency of Cardiac Myosin Binding Protein-C in the Development of Hypertrophic Cardiomyopathy

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Haploinsufficiency of Cardiac Myosin Binding Protein-C in the Development of Hypertrophic Cardiomyopathy Loyola University Chicago Loyola eCommons Dissertations Theses and Dissertations 2014 Haploinsufficiency of Cardiac Myosin Binding Protein-C in the Development of Hypertrophic Cardiomyopathy David Barefield Loyola University Chicago Follow this and additional works at: https://ecommons.luc.edu/luc_diss Part of the Physiology Commons Recommended Citation Barefield, David, "Haploinsufficiency of Cardiac Myosin Binding Protein-C in the Development of Hypertrophic Cardiomyopathy" (2014). Dissertations. 1249. https://ecommons.luc.edu/luc_diss/1249 This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Dissertations by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 2014 David Barefield LOYOLA UNIVERSITY CHICAGO HAPLOINSUFFICIENCY OF CARDIAC MYOSIN BINDING PROTEIN-C IN THE DEVELOPMENT OF HYPERTROPHIC CARDIOMYOPATHY A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL IN CANDIDACY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY PROGRAM IN CELL AND MOLECULAR PHYSIOLOGY BY DAVID YEOMANS BAREFIELD CHICAGO, IL AUGUST 2014 Copyright by David Yeomans Barefield, 2014 All Rights Reserved. ii ACKNOWLEDGEMENTS The completion of this work would not have been possible without the support of excellent mentors, colleagues, friends, and family. I give tremendous thanks to my mentor, Dr. Sakthivel Sadayappan, who has facilitated my growth as a scientist and as a human being over the past five years. I would like to thank my dissertation committee: Drs. Pieter de Tombe, Kenneth Byron, Leanne Cribbs, Kyle Henderson, and Christine Seidman for their erudite guidance of my project and my development as a scientist. I would like to thank the Department of Cell and Molecular Physiology for seeing some potential in me and for giving me the opportunity to grow scientifically and personally. I thank Dr. Diederik Kuster and Dr. Ramzi Khairallah for their mentorship and motivation. I am grateful for the support and camaraderie of my lab mates, Dr. Suresh Govindan, Dr. Xiang Ji, Brian Lin, and Thomas Lynch. I am truly lucky that I entered this program with Stefan Mazurek, with whom I have had the privilege of weathering all the trials of the past five years. I also would like to acknowledge all of the excellent people who shared a lunch table with me for providing a daily respite; I am both sorry and grateful that there are too many of you to name. iii Finally, I thank my wife Lisa, whose decade of partnership and support has made all this possible. I am also deeply thankful for the endless support of my parents, Robert and Anne Barefield, my sister Rose, and my brother Mark. iv For My Wife and Parents TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................... iii LIST OF TABLES ............................................................................................... ix LIST OF FIGURES ............................................................................................... x LIST OF ABBREVIATIONS ............................................................................... xii CHAPTER ONE: INTRODUCTION ...................................................................... 1 CHAPTER TWO: LITERATURE REVIEW ........................................................... 3 Cardiovascular Physiology in Health and Disease ...................................... 3 The Healthy and Sick Heart ........................................................................... 5 Cardiac Anatomy and Function ..................................................................... 6 The Pressure-Volume Relationship of the Left Ventricle ............................. 12 Electrical Regulation of Contraction ............................................................ 16 Regulation of Cardiac Function ................................................................... 18 Excitation-Contraction Coupling .................................................................. 19 Molecular Mechanisms of Sarcomere Contraction ...................................... 22 Etiology of Hypertrophic Cardiomyopathy ................................................... 24 Heart Failure ................................................................................................ 31 HCM: A Disease of the Cardiac Sarcomere ................................................ 34 The Z-Disks ................................................................................................. 36 The Thick Filament ...................................................................................... 37 The Thin Filament ....................................................................................... 41 Regulation of Sarcomere Function .............................................................. 42 Sarcomere Protein Mutations ...................................................................... 44 The Poison Polypeptide Mechanism ........................................................... 46 The Haploinsufficiency Mechanism ............................................................. 47 Other Pathological Mechanisms in Sarcomere HCM Mutations .................. 48 Discovery of New HCM Linked Mutations ................................................... 48 Cardiac Myosin Binding Protein-C .............................................................. 50 Molecular Structure and Localization of cMyBP-C ...................................... 52 Phosphorylation Mediated Regulation ......................................................... 53 Experimental Investigation of MYBPC3 Mutations in HCM ......................... 58 vi Mouse models of MYBPC3 mutations ......................................................... 62 CHAPTER THREE: AIMS AND HYPOTHESIS ................................................. 66 CHAPTER FOUR: CONTRACTILE DYSFUNCTION IN HETEROZYGOUS MYBPC3 MICE ................................................................................................. 68 Abstract ......................................................................................................... 68 Introduction ................................................................................................... 69 Results ........................................................................................................... 71 Reduced MYBPC3 Transcript with Preserved cMyBP-C in Het Hearts ....... 71 Phosphorylation of cMyBP-C Regulates Sarcomere Function. ................... 74 Heterozygous Mice do not Exhibit Gross Morphological Changes .............. 76 Heterozygous Cardiomyocytes Show Reduced Force Generation .............. 78 MYBPC3 Heterozygous Mice Have Altered Cardiac Function in vivo ......... 80 Discussion ..................................................................................................... 86 CHAPTER FIVE: HAPLOINSUFFICIENCY OF MYBPC3 IN THE DEVELOPMENT OF HYPERTROPHIC CARDIOMYOPATHY Abstract ......................................................................................................... 93 Introduction ................................................................................................... 94 Results ........................................................................................................... 95 Increased Hypertrophy in Heterozygous Mice Following TAC ..................... 95 Gene Expression Profile Shows Hypertrophy Post-TAC ............................. 99 Decreased cMyBP-C Content in Heterozygous Sarcomeres .................... 101 Reduced Contractility in Het Cardiomyocytes ........................................... 106 Reduced Systolic Function in Heterozygous Hearts Post-TAC ................. 115 Alterations in Gene Expression in Heterozygous Hearts. .......................... 121 Discussion ................................................................................................... 130 CHAPTER SIX: TURNOVER OF cMyBP-C..................................................... 137 Introduction ................................................................................................. 137 Results ......................................................................................................... 139 Knock-Down of MYBPC3 .......................................................................... 139 Determination of Gene Expression on Protein Turnover ........................... 142 MYBPC3 Gene Dosage Directly Alters Force Development ..................... 144 Discussion ................................................................................................... 147 CHAPTER SEVEN: FUTURE DIRECTIONS AND CONCLUSIONS ............... 148 Altered Function with Normal Protein Level ............................................. 148 vii Haploinsufficiency vs Poison Polypeptide ............................................... 149 Decreased Transcript as a Mechanism of Dysfunction ........................... 151 Mutations as Modifiers of Cardiovascular Disease ................................. 153 Conclusion .................................................................................................. 154 CHAPTER EIGHT: METHODS AND MATERIALS .......................................... 155 Mouse model of MYBPC3 haploinsufficiency .......................................... 155 In vivo TAC in Mice ....................................................................................
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