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Contractile Dysfunction in Heart Failure and Familial Hypertrophic Cardiomyopathy

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Contractile Dysfunction in Heart Failure and Familial Hypertrophic Cardiomyopathy CONTRACTILE DYSFUNCTION IN HEART FAILURE AND FAMILIAL HYPERTROPHIC CARDIOMYOPATHY By YI-HSIN CHENG Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Adviser: Dr. Julian E. Stelzer Department of Physiology and Biophysics CASE WESTERN RESERVE UNIVERSITY January, 2014 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of __________Yi-Hsin Cheng ________________________________ candidate for the _________Doctoral_______________________degree *. (signed)__________Corey Smith_______________________ (chair of the committee) ___________Julian Stelzer ______________________ ___________Thomas Nosek _____________________ ___________David Van Wagoner _________________ ___________Brian Hoit _________________________ ___________Xin Yu ___________________________ (date) ______September 10th, 2013________________ *We also certify that written approval has been obtained for any proprietary material contained therein. ii DEDICATION To my family overseas, who have supported me and allowed me to pursue anything I want. To my boyfriend, who has helped me through the conflicts and struggles due to the nature of this work and led me on the right path. To my vegan mentors and community, who continue to give me hope. iii TABLE OF CONTENTS List of Tables ix List of Figures x Acknowledgements xii List of Abbreviations xv Abstract xx Chapter 1: Cardiac Health and Disease 22 1.1 Introduction 22 1.2 Cardiac Structure and Functions 23 1.2.1 Heart Structure and Functions 23 1.2.2 Cardiomyocyte and Myofilament Structure and Functions 26 1.2.3 The Electrical Properties of the Heart 37 1.2.4 Ca2+ Handling 39 1.2.5 Autonomic and -adrenergic Regulation 41 1.3 Cardiac Metabolism 44 1.4 Cardiac Dysfunction 47 1.4.1 LV Dysfunction 47 1.4.2 LV Remodeling 48 1.4.3 Heart Failure 51 1.4.4 Familial Hypertrophic Cardiomyopathy 59 1.5 Rationale and Hypothesis 66 iv Chapter 2: Changes in Myofilament Proteins, but not Calcium Regulation, are Associated with a High Fat Diet-induced Improvement in Contractile Function in Heart Failure 68 2.1 Introduction 69 2.2 Materials and Methods 71 2.2.1 Experimental Model 71 2.2.2 Plasma Metabolic Substrates 72 2.2.3 Echocardiography 72 2.2.4 Hemodynamic Measurements 73 2.2.5 Histological Assessment of Cardiac Morphology 73 2.2.6 Cardiomyocyte Isolation 74 2.2.7 In Vitro Cardiomyocyte Shortening and Ca2+ Transients 75 2.2.8 Protein Expression by Western Blot Analysis 76 2.2.9 L-type Ca2+ Current Measurements 77 2.2.10 Myosin Heavy Chain Protein Expression 77 2.2.11 Myofilament Protein Phosphorylation and Expression 78 2.2.12 Statistics 79 2.3 Results 79 2.3.1 Body Weight and Metabolic Substrates 79 2.3.2 Cardiac Morphology 80 2.3.3 Echocardiography and Hemodynamic Function 81 2.3.4 Cell Shortening 83 2.3.5 Ca2+ Transients 83 v 2.3.6 Ca2+ Regulatory Protein Expression 86 2.3.7 Correlations between in Vivo and in Vitro Function 86 2.3.8 ICa 88 2.3.9 Myofilament Protein Composition and Phosphorylation 89 2.4 Discussion 90 Chapter 3: Impaired Contractile Function Due to Decreased Cardiac Myosin Binding Protein C Content in the Sarcomere 96 3.1 Introduction 97 3.2 Materials and Methods 100 3.2.1 Ethical Approval and Experimental Model 100 3.2.2 Echocardiography 100 3.2.3 Hemodynamic Measurements 101 3.2.4 Electrocardiogram 101 3.2.5 High Resolution Optical Mapping 102 3.2.6 Histological Assessment of Cardiac Morphology 103 3.2.7 Myocyte Isolation and in Vitro Shortening and Ca2+ Transients 103 3.2.8 Myofilament Contractile Function 104 3.2.9 Quantification of Protein Expression and Phosphorylation 109 3.2.10 Phosphatase Activity Assay 112 3.2.11 Statistical Analysis 112 3.3 Results 112 (Comparison between WT and MyBP-C−/− Models) 112 3.3.1 Myofilament Protein Content and Phosphorylation 112 vi 3.3.2 Isolated Myocyte Contractile Properties 114 3.3.3 Myocyte Ca2+ Handling Properties 116 3.3.4 Expression of Ca2+ Handling Proteins and PP1 Activity 116 3.3.5 Echocardiography 118 3.3.6 In Vivo Hemodynamic Function at BL and Following DOB Challenge 119 3.3.7 Optical Mapping and Cx43 Protein Expression and Phosphorylation 121 3.3.8 ECG 123 (Comparison between WT and MyBP-C+/− Models) 124 3.3.9 Myofilament Protein Content and Phosphorylation 124 3.3.10 Steady-state Force and Dynamic Cross-bridge Kinetics Prior to and Following PKA Treatment 126 3.3.11 Isolated Myocyte Contractile Properties 131 3.3.12 Myocyte Ca2+ Handling Properties 132 3.3.13 Expression and Phosphorylation of Ca2+ Handling Proteins 133 3.3.14 In Vivo Hemodynamic Function 134 3.3.15 Echocardiography 135 3.3.16 Histological Assessment of Cardiac Morphology 136 3.3.17 ECG 137 3.4 Discussion 140 3.4.1 In Vitro Contractile Function and Ca2+ Handlingin MyBP-C−/− Myocytes 142 3.4.2 In Vivo Contractile and Hemodynamic Function in MyBP-C−/− Hearts 144 3.4.3 Electrical Conduction and ECG Recordings in MyBP-C−/− Hearts 145 vii 3.4.4 Myofilament Contractile Function in MyBP-C+/− Hearts 146 3.4.5 In Vitro Contractile Function and Ca2+ Handlingin MyBP-C+/− Myocytes 151 3.4.6 In Vivo Contractile and Hemodynamic Function in MyBP-C+/− Hearts 152 3.4.7 ECG Recordings in MyBP-C+/− Hearts 154 Chapter 4: Summary and Future Directions 156 4.1 Summary and Future Directions of “Changes in Myofilament Proteins, but not Calcium Regulation, are Associated with a High Fat Diet-induced Improvement in Contractile Function in Heart Failure” 156 4.1.1 Potential Factors Contributing to MHC Changes 156 4.1.2 Other Potential Mechanisms for Improved Contractility in HFSAT Animals 160 4.1.3 Limitations 165 4.1.4 Fat vs. Carbohydrate 167 4.2 Summary and Future Directions of “Impaired Contractile Function Due to Decreased Cardiac Myosin Binding Protein C Content in the Sarcomere” 170 4.2.1 Regional Differences in Expression of MyBP-C 171 4.2.2 Cardiac Energetic Alterations 174 4.2.3 Autonomic Regulations 175 4.2.4 Beyond Mutations? 178 Reference List 181 viii LIST OF TABLES Table 1-1. Summary of Hypertrophic Cardiomyopathy Susceptibility Genes 61 Table 2-1. Plasma Substrates, Echocardiography and Hemodynamic Function 80 Table 3-1. Gravimetric Measurements and in Vivo LV Function by Echocardiography between WT and MyBP-C−/− Models 119 Table 3-2. LV Hemodynamic Function at BL and Following -adrenergic Stimulation between WT and MyBP-C−/− Models 120 Table 3-3. ECG at BL and Following -adrenergic Stimulation between WT and MyBP-C−/− Models 123 Table 3-4. Steady-state Mechanical Properties of Skinned Fibers Isolated from WT and MyBP-C+/− Myocardium 127 Table 3-5. Cross-bridge Kinetics of Skinned Fibers Isolated from WT and MyBP-C+/− Myocardium 129 Table 3-6. LV Hemodynamic Function at Baseline and Following -adrenergic Stimulation between WT and MyBP-C+/− Models 135 Table 3-7. LV Morphology and in Vivo Function Measured by Echocardiography between WT and MyBP-C+/− Models 136 Table 3-8. Electrocardiographic Data Acquired by Radio Telemetry between WT and MyBP-C+/− Models 138 Table 3-9. Comparisons between MyBP-C−/− and MyBP-C+/− Models. 139 Table 4-1. A List of Pathophysiological Stimuli Controlling mRNA and/or Protein Expression of Cardiac MHC Isoforms 158 ix LIST OF FIGURES Figure 1-1. LV Pressure-volume Loop 24 Figure 1-2. Structure of the Sarcomere 27 Figure 1-3. Components of the Filaments 28 Figure 1-4. Cardiac Muscle Cross-bridge Cycling 29 Figure 1-5. Slack-restretch and stretch activation responses 33 Figure 1-6. General Domain Structure of Mouse MyBP-C 36 Figure 1-7. Cardiac Ionic Currents, Action Potentials, and ECGs in Humans versus Mice 38 Figure 1-8. Ca2+ Transport in Ventricular Myocytesduring ECC 40 Figure 1-9. Determination of Resting Cardiac Parasympathetic and Sympathetic Tone 44 Figure 1-10. Cardiac Energy Metabolism 46 Figure 1-11. Patterns of LV Remodeling Based on EDV, Wall Mass, and RWT 50 Figure 2-1. Histological Assessment of Infarct Size and Myocyte Cross-sectional Area 82 Figure 2-2. LV Cardiomyocyte Shortening 84 Figure 2-3. LV Cardiomyocyte Ca2+ Regulation 85 Figure 2-4. Correlations between in Vivo and in Vitro Functional Measurements 87 2+ Figure 2-5. L-type Ca Currents (ICa) 88 Figure 2-6. Myofilament Protein Composition and Phosphorylation 89 Figure 3-1. Stretch Activation Responses in Murine Myocardium 108 x Figure 3-2. Protein Expression and Phosphorylation of Myofibrillar Proteins between WT and MyBP-C−/− Models 113 Figure 3-3. Ventricular Cardiomyocyte Sarcomere Shortening between WT and MyBP-C−/− Models 115 Figure 3-4. Ventricular Cardiomyocyte Ca2+ Transients between WT and MyBP-C−/− Models 117 Figure 3-5. Protein Expression of Ca2+ Handling Proteins and PP1 Activity 118 Figure 3-6. Optical Mapping and Protein Expression of Cx43 122 Figure 3-7. Expression and Phosphorylation of Myofilament Proteins between WT and MyBP-C+/− Models 125 Figure 3-8. Rate of Force Development and Fiber Stiffness 130 Figure 3-9. Ventricular Cardiomyocyte Sarcomere Shortening between WT and MyBP-C+/− Models 132 Figure 3-10. Ventricular Cardiomyocyte Ca2+ Transients between WT and MyBP-C+/− Models 133 Figure 3-11. Morphology of the Hearts and Cardiomyocytes 137 Figure 3-12. ECG Waveforms 138 Figure 4-1. Regional Differences in Expression of MyBP-C 173 Figure 4-2. Sympathetic and Parasympathetic Tone 176 xi ACKNOWLEDGEMENTS I have a special thank you for Cathy Carlin, who protected me through a conflict with a faculty member during my very first week in the program. I have followed her advice ever since and found it helpful, and I want to thank her for that.
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