Characterization of TRP Ion Channels in Cardiac Muscle A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy By Spencer R. Andrei May 2017 © Copyright All rights reserved Except for previously published materials Dissertation written by Spencer R. Andrei B.S., University of Mount Union, 2012 Ph.D., Kent State University, 2017 Approved by _____________________, Chair, Doctoral Dissertation Committee Derek S. Damron, Ph.D. _____________________, Member, Doctoral Dissertation Committee Ian N. Bratz, Ph.D. _____________________, Member, Doctoral Dissertation Committee Colleen Novak, Ph.D. _____________________, Member, Doctoral Dissertation Committee Soumitra Basu, Ph.D., MBA _____________________, Graduate Faculty Representative Hanbin Mao, Ph.D. Accepted by _____________________, Director, School of Biomedical Sciences Ernest J. Freeman, Ph.D. _____________________, Dean, College of Arts and Sciences James L. Blank, Ph.D. Table of Contents LIST OF FIGURES……………………………………………………………………...v LIST OF TABLES……………………………………………………………………..vii LIST OF ABBREVIATIONS…………………………………………………………viii ACKNOWLEDGMENTS………………………………………………………..…..….x CHAPTER ONE: BACKGROUND........……………………………………………...1 Heart Failure Epidemiology…………………………………………………….1 Contractile Machinery of the Heart……………………………………………2 The Cardiac Cycle………………………………………………………3 Ventricular Cardiomyocytes……………………………………………6 Cross-Bridge Cycling and the Sliding Filament Theory……………7 Excitation-Contraction Coupling…………………………………….10 2+ [Ca ]i and Myofilament Sensitivity in Myocardial Contractility Regulation…………………………………………………………..…16 Heart Failure Pathophysiology…………………………………………..….17 Current Treatment Modalities of Heart Failure…………………….21 TRP Ion Channels Super Family……………………………………………22 TRPA1………………………………………………………………….25 TRPV1……………………………………………………………...…..26 TRPA1 and TRPV1 Interactions………………………………...…..26 TRP Channels and the Cardiovascular System………...………..27 iii Summary of TRPA1 and TRPV1 in Heart Failure…………………29 CHAPTER TWO: TRPA1 is functionally co-expressed with TRPV1 in cardiac muscle: Co-localization at z-discs, costameres and intercalated discs...…31 Introduction..……………………………………………………………………31 Materials and Methods………………………………………………………..34 Results………………………………………………………………………….41 Discussion………………………………………………………………………56 CHAPTER THREE: Stimulation of TRPA1 and TRPV1 Ion Channels Increase Intracellular Ca2+ Transients and Contraction in Mouse Ventricular Myocytes……………………………………………………………………………….65 Introduction.…………………………………………………………………….65 Materials and Methods ……………………………………………………….67 Results………………………………………………………………………….72 Discussion…………………………………………………………………...…96 CHAPTER FOUR: The role of TRPA1 in myocardial infarction (MI) and ischemia-induced cell death……………………………………………………...103 Introduction…………………………………………………………………...103 Materials and Methods………………………………………………………105 Results………………………………………………………………………...110 Discussion…………………………………………………………………….118 CHAPTER FIVE: CONCLUSIONS………………………………………………...127 REFERENCES……………………………………………………………………….129 iv List of Figures Figure 1. Wigger’s diagram…………………………...…………………………...…5 Figure 2. Structural Arrangement of Contractile Filaments in a Cardiac Myofibril and Sarcomere……………………………………………………………..8 Figure 3. Myosin Cross-bridge Cycling During a Normal Contraction Cycle…..11 Figure 4. Ca2+ Cycling During Contraction and Relaxation in a Cardiomyocyte…………………………………………………………….14 Figure 5. A Topological Structure of TRP Channels……………………………..24 Figure 6. TRPA1 and TRPV1 are expressed in CMs obtained from wild-type (WT) mice………………………………………………………………….42 Figure 7. TRPA1 and TRPV1 colocalize throughout the different layers of cardiac muscle………………………………………………………….…………..44 Figure 8. TRPA1 and TRPV1 localize at the costameres and Z-discs in cardiac myofibers…………………………………………………………….…….45 Figure 9. TRPA1 and TRPV1 colocalize at the Z-disc, costameres and intercalated discs in CMs………………………………………….……..48 Figure 10. TRPA1 and TRPV1 stimulation elicits transient increases in intracellular free calcium concentration in quiescent CMs……….....51 Figure 11. AITC and capsaicin induce dose-dependent increases in intracellular free calcium concentration in WT CMs through mechanisms dependent upon TRPA1 and TRPV1, respectively………………..….54 2+ Figure 12. Allyl isothiocyanate (AITC) increases [Ca ]I and shortening in CMs………………………………………………………………………...73 Figure 13. AITC increases fractional shortening, maximum velocity of shortening and maximum velocity of relengthening in CMs………………………75 v 2+ 2+ Figure 14. AITC increases peak [Ca ]I and accelerates time to peak [Ca ]I and 2+ the rate of [Ca ]I decay in CMs…….…………………………………...78 2+ Figure 15. Capsaicin increases [Ca ]I and contractile function in CMs…….…...82 2+ Figure 16. AITC has no effect on [Ca ]I and shortening in CMs obtained from TRPA1 null mice……………………………………….………………….85 2+ Figure 17. Capsaicin has no effect on [Ca ]I and shortening in CMs obtained from TRPV1 null mice……………………….……………………………88 2+ Figure 18. Treatment with HC030031 or SB366791 Does Not Alter [Ca ]i Dynamics or Contractile Function in CMs……………………………..91 Figure 19. TRPA1 activation with AITC dose-dependently increases ejection fraction in wild-type murine hearts……………………………………...95 Figure 20. TRPA1 gene deletion leads to exaggerated scar formation following myocardial infarction in mice……………….…………………………..111 Figure 21. TRPA1-/- mice exhibit deteriorated cardiac function following MI………………………………………………………….………………114 Figure 22. AITC attenuates ischemia-induced CM cell death…........................116 vi List of Tables Table 1. Comparison of AITC-, capsaicin- and ISO-induced changes in CM 2+ [Ca ]i and contractile function…………………………………………..93 Table 2. TRPA1-/- mice exhibit deteriorated cardiac function following MI……..114 vii List of Abbreviations ACEi – Angiotensin converting enzyme inhibitor ADP – Adenosine diphosphate AITC – Allyl isothiocyanate AngII – Angiotensin II ATP – Adenosine triphosphate β-AR – Beta-adrenergic receptor 2+ Ca - Calcium ion 2+ [Ca ]I – Intracellular free calcium concentration CA – Cinnamaldehyde cAMP – cyclic adenosine monophosphate CICR – Calcium-induced calcium release CM – Adult mouse ventricular cardiomyocyte DRG – Dorsal root ganglion ECC – Excitation-contraction coupling ECG – Electrocardiogram eNOS – endothelial nitric oxide synthase HF – Heart failure HFpEF – Heart failure with preserved ejection fraction HFrEF – Heart failure with reduced ejection fraction ISO - Isoproterenol K+ - Potassium ion viii LAD – left anterior descending artery LTCC – L-type calcium channel LV – Left ventricle MCU – Mitochondrial calcium uniporter MI – Myocardial infarction MLC2 – Myosin light chain 2 Na2+ - Sodium ion NCX – Sodium/calcium exchanger NO – Nitric oxide PKA – Protein kinase A PKCε – Protein kinase C epsilon PLB – Phospholamban RAAS – Renin-angiotensin-aldosterone system RYR – Ryanodine receptor SERCA – Sarcoplasmic reticulum calcium ATPase SNS – Sympathetic Nervous System SR – Sarcoplasmic reticulum Tn(C, I, T) – Troponin (C, I, T) TRPA1 – Transient receptor potential ankyrin channel subtype-1 TRPA1-/- - TRPA1 knockout TRPV1 – Transient receptor potential vanilloid channel subtype-1 TRPV1-/- - TRPV1 knockout WT – wild-type ix Acknowledgments This dissertation would not have been possible without the tremendous support I have received over the course of the past several years. First, I would like to thank my advisor, Dr. Derek Damron. I am truly appreciative and incredibly fortunate to have served as an understudy of Dr. Damron. I am grateful for his valuable insight, guidance, criticisms and support throughout my doctoral studies. I would also like to thank Dr. Ian Bratz. Dr. Bratz has served as an extraordinary source of knowledge, advice and guidance over the course of the past few years. I will never be able to put into words the amount of respect I have for both of these men, but this short paragraph will have to suffice. They have prepared me extensively for my future endeavors and have placed me on a trajectory where failure is not an option. I will forever be thankful for the mentorship and friendship of both Dr. Damron and Dr. Bratz. I’d also like to send my sincerest thanks and appreciation to the members of my doctoral committee, Dr. Colleen Novak and Dr. Soumitra Basu, for their time and energy, as well as their valuable insight into our research and willingness to collaborate. I’d like to thank past and present members of the Damron and Bratz labs including Dr. Pritam Sinharoy, Dr. Daniel Dellostritto, Dr. Loral Showalter, Monica Ghosh and John Kmetz for the amazing experience I’ve had in my doctoral studies. I’d also like to thank Dr. Gary Meszaros, Dr. Charles Thodeti, Dr. Daniel Luther, Dr. Roslin Thoppil, Dr. Holly Cappelli and Ravi Adapala for valuable experience in microsurgery and associated procedures. I would like to acknowledge the Faculty of Biological and Biomedical Sciences and x Kent State University for funding my doctoral work without which none of this could be possible. Last, and certainly not least, I’d like to thank my friends and family for their unconditional love, support and sacrifice. I am truly grateful for my mom, dad, sister and nephew who are my biggest supporters and will always be my inspiration to do great things. To my friends, I’d like to express my appreciation for their patience, faith and for dealing with my moodiness when research stressed me out. This dissertation is a dedication to my family, friends, mentors, colleagues