Role of Mechanosensitive Ion Channel Trpv4 in Cardiac
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ROLE OF MECHANOSENSITIVE ION CHANNEL TRPV4 IN CARDIAC REMODELING A dissertation submitted to Kent State University in collaboration with Northeast Ohio Medical University in partial fulfillment of the requirements for the degree of Doctor of Philosophy By Ravi Kumar Adapala May 2018 © Copyright All rights reserved Except of previously published material Dissertation written by Ravi Kumar Adapala B.S., Sri Venkateswara University, 2003 M.S., University of Hyderabad, 2006 Ph.D, Kent State University, 2018 Approved by ____________________________, Chair, Doctoral Dissertation Committee Charles K. Thodeti, Ph.D. ____________________________, Members, Doctoral Dissertation Committee William M. Chilian, Ph.D. ____________________________, Liya Yin, M.D., Ph.D. ____________________________, Moses O. Oyewumi, Ph.D. ____________________________ Gary. Koski, 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 ACKNOWLEDGEMENTS .............................................................................................. xi CHAPTER I: INTRODUCTION ........................................................................................ 1 1.1. Significance ....................................................................................... 1 1.2. Cardiac remodeling following injury/insult ........................................... 2 1.3. Cardiac fibrosis-heart failure target ..................................................... 6 1.4. Cardiac fibroblast differentiation- A key to cardiac fibrosis ................ 14 1.5. TRPV4 ............................................................................................. 25 1.6. TRPV4-Cardiac fibroblast differentiation .......................................... 27 1.7. Significance of the Present Study ..................................................... 32 SPECIFIC AIMS ............................................................................................................ 33 CHAPTER II: MATERIALS AND METHODS ................................................................ 36 iii TABLE OF CONTENTS (continued) CHAPTER III: RESULTS .............................................................................................. 50 3.1.1. Absence of TRPV4 preserves cardiac structure and function post-transverse aortic constriction surgeries ............................ 50 3.1.2. TRPV4 deletion attenuates cardiac fibrosis induced by TAC ... 55 3.2.1.TRPV4 regulates profibrotic gene expression in cardiac fibroblasts.........................................................................…......57 3.2.2. TRPV4 mediates CF differentiation through Rho /Rhokinase/ MRTF-A pathway……………………………………...........…......61 3.3.1. TRPV4 deletion protects myocardium from ischemia-induced pathological remodeling following acute MI ............................. 69 3.3.2. Absence of TRPV4 reduces myocyte apoptosis and enhances coronary angiogenesis, post-MI…………………………….…….75 CHAPTER IV: DISCUSSION…………………………………………………………………79 Conclusions……………..…………………………………………... 94 Future Directions ............................................................................ 96 REFERENCES ................................................................................................... 97 iv LIST OF FIGURES Figure 1. Schematic representation of pathways involved in pathological stimuli- induced heart failure ............................................................................................. 4 Figure 2. Schematic representation of cardiac fibrosis following injury/insult ..... 8 Figure 3. Therapeutic targets in key events of cardiac fibrosis ......................... 13 Figure 4. Multiple functions of fibroblasts in the heart ........................................ 15 Figure 5. Cardiac fibroblast differentiation into myofibroblast is a key event in fibrosis ................................................................................................................ 18 Figure 6. Schematic representation of possible mechanisms by which TRP channels regulate CF differentiation ................................................................... 23 Figure 7. Pictorial representation of monomeric TRPV4 structure ..................... 26 Figure 8. TRPV4 is required for TGF-1 induced CF differentiation into MF ..... 29 Figure 9. TRPV4 channels mediate TGF-1-ECM stiffness induced differentiation……………………………………………...……………………………31 Figure 10. Transverse Aortic Constriction (TAC) ............................................... 38 Figure 11. Electrocardio gram showing myocardial Infarction ........................... 40 Figure 12. Echocardio graphic analysis of cardiac function ............................... 42 Figure 13. TRPV4 deletion maintains myocardial integrity following TAC ......... 52 v LIST OF FIGURES (Continued) Figure 14. Absence of TRPV4 preserves cardiac function after TAC induced pressure overload ............................................................................................... 54 Figure 15. TRPV4 deletion reduces cardiac fibrosis post-TAC .......................... 56 Figure 16. Functional characterization of TRPV4 in mouse CF ........................ 58 Figure 17. TRPV4 required for profibrotic gene expression and mouse cardiac fibroblast differentiation into myofibroblast. ........................................................ 60 Figure 18. TRPV4 mediates CF differentiation via Rho/Rho kinases pathway .. 62 Figure 19. TRPV4 mediates TGF-1 induced MRTF-A activation ..................... 64 Figure 20. Silencing of MRTF-A using MRTF-A specific siRNAs. ...................... 65 Figure 21. TRPV4 mediates mCF differentiation through MRTF-A pathway ..... 66 Figure 22. MRTF-A acts downstream of Rho kinase during CF differentiation .. 68 Figure 23. TRPV4 deletion preserves myocardial function post-MI .................. 70 Figure 24. Absence of TRPV4 preserves myocardial structure and integrity following MI…………………………………………..…………………………..…….72 Figure 25. TRPV4KO hearts exhibit reduced cardiac fibrosis 8 weeks post MI .73 Figure 26. Myocyte apoptosis is significantly reduced in TRPV4KO hearts following 7days post MI………………………………………………………...……..76 vi LIST OF FIGURES (Continued) Figure 27. TRPV4KO hearts exhibit increased angiogenesis, 7 days post MI ... 77 Figure 28. Schematic representation of molecular mechanism by which TRPV4 mediate TGF-1 induced CF differentiation into MF…………………….....……..89 Table 1. List of primers .................................................................................... 49 vii LIST OF ABBREVIATIONS AA - Arachidonic acid AB1 – AB159908 ACE – Angiotensin converting enzyme AF – Atrial fibroblast ANOVA – Analysis of variance ARB – Angiotensin II receptor blocker ARD – Ankyrin rich domain Asp – Aspartic acid ATP – Adenosine triphosphate AZIN1 - Antizyme inhibitor 1 bFGF – Basic fibroblast growth factor CCG – CCG1423 CD31 – Cluster of differentiation 31 CF – Cardiac fibroblasts CCN2 – Connective tissue growth factor CVD- Cardiovascular diseases EC – Endothelial cell ECM – Extracellular matrix ED-A fibronectin – Extra domain A fibronectin EET – Epoxyeicosatrienoic acid EF – Ejection fraction EGFP – Enhanced green fluorescent protein EKG – Electro cardiogram EMT – Epithelial to mesenchymal transition EndoMT – Endothelial to mesenchymal transition ERK1/2 – Extracellular regulated kinase 1/2 viii FGF – Fibroblast growth factor FGFR – Fibroblast growth factor receptor FS – Fractional shortening FSP-1 – Fibroblast specific protein 1 GPCR – G-protein coupled receptor GSK – GSK1016790A GSK2 – GSK2193874 IGF – Insulin growth factor JNK - c-Jun N-terminal kinases LAD – Left anterior descending artery LOX – Lysysl oxidase MAP7 - Microtubule-associated protein 7 mCF– Mouse cardiac fibroblasts Met – Methionine MF – Myofibroblasts MI – Myocardial Infarction MMP – Matrix metalloproteinases MRTF-A – Myocardin related transcription factor-A OTRPC4 – Osmosensitive transient receptor potential channel 4 PDGF – Platelet-derived growth factor PSR – Picrosirius red PDGFR – Platelet-derived growth factor receptor PRD – Proline rich domain RAAS – Renin Angiotensin Aldosterone System rCF – Rat cardiac fibroblasts SMA – Smooth muscle actin SRF – Serum response factor SEM – Standard error mean ix TAC – Transverse aortic constriction TAK1 – TGF- activated kinase 1 TGF-Transforming growth factor-beta TGF-r – Transforming growth factor receptor TIMP – Tissue inhibitors of metalloproteinases TRP – Transient receptor potential TRPM7 – Transient receptor potential melastatin 7 TRPV4 – Transient receptor potential vanilloid 4 TRPV4KO – Transient receptor potential vanilloid 4 knockout TUNEL – Terminal deoxynucleotidyl transferase dUTP nick end labeling VEGF – Vascular endothelial growth factor VEGFR2 – Vascular endothelial growth factor receptor 2 VRAC – Volume-regulated anion channel VR-OAC – Vanilloid receptor-related osmotically activated channel WGA – Wheat germ agglutinin WT – Wild type x ACKNOWLEDGEMENTS I would like to thank the Kent State Biomedical Science Program and NEOMED Integrative