Role of Mechanosensitive Ion Channel Trpv4 in Cardiac

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

Ravi Kumar Adapala

May 2018

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

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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

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

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

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

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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

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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

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

ACKNOWLEDGEMENTS

I would like to thank the Kent State Biomedical Science Program and NEOMED

Integrative Medical Science Department for giving me the opportunity to complete my Ph.D. I foremost would like to thank my graduate advisor, Dr Charles Thodeti willingness and support, both as a mentor, and a friend over the past 6 years. I treasure your mentorship and ability to provide the opportunities which allowed me to obtain a valuable set of skills both inside and out of the lab that will serve as a springboard towards my future endeavors. I will forever be thankful for your fatherly advice that served as a crutch throughout my graduate research and will continue to guide me through life. Without your support, my achievements through this work could not have been possible. Thank you.

To my committee members, Dr. Chilian, and chair of Integrative Medical Sciences who gave every graduate student an opportunity to shine and better themselves and learn a lot of things that being in the lab alone doesn’t teach us. thank you for your guidance and support over the years and to Dr. Liya Yin, Dr. Oweyumi and

Dr. Koski for taking the time out to read, critique and provide insight into my thesis.

I also would like to thank Dr. Meszaros for being an excellent moderator and continues support during my NEOMED life.

I would like to thank Dr. Eric small who gifted the -SMA and col11 luciferase constructs which helped me to finish some of the my work in the this thesis. I would like to thank for Dr. Ohanyan for teaching me rodent surgeries and his workshop

which given me immense experience on rodent microsurgeries and our long lasting discussions during the surgeries. He made me the good rodent surgeon.

To all the administrative and research staff, especially Dr. Walter Horton, Dr. Eric

Mintz, Karen Greene, Carolyn Miller, Deb Enos, Sharon Usip, Ruby Pahls, Dori

Parker, Corey Robinson, Margaret Weakland, Cheryl Hodnichak, Judy Wearden

(KSU) and Dona warner (KSU) - thank you for all your help, encouragement and for all the fun banter!

To my parents, papa and mummy- thank you for all that you have done. This work is dedicated to the sacrifices you made to see me succeed. For the love, acceptance and freedom you have given me, I am eternally grateful and also thank you to my brother and sister, for their unconditional support and love and wise words shared with me throughout these years.

To the most important person of my life, my best friend and better half, Tejaswi, thank you for the best 3 years of my life, for every up and down we have shared throughout graduate school and life outside, I have learnt so much and become a better person because of it. I would like to thank my stress buster and daughter

Mayukha for providing me cute and innocent smile which took alternate universe of absolute silliness and complete joy, where all my problems somehow cease to exist.

Last but not the least, to all my friends I know and here at NEOMED; specially

Sanjay and his family whom I have known for 10 years, what an amazing friendship we have, thank you. Roslin, my sister and friend, who stand my side and helped

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me in the lab and outside of the lab. To my personal editor, Holly Cappelli, I have no words to describe what a miracle you are! Anantha (Koti), thank you for being with me and helped me to improve my thesis with your timely critiques and efforts in the lab and outside of the lab. I would like to thank our surgeon and friend Ashot for his efforts and came in right time to lab which could made me to finish my thesis.

I would like to thank my lab members Nina, Sasha and other summer students for their time and helping me during my Ph.D. time.

Finally, I would like thank all my friends Ajay, Lakshman, Bindu, Nirupama Preethi,

Ritu, Soumyadip, Anurag, Danielle, Luther, Kavitha and Vinay and his family for your help and joy in my life.

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CHAPTER I

Introduction

1.1. Significance Cardiovascular diseases (CVD) are one of the underlying causes of mortality in the world. In the United States alone, the mortality rate is nearly

801,000 deaths per year and approximately 1 death every 40 Sec. An estimate of

92.1 million Americans are living with at least one kind of CVDs, which encompasses more deaths than cancer. Of those deaths resulted by CVD, heart attacks contributed the largest percentage (45.1%) followed by other CVDs like heart failure (8.5%) and high blood pressure (9.1%) and these mortalities are expected to grow to 23.6 million by 2030. Although current interventional therapies reduced the rate of hospitalization in the US, the early post-discharge mortality and readmissions were largely unchanged and may even be worsening (Benjamin et al., 2017; Go et al., 2013; Mozaffarian et al., 2015).

Besides these overwhelming statistics, improvements in patient care have increased and the patient will survive with heart failure, often leaving the patient with the maladaptive structural responses during the wound healing process

(Fraccarollo, Galuppo, & Bauersachs, 2012). These structural changes that are caused due to injury or insult are referred as cardiac remodeling. Normally, cardiac remodeling is an adaptive response following injury/insult and improves heart performance, however, the abnormal remodeling can lead to heart failure (Beltrami et al., 1994). Current therapies are mostly targeting cardiac remodeling that is based on soluble factor signaling but their success has been limited at the clinical level (Z. Fan & Guan, 2016; Fraccarollo et al., 2012). Despite advanced therapeutics cardiac remodeling based mortality remains high, therefore, extensive research is required to understand the pathophysiology and to find novel targets in the remodeling process.

1.2. Cardiac remodeling following injury/insult

One of the main challenges in cardiovascular medicine is to understand the molecular basis of the remodeling process. Hochman and Bukley were first to use the term cardiac remodeling following the observation of dilation and thinning of the infarcted area after a myocardial infarction (MI) and later Pfeffer used the term to describe the volume increase of the left ventricular (LV) chamber after MI

(Hochman & Bulkley, 1982; Pfeffer, Pfeffer, & Braunwald, 1985). Cardiac remodeling, defined by Oparil in 1985, as an enlargement of the myocytes and non-myocyte cell hyperplasia that are caused in response to alterations in workload and neurohormonal factors during hypertrophy. Recently, any architectural and complex alterations in the myocardium in response to physiological or pathological changes is referred to as ventricular remodeling

(Oparil, 1985).

Cardiac remodeling is further classified as a physiological and pathological remodeling process. Physiological remodeling associated with growth, exercise, and pregnancy to maintain the normal function, which results in decreased wall stress, increase in pumping, and improved vascularization (Hill & Olson, 2008).

Molecular level signaling pathways of the physiological remodeling is well orchestrated including, vascular endothelial growth factor B (VEGF-B), insulin growth hormone (IGH), and insulin-like growth factor 1 (IGF1), as well as the thyroid hormone triiodothyronine (T3), which control the myocyte contractility and remodeling, survival, electrical remodeling and angiogenesis (Bernardo &

McMullen, 2016; Eghbali et al., 2005; Spaich, Katus, & Backs, 2015).

In contrast, pathological cardiac remodeling is usually triggered by several pathophysiological stimuli such as pressure overload, volume overload, hypertension, myocardial infarction, genetic anomalies, and neuroendocrine activation. Pathological cardiac remodeling was initially thought to develop as a compensatory mechanism to reduce wall stress and preserves the cardiac function, however, this eventually led to devastating maladaptive alterations that result in heart failure and death (Figure 1). In pressure overload diseases such as aortic stenosis or hypertrophy, the heart develops concentric hypertrophy, characterized by an increase in myocyte thickness as compared to the length of the cells and parallel assembly of sarcomeres, which induces a reduction in wall stress and an increase in the free wall and septal thickness (Grossman, Jones, &

McLaurin, 1975). In contrast, in volume overload diseases like MI or dilated cardiomyopathy, the loss of cardiac myocytes and surviving myocyte

Figure 1. Schematic representation of pathways involved in pathological stimuli-induced heart failure: Pathological stimuli such as ischemia/reperfusion or excessive mechanical load can induce ischemic and hypertrophic phenotypes, eventually leading to heart failure through various mechanisms including: cardiomyocyte loss through apoptosis and necrosis, cardiomyocyte hypertrophy via mechanical and neurohormonal factors, cardiac fibrosis via activated fibroblasts and arrhythmic phenotype due to damage in the structure and changes in ion transport (Adapted from Jana S. Burchfield et al. Circulation. 2013; 128:388-

400).

rearrangements lead to eccentric hypertrophy in which lengthening of the myocytes induces the addition of sarcomeres to accommodate the greater volume and after dilatation (I. Shimizu & Minamino, 2016; Tham, Bernardo, Ooi, Weeks, &

McMullen, 2015). These alterations, in turn, changes the extracellular matrix

(ECM) organization and vasculature as well as cellular cross-talks. In addition to

LV hypertrophic changes, cardiac fibrosis also affects the contractility and relaxation, which orchestrated together leads to detrimental cardiac remodeling. It has been demonstrated that injury/insult induces inflammation, oxidative stress and changes in energy metabolism trigger the hypertrophic and pro-fibrotic signaling pathway resulting in progressive myocyte loss (Figure 1) (Schirone et al., 2017).

Likewise, alterations in calcium transport proteins and calcium homeostasis are responsible for reduced contractility through decreases in systolic and increases in diastolic calcium release (Lehnart, Maier, & Hasenfuss, 2009).

Additionally, mechanical wall stress activates the mechanosensitive ion channels, which senses the acute changes in ECM through integrins, surface proteins connected to the ECM, signals through AKT pathway (Lehnart et al., 2009; Matsui et al., 2002). Mechanical stretch also increases the release of angiotensin II and endothelin which activates the G-protein coupled receptor (GPCR) signaling through Gq. Modulation of Gq subunits are associated with LV hypertrophy and fibrosis (Harvey & Leinwand, 2011; X. Wang, Bosonea, Odenbach, & Fernandez-

Patron, 2012). Moreover, changes in mechanical stiffness activate latent TGF- to an active TGF-1 form, a key event in the deposition of ECM proteins (Hinz, 2009;

Wipff, Rifkin, Meister, & Hinz, 2007). Consequently, prolonged exposure to these factors leads to ECM deposition in intestinal spaces by activated cardiac fibroblasts (CFs) and leads to compromised heart performance.

1.3. Cardiac fibrosis-heart failure target

Mammalian heart is a complex arrangement of cellular and acellular components such as myocytes, endothelial cells, fibroblasts, and ECM scaffold, respectively. The cardiac ECM is comprised of structural adhesion proteins which form a scaffold structure to adhere the cellular components of tissues and contribute to biochemical, mechanical signaling and restrict the propagation of electrical activity. The composition of the ECM depends on the origin of the tissue, however, the ECM in the heart includes collagens (type I, III, IV, V and VI), fibronectin, laminin, versican, biglycan, hyaluronic acid and various proteases, growth factors and cytokines (Zamilpa & Lindsey, 2010). In addition to structural support to the myocardium, Type I and III collagens provide mechanical support by stiffening the myocardial wall which helps in transmitting the mechanical force of the contraction (Horn & Trafford, 2016; Talman & Ruskoaho, 2016). In healthy cardiac tissue, the homeostatic control of the ECM involves balancing synthesis and degradation. Imbalance in this tightly regulated process results in structural and functional abnormalities in the heart.

In normal and pathological conditions, CF are predominantly maintaining homeostasis of the ECM through synthesizing the ECM proteins as well as extracellular proteases known as matrix metalloproteases (MMP) and their inhibitors, Tissue inhibitors of metalloproteases (TIMPS). Under pathological

conditions, resident CF and infiltrated leukocytes secrete MMPs in damaged tissue to facilitate cell migration by degrading ECM proteins and creating a path for the growth of new blood vessels (Figure 2) (Birkedal-Hansen et al., 1993). MMP-1 is the initial proteolytic enzyme which degrades the larger fibrillar collagen into smaller proteins, additional degradation completed by MMP2, 3 and 9. Several studies in mice showed the MMP-1, 3 and 9 expressions are increased as soon as two days after the injury (Chen et al., 2005). The shift of collagen degradation initiates the increased production of the ECM proteins and TIMPS at damaged tissue via differentiating the CF into myofibroblasts (MF). The overproduction of fibrillary collagens in the infarcted area forms a scar which lends the mechanical support and decreases the rupture of the myocardium. Although, overproduction of ECM deposition is beneficial at the infarcted area, deposition of ECM at remote zone is detrimental for the heart performance (Sun & Weber, 2000). Thus, the excessive accumulation of ECM in the myocardium following injury or insult referred as cardiac fibrosis.

The cardiac fibrosis following injury increases the stiffness and decreases compliance of the damaged tissue and negatively affects the contraction and relaxation of the myocardium leading to heart failure (D. Fan, Takawale, Lee, &

Kassiri, 2012). Based on the cause, cardiac fibrosis is further divided as replacement fibrosis and reactive fibrosis. Upon myocardial injury, the necrotic myocytes form a fibrin clot, which is replaced with a temporary collagenous scar referred as replacement fibrosis by MF (Figure 2). During maturation of the scar

Figure 2. Schematic representation of cardiac fibrosis following injury/insult.

(Left) Activation of fibroblasts due to changes in mechanical loading induces reactive interstitial and perivascular fibrosis, if prolonged this can lead to myocyte loss and contribute to replacement fibrosis. (Right) Schematic representation of myocyte loss due to infarction via necrosis, apoptosis leading to fibroblasts activation and scar formation (replacement fibrosis). Myocytes (red), myocyte loss

(brown), fibrillar collagen (blue strands), and fibroblasts (blue spindle-shaped cells) are depicted in the picture. (Adopted from Erik B. Schelbert et al. JACC 2014;

63:2188-2198)

at the infarct site, the growth factors promote depletion of myofibroblast activity and are removed from the infarcted area possibly through apoptosis. Additionally, the vasculature is disintegrated, and vascular cells die and are replaced with the collagens. Finally, temporary collagen III is replaced with collagen I by remaining surviving MF (Schelbert, Fonarow, Bonow, Butler, & Gheorghiade, 2014; Sun &

Weber, 2000). The enhanced expression of LOX (Lysyl oxidase) catalyzes the collagen I cross-linking to significantly form mature collagen leading to an increase in the tensile strength and contraction of the scar (Cleutjens, Blankesteijn,

Daemen, & Smits, 1999; D. Fan et al., 2012). These changes in the scar alter the chamber geometry and contribute to deposit the ECM in interstitial spaces of myocytes at remote area of the myocardium (Figure 2). During normal wound healing process, myocytes undergo apoptosis, however, in the heart the persistent activation of MF may be required for continuous replacing of the ECM and this may be because of continuous contraction and it reduces the rupture of the scar and leads to systolic dysfunction (Cleutjens et al., 1999).

The alterations in function and geometry in the myocardium due to injury, deposition of the ECM in normal region of the myocardium referred as reactive fibrosis. In contrast to replacement fibrosis, reactive fibrosis is not often associated with the loss of myocytes observed majorly in chronic remodeling accompanied by the hypertrophic growth of the cardiomyocytes as they compensate the increased workload to increase the cardiac function and meet the metabolic demand of the body (Beltrami et al., 1994). The progression of the growth of the hypertrophy and stiffness is attributable to excessive cross-linking of the collagen and tonic

contraction of the fibrous tissue by MF leading to compromised diastolic function

(Burlew & Weber, 2002; Janicki, 1992; Kass, Bronzwaer, & Paulus, 2004). The regulation and molecular mechanisms of the reactive fibrosis in non-infarcted regions and chronic injury are poorly understood. Recent findings demonstrated that increases in mechanical stress in the remote non-infarcted areas following MI or afterload due to hypertension and aortic stenosis and this stress induces the latent TGF-1 activation (Hinz, 2009). Additionally, continuous secretion of pro- fibrotic proteins in the infarcted areas from persisting myofibroblasts permeates towards the non-infarcted regions and increases the proliferation of the local fibroblasts and leading to increased deposition of ECM in interstitial spaces.

Irrespective of the origin of the injury, MF are the principle phenotype involved in both reparative and reactive cardiac fibrosis. Multiple studies have reported that the accumulation of the MF into interstitial spaces increased following injury, including myocardial infarction, pressure and volume overload myopathies, aging, and alcoholic myopathies (Kong, Christia, & Frangogiannis, 2014). The major source of the myofibroblasts are the resident fibroblasts, however, several studies suggested that the significant origin from other cells such as bone marrow- derived circulating fibrocytes (Mollmann et al., 2006), endothelial cells (Zeisberg et al., 2007). Recently, Morris et al. using cell lineage tracing demonstrated that the accumulation of myofibroblasts derived from two distinct populations of epicardium and endocardium during pressure overload-induced injury (Moore-

Morris et al., 2014). The abundance of the pro-fibrotic mediators induces the differentiation of resident fibroblasts into myofibroblasts following injury may

suggest the resident fibroblasts are the most important source of the myofibroblasts. On the other hand, using bone marrow cells labeled with eGFP, it was demonstrated that the fibrocytes play significant role in scar formation after acute MI (Haudek et al., 2006; Mollmann et al., 2006). Moreover, Zeisberg et al. using mice where in endothelial cells are marked with LacZ and FSP-1 found that the substantial number of fibroblasts are derived from endothelial origin following cardiac injury (Zeisberg et al., 2007). Regardless of the origin, prolonged activation of the myofibroblasts deposit excessive ECM into interstitial spaces, which disrupts the coordination of myocardial excitation-contraction coupling in systole and diastole and may impair the systolic and diastolic function through several mechanisms. First, fibrillar collagen loss may result in uncoordinated contraction of the cardiac myocyte through impaired myocardial force development (Baicu et al., 2003). Second, ECM proteins and their receptors on cells play a key role in cardiomyocyte homeostasis, for example; collagen VI knockout mice show improved cardiac remodeling following acute MI (Luther et al., 2012) and laminin4 deficient mice exhibit systolic dysfunction due to vascular remodeling abnormalities (J. Wang et al., 2006). Finally, fibrosis induced myocyte sliding displacement leading to many myocyte layers in the myocardial wall and follows to ventricular dilatation (Beltrami et al., 1994). Therefore, it is crucial to understand the key regulators of the fibrosis in the progression of the disease and may be a potential therapeutic target to aim at altering cardiac fibrosis and cardiac function following injury.

Current antifibrotic therapies mainly focused on decreasing secretion and activity of the growth factors and cytokines, these including anti-fibrotic agents, localized delivery of stem cells in biomaterials (Figure 3) (Z. Fan & Guan, 2016).

However, emerging data suggest that both preventing and ameliorating ECM expansion from the overproduction of collagen is most critical to succeed in anti- fibrotic therapies. Indeed, individual or combination treatment with ACE

(Angiotensin Converting Enzyme) inhibitors, ARBs (Angiotensin II receptor blockers), RAAS system, -blocker for heart failure have shown modest improvements in cardiac function and remodeling following heart failure (Z. Fan &

Guan, 2016). Some of the recent antifibrotic therapies developed to target the mechanism and regulators of the cardiac fibrosis such as TGF-1, Ang II,

Endothelin-1, PDGF, and CCN2 (Leask, 2010). Losartan an antihypertensive angiotensin II type 1 receptor blocker was shown to reduce the aortic wall thickness in fibrillin-1 mutation induced morfan syndrome by suppressing circulating TGF- availability and its signaling suggesting that TGF- as another potential therapeutic target (De Mello & Specht, 2006; Shibasaki et al., 2005).

Pirfenidone is an orally active small molecule that inhibits the collagen synthesis through reducing activation of latent TGF- by antagonizing the TGF- activating convertase enzyme (Burghardt et al., 2007). In a multicenter randomized clinical trial of pirfenidone treatment for idiopathic pulmonary fibrosis has been shown that confusing and uncertain improvement in pulmonary function (Gan, Herzog, &

Gomer, 2011). Moreover, plant alkaloid halofuginone inhibited collagen synthesis through TGF- signaling by reducing smad3 phosphorylation

Figure 3. Therapeutic targets in key events of cardiac fibroblast differentiation. Stimulators and their respective targets during cardiac fibroblast differentiation into myofibroblasts. Soluble factors as well as mechanical factors induce CF differentiation. TGF1 or cytokine inhibitors, antagonizing ECM protein activity by antibodies and anti-fibrotic growth factors are possible therapeutic targets during CF differentiation. Myofibroblasts activity may be hindered by targeting HADACs and TGF- activation using miRNAs and inhibitors. (Adapted from Hinz B et al. American journal of pathology, 2012; 180:1340-1355).

(Halevy, Nagler, Levi-Schaffer, Genina, & Pines, 1996; Nagler et al., 1996).

Addition to biochemical factors, microRNAs (miRs), small noncoding RNA molecules is being considered not only as diagnostic biomarkers but also therapeutic targets. For example, a recent study from Lichan Toa et al. found that miR-433 exhibit increased expression in the myocardium and fibrosis and miR-433 may protect the myocardium via targeting JNK and AZIN1 genes (Figure 3) (Tao et al., 2016). Collectively, there is a wide range of possibilities for anti-fibrotic therapies based on biochemical factors and miRNA are at different developmental stages. Although therapies targeting biochemical factors are promising at the in vivo level, more data is necessary to evaluate the efficiency at the clinical level.

However, most of the therapies to date are targeting the soluble factor signaling, the efficiency of these drugs is modest in reducing the cardiac fibrosis following heart failure. Therefore, an urgent need of developing alternative novel targets to treat the fibrosis and LV dysfunction.

1.4. Cardiac fibroblast differentiation - A key to cardiac fibrosis

Cardiac fibroblasts (CF) are one of the largest cell types among the non- myocyte cell population in the mammalian heart. Although the actual number of

CF in the heart remains unknown, it has been stated that the adult mouse heart constitutes 55% myocyte and 27% of CFs. Whereas, Rat heart consists of 30% myocytes and 46.9% of CFs (Banerjee, Fuseler, Price, Borg, & Baudino, 2007).

CF are the predominant phenotype that help in maintaining the heart structural integrity by balancing the synthesis and degradation of ECM proteins and

Figure 4. Multiple functions of fibroblasts in the heart. Cardiac fibroblasts maintain the structural integrity of myocardium by balancing the ECM synthesis and degradation. Upon activation, these cells migrate to wound area, proliferate and differentiates into myofibroblasts. Cardiac fibroblasts synthesize various cytokines and chemokines which enhance CF proliferation and migration.

(Adapted from Porter and Turner (2009) Pharmacol Therap. 123: 255-278).

secreting cytokines and proteases. The primary function of the CF is migration to wound area and to remodel and repair the site of the injury by forming a collagenous scar. Upon injury or insult such as myocardial infarction, atrial fibrillation, pressure and volume overload-induced hypertrophy, CFs proliferate, migrate toward injury, and differentiates into MF (Figure 4) (Porter & Turner,

2009). The morphological features of MF are spindle-shaped appearance, dendritic-like protruding, elongated nuclei, extended Golgi and endoplasmic reticulum and electron dense contractile bodies contain embryonic smooth muscle myosin and smooth muscle actin (SMA). The matured myofibroblasts identified with the incorporation of -SMAinto stress fibers (Hinz et al., 2007). The defining markers of MF in vivo are abundant expression of ECM proteins like periostin, collagen-I and III, fibronectin, ED-A isoform of fibronectin, SM22 and caldesmon.

MF are absent in a healthy heart, but injury or stress induces their appearance in as quickly as in two days. Diverse origin of cardiac myofibroblasts such as locally residing fibroblasts, circulating fibrocytes from hematopoietic lineage and resident endothelial cells or epithelial cells via endothelial to mesenchymal transition (EndoMT) (Zeisberg et al., 2007) or epithelial to mesenchymal transition (EMT) (Moore-Morris et al., 2014) respectively. The source of CF is arguable because of the lack of definitive CF specific marker and variability among current lineage tools, it is still believed that the fibroblasts in the interstitium of the myocardium derived from the mesenchymal cells in the embryonic proepicardium which developed to epicardium in later stages (Kong et

al., 2014; Moore-Morris et al., 2014). The epicardium-derived cells undergo epithelial to mesenchymal transition with the influence of growth factors such as

TGF-1, PDGF, FGF differentiate into fibroblast phenotype (Olivey, Mundell,

Austin, & Barnett, 2006). Additionally, various cytokines like TGF-, PDGF and

Wnt effects the resident endothelial cells to delaminate and undergo endothelial to mesenchymal transformation and migrate to injury site and mature into fibroblastic phenotype (Armstrong & Bischoff, 2004; de Lange et al., 2004; Krenning, Zeisberg,

& Kalluri, 2010). Even though developed from various sources with a different marker, some of them were passed down to myofibroblasts such as TCF21, platelet-derived growth factor -1 (PDGF-), Thymus antigen 1(thy1) wilm’s tumor gene 1 (WT1), but they fail to mark a sole population of the fibroblasts. For example, a small fraction of the WT1+ cells and Thy1+ cells became -SMA positive in response to pressure overload-induced hypertrophy. Recently TCF21 a transcription factor has promising effects with the labeling of the resident fibroblasts following injury (Acharya et al., 2012). The limitation of uniform labeling of the mature myofibroblasts presents a huge hurdle to targeting activated cardiac fibroblasts in in vivo (Ali et al., 2014; Krenning et al., 2010).

Besides their heterogeneous origin, CFs responds to biochemical, mechanical and electrical stimulation and balancing the rate of synthesis and degradation of ECM. Dynamic fluctuation of the biochemical, mechanical and electrical environment in the injured myocardium trigger the differentiation of

Figure 5. Cardiac fibroblast differentiation into myofibroblast is a key event in fibrosis. Both soluble factors (TGF-1 and Ang II, EDA fibronectin) and mechanical factors (stiffness and wall stress) are required for cardiac fibroblast differentiation into hyper-secretory and hyper-contractile phenotype myofibroblasts. (-SMA (green) expression and incorporation into stress fibers is a known marker of myofibroblasts, Nuclei were stained with DAPI (blue).

resident CF to MF. Along with biochemical factors including TGF-1, Endothelin-

1, PDGF-1, mechanical factors also involve in CF differentiation and the molecules that integrate soluble and mechanical signaling during CF differentiation into MF (Sun & Weber, 2000) (Figure 5). TGF-1 is one of the predominant soluble factors implicated in CF differentiation to MF and fibrotic response. Typically, TGF-

1 secreted as inactive latent TGF-1 into pools of ECM, which becomes activated upon enzymatic cleavage or mechanical liberation. Although there are three TGF-

 ligands, TGF-1 has mostly studied in fibroblast differentiation, which binds to heteromeric TGF-1 and 2 receptors, which activates the canonical SMAD2/3 or non-canonical pathway through MAP kinases, RhoGTPase-Actin, PI3K/protein kinase B and TNF receptor-associated factor 4 &6. In canonical pathway, SMAD2/3 is phosphorylated and complex with

SMAD4, which is translocated into the nucleus and induce fibrotic gene expression

(Leask & Abraham, 2004). Indeed, the SMAD3 inhibition in vitro in fibroblasts attenuated myofibroblasts regulated ECM associated genes including Col11,

Col12, Col31, Col52, Col61, Col63, fibronectin and TIMP-1 (Dobaczewski et al., 2010; Y. E. Zhang, 2009). Even though SMAD3 knockout exhibited reduced fibrosis following myocardial infarction, a significant amount of -SMA positive cells found in the infarcted areas shows that may another pathway also involved in CF differentiation. Recent evidence demonstrates that glycogen synthesis kinase3- regulate increases the -SMA positive cells via SMAD3 hyperactivation, which promotes increased fibrotic scar, however, these effects were reduced

following treatment with SMAD3 inhibitor supporting that SMAD3 regulate CF differentiation directly or indirectly in the cardiac injury (Dobaczewski et al., 2010).

In contrast to SMAD3, growing evidence prove that SMAD2 does not contribute to

CF differentiation following injury. The inhibitory SMAD 6 and 7 prevents CF differentiation through inhibiting SMAD2/3 phosphorylation (Schmierer & Hill,

2007).

Contrast to classical pathway, non-classical TGF-1 signaling activates

TGF-r2 implicated as a regulator of CF differentiation through initiating several downstream molecular mechanisms by various intermediate signaling molecules.

TGFr2 also involved in activating alternative classical pathway through ERK and

Rho kinases. In non-classical pathway, TGF-r2 activates TAK1, which promotes phosphorylation of p38 MAPK, JNK signaling resulted in enhanced fibrotic gene expression. TGFr2 knockdown underscores the nonclassical pathway induced cardiac remodeling, due to the dual specific activity of MKK 3 and 6 transduce signals from TAK1 to p38 enhancing the ECM gene expression. The strategic inhibition of p38 using pharmacological antagonists blocks fibrotic responses reduction in collagen and -SMA positive cells following cardiac injury (Kim et al.,

2007). These results suggest that p38 MAPK regulate downstream TGFr2 signaling of regulating CF phenotypic changes. Taken together, TGF-1 induces

CF differentiation into myofibroblasts through SMAD as well as various intermediate signaling pathways.

In addition to soluble factors, mechanical factors such as stretch and matrix stiffness also control CF differentiation into MF (Figure 5). CF secrete various

ECM proteins such as collagen, periostin, fibronectin and ED-A fibronectin which were shown to regulate proliferation, migration and differentiation during injury.

Periostin, collagen 1 and III has been shown to promote myofibroblast proliferation and collagen synthesis (Lighthouse & Small, 2016). ED-A fibronectin plays a key role in increasing CF contractility and maintaining structural integrity through connecting ECM to integrins and stress fibers and collagen synthesis (Serini et al.,

1998). In addition to signaling provided by ECM proteins, mechanical stress induces CF proliferation and differentiation. CF exposed to cyclic stretch increased

-SMA and collagen 1 expression indicating a role for mechanical stretch in CF differentiation. Moreover, effects of matrix stiffness have evaluated using 3D encapsulation of collagen gels suggest that free-floating disc shows reduced -

SMA expression whereas constrained gels offered increased -SMA expression and incorporation suggesting that matrix stiffness controls CF differentiation (Yong et al., 2015). Both mechanical and soluble factor signaling are interdependent during CF differentiation. Mechanical forces generated by ECM transmitted to cells through integrins and regulate cell fate through activating soluble factors and vice versa. For example, matrix stiffness activates integrins which inturn release active

TGF-1 from ECM beds (Hinz, 2009; Wipff et al., 2007). Though biochemical and mechanical factors are dependent on each other, the molecules and their signaling mechanisms that integrates both of them are unknown.

Calcium, a second messenger plays a central role in intercellular communication, not only regulates myocyte contraction but also mediates the intra and extracellular signals. Mechanical stimuli and contractile forces applied onto cell membrane enhance the calcium entry or exit throughout the plasma membrane, which further modulate additional activation of multiple pathways and gene expression. However, identity of mechanosensitive calcium ion channels is not yet clear. Interestingly, emerging evidence suggests that TRP (transient receptor potential) cation channels are involved in controlling various pathophysiological conditions in heart failure. TRPs are categorized into following subcategories TRPC (canonical), TRPM (melastatin), TRPV (vanilloid), TRPP

(polycystin), TRPA (ankyrin) and TRPML (mucolipin). Till date, TRPC1, TRPC6,

TRPC3, TRV4, and TRPM7 are implicated in CF proliferation and differentiation, however, exact mechanisms are not well known. Importantly, Davis et al demonstrated that TRPC6 required for TGF-1, or Ang II induced CF differentiation into myofibroblasts through activating calcineurin/ NFAT pathway to induce -SMA expression (Davis, Burr, Davis, Birnbaumer, & Molkentin, 2012) . On the other hand, Du et al. demonstrated that TRPM7 mediates atrial fibroblast differentiation.

Notably, we have recently shown that a mechanosensitive ion channel, TRPV4 regulates TGF-1-induced CF differentiation through integrating biochemical and mechanical factor signals (Figure 6) (Adapala et al., 2013; Du et al., 2010).

Figure 6. Schematic representation of possible mechanisms by which TRP channels regulate CF differentiation. TGF-β and Ang II were demonstrated to induce expression of TRP channels via p38 MAPK. TRP channels, then induce fibroblast proliferation (TRPC3) and differentiation (TRPC6; α-SMA expression) through ERK1/2 and CnA/NFAT signaling, respectively. TRPV4 channels, on the other hand, integrate both soluble (TGF-) and mechanical signals (ECM stiffness/integrin/TRPV4/Rho-ROCK) a leading to synthesis and incorporation α-

SMA into actin stress fibers. The increased cell contraction and tensional forces

may activate additional integrins (red arrows) leading to the increased ECM stiffness and activation of latent TGF-β. Ang II = angiotensin II; CnA=calcineurin;

NFAT=Nuclear factor of activated T-cells; SRF, serum responsive factor; α-SMA,

α-smooth muscle actin; TGFβ = transforming growth factor β;. Modified or adopted from Thodeti et al. Channels (Austin). 2013 May-Jun;7(3):211-4.

1.5. TRPV4

A growing evidence has been implicated that the members of transient receptor potential ion channel (TRP) family including TRPC (canonical), TRPM

(melastatin) and TRPV (Vanilloid) may be a key regulator of mechano-transduction

(Christensen & Corey, 2007; Liedtke & Kim, 2005; O'Neil & Heller, 2005; Pedersen

& Nilius, 2007). TRPV4 has expressed ubiquitously including cardiovascular system, (Earley, Heppner, Nelson, & Brayden, 2005; Earley et al., 2009; Hatano,

Suzuki, Itoh, & Muraki, 2013; Watanabe et al., 2002), digestive system (Egbuniwe et al., 2014; Skrzypski et al., 2013; Yamawaki et al., 2014; L. P. Zhang, Ma,

Abshire, & Westlund, 2013), respiratory system (Alvarez et al., 2006; Andrade,

Fernandes, Lorenzo, Arniges, & Valverde, 2007; Dahan et al., 2012; Fernandez-

Fernandez et al., 2008; Hamanaka et al., 2007; Jia et al., 2004), and urinary system

(Berrout et al., 2014; Birder et al., 2007; Janssen et al., 2011). TRPV4 function is activated by a variety of physical and chemical factors such as hypotonicity, heat, stiffness, cyclic stretch, phorbol esters, arachidonic acid (AA) and epoxy eicosanoid acids (EETs).

Structurally, TRPV4 assemble as a homotetramer or hetero tetramer with other channels, each monomer comprised of 874 amino acids and broken into three components such as six transmembrane domain, -NH2 terminus, and -

COOH terminus (Figure 7) (Auer-Grumbach et al., 2009). Among six transmembrane domains, S5 and S6 forms pore like structure and allow the influx of ions (Xu, Fu, Tian, & Cohen, 2006). NH2 terminus remains in cytoplasm contains

Figure 7. Pictorial representation of monomeric TRPV4 structure: TRPV4 comprised of six transmembrane domains, in which 5th and 6th form pore like structure. N terminus lies inside the cytoplasm with proline rich ankyrin domains.

C terminus lies close to membrane and it has calmodulin binding domain. Adapted from Auer-Grumbach, Olschewski et al. Nature genetics, 2010; 42:160-164.

six proline-rich ankyrin repeat domain (ARD), which implicated in mechanosensitivity of the TRPV4 channels. The interaction between residues

P142, 143 and 144 of ARD with pacsin3 implicated in vesicular membrane transport, endocytosis, and cytoskeletal reorganization (Cuajungco et al., 2006;

D'Hoedt et al., 2008). TRPV4-COOH terminus resides close to surface in the cytoplasm and composed with highly conserved TRP domain and calmodulin binding domain, in which TRP domain required functional tetramerization (Garcia-

Sanz et al., 2007) and calmodulin binding domain essential for calcium influx during activation (Strotmann, Schultz, & Plant, 2003). Moreover, the interaction of

TRPV4 COOH terminus and microtubule-associated protein (MAP7) helps in the

TRPV4 membrane and participate in cytoskeletal linkage (Suzuki, Hirao, & Mizuno,

2003).

1.6. TRPV4- Cardiac fibroblast differentiation

Given the ubiquitous expression and diverse range of activities in physiology, TRPV4 channels have got significant attention due to their involvement in mechanotransduction. Therefore, it’s not surprising that mutation or changes in expression and activities contribute to several disabling and lethal human pathologies, including skeletal dysplasia's and neuropathies (Lamande et al.,

2011; Loukin, Zhou, Su, Saimi, & Kung, 2010; McNulty, Leddy, Liedtke, & Guilak,

2015; Nilius & Voets, 2013). Further, TRPV4 knockdown in mice display minor phenotypes including changes in osmosensation, vascular function, thicker bones, impaired bladder wall stretching and impaired pressure, hear sensation (Cortright

& Szallasi, 2009; Earley et al., 2005; Gevaert et al., 2010; Lechner et al., 2011;

Sonkusare et al., 2012; Vriens et al., 2005).

Using genome-wide analysis Zhou et al first time reported that TRPV4 might have a potential role in myocardial infarction (Zhou et al., 2011). Later, Hatano et al showed expression of TRPV4 in rat cardiac fibroblasts (Hatano, Itoh, & Muraki,

2009). We and others have established TRPV4 activation by mechanical forces such as cyclic stretch in endothelial cells, cell swelling in endothelial and epithelial cells, and shear stress in endothelial and renal epithelial cells (Arniges, Vazquez,

Fernandez-Fernandez, & Valverde, 2004; Thodeti et al., 2009). Since, mechanical factors are critical for cardiac fibroblast differentiation and TRPV4 was a mechanosensitive ion channel, we investigated the role TRPV4 in cardiac fibroblasts differentiation into myofibroblasts. To determine this, we measured

TRPV4 expression using RT-PCR and calcium imaging and found that CF functional express of TRPV4 (Figure 8A, B). Further, we found that TGF-1 induced robust differentiation of CF to MF as indicated by incorporation of -SMA into stress fibers. Importantly, pharmacological inhibition of TRPV4 with AB159908

(Figure 8C) or siRNA knockdown of TRPV4 significantly attenuated TGF-1- induced CF differentiation. Next, we investigated whether TRPV4 regulates TGF-

1 induced CF differentiation though mechanical signaling. To explore this, we cultured CF on ECM gels with varying stiffness (low-98 Pa; intermediate-370 Pa and high-2280 Pa) and stimulated with TGF-1 in presence or absence of

AB159908. We found that TGF-1-induced

Figure 8. TRPV4 is required for TGF-1 induced CF differentiation into MF.

A&B) Representative PCR and western blot analysis showing the expression of

TRPV4 in rat CFs. C) Immunofluorescence images (20X) of fibroblast differentiation as demonstrated by the increase in expression and incorporation of

α-SMA (green) in to stress fibers. Nuclei were stained with DAPI (blue). CFs were serum starved and stimulated with TGF-β1 for 24 h in the presence or absence of

TRPV4 antagonist AB159908 (AB1). Adapted from Adapala et al J Mol Cell

Cardiol. 2013 Jan; 54: 45–52.

differentiation of CF cultured on high stiffness gels but not on low stiffness (98Pa,

370Pa) gels (Figure 9A). Further, pharmacological inhibition of TRPV4 is significantly diminished the TGF-1-induced CF differentiation on high stiffness gels (Figure 9B). Taken together, these findings indicate that TRPV4 mediates CF differentiation by integrating soluble (TGF-1) and mechanical (stiffness) signaling.

Additionally, we found that TGF-1 stimulation increased TRPV4 expression and function (Adapala et al., 2013). Recently, TRPM7 was shown to mediate atrial fibroblast differentiation, though AF exhibit robust TRPV4 expression (Du et al.,

2010). Interestingly, we found that ventricular fibroblasts show high expression of

TRPM7 but pharmacological inhibition of TRPM7 with carvacol failed to inhibit

TGF-1 induced ventricular CF differentiation. These results suggest that TRPM7 does not play a role in ventricular fibroblasts differentiation and TRPV4 is the mechanosensor that integrates both soluble and mechanical signaling in CF differentiation (Adapala et al., 2013). In the present dissertation, I have addressed the physiological significance of TRPV4 following injury/insult and molecular mechanisms by which TRPV4 regulate CF differentiation into MF.

Figure 9. TRPV4 channels mediate TGF-β1-ECM stiffness-induced CF differentiation: A) Immunofluorescence images of CF differentiation (the incorporation of α-SMA in to stress fibers (green) on gelatin hydrogels of varying stiffness (98–2280 Pa). B) Immunofluorescence images depicting TRPV4- dependent differentiation of CFs on high stiffness gels in the presence or absence of TRPV4 antagonist AB159908. Adapted from Adapala et al J Mol Cell Cardiol.

2013 Jan; 54: 45–52.

1.7. Significance of the present study Ischemic heart diseases emerged as the leading cause of death in the world. Although advanced interventional therapies improving the patient survival, it poses new challenges for after care as maladaptive remodeling of the heart

(cardiac fibrosis) in these patients may lead to heart failure and death. Current therapies for the treatment of pathological remodeling are mostly based on targeting soluble factor signaling mechanisms. Even though, these therapies are efficient at preclinical level, but showed modest results at clinical settings (Z. Fan

& Guan, 2016). Thus, alternative approaches are needed to identify novel therapeutic targets. Since the heart is continuously exposed to the mechanical stretch, it is conceivable that mechanical forces play a key role in cardiac fibrosis.

In fact, our recent results demonstrated that TRPV4 regulates CF differentiation to

MF via integration of soluble and mechanical signaling (Adapala et al., 2013).

However, the physiological significance of TRPV4 in cardiac remodeling following injury/insult and the underlying molecular mechanism by which TRPV4 integrates soluble and mechanical signaling is not known. Therefore, investigating the physiological role of TRPV4 and dependent mechanotransduction may aid in the development of better therapeutic targets to treat fibrosis in heart and other organs.

Specific Aims

We hypothesize that TRPV4 regulates CF differentiation via integration of

soluble and mechanical signaling and deletion of TRPV4 reduces cardiac fibrosis

and protects the myocardium following injury/insult. To test this hypothesis, we

propose the following specific aims:

1. Determine the functional significance of TRPV4 in cardiac remodeling following

pressure overload-induced hypertrophy.

2. Delineate the molecular mechanisms by which TRPV4 regulate cardiac fibroblasts

differentiation into myofibroblasts.

3. Ascertain the functional role of TRPV4 in cardioprotection following myocardial

infarction.

Specific Aim 1. Determine the physiological significance of TRPV4 in cardiac

remodeling following pressure overload-induced cardiac hypertrophy: We

hypothesized that absence of TRPV4 preserves cardiac function and reduces

collagen deposition following pressure overload-induced hypertrophy.

1a. The absence of TRPV4 modifies cardiac structure and function post-transverse

aortic constriction surgeries: WT and TRPV4KO mice were subjected to pressure

overload by transverse aortic constriction (TAC). Cardiac function was measured

non-invasively using 2D echocardiography before and 28 days after TAC

surgeries. Cardiac hypertrophy was analyzed using wheat germ agglutinin immune

staining.

1b. TRPV4 deletion attenuates the deposition of interstitial fibrosis following pressure overload-induced hypertrophy: Heart tissues sections were analyzed for fibrosis using Masson’s trichrome and Picro Sirus Red (PSR) staining.

Specific Aim 2. Delineate the molecular mechanisms by which TRPV4 regulate CF differentiation into myofibroblasts:

Previously, we demonstrated that CFs express functional TRPV4 and these channels are required for integrating TGF-1(soluble) and matrix stiffness

(mechanical) induced CF differentiation (Adapala et al., 2013). However, underlying molecular mechanisms by which TRPV4 regulate CF differentiation is not known. Previous studies from our lab have shown that TRPV4 regulate cyclic stretch-induced endothelial cell reorientation through modulating Rho/Rho kinase pathway (Ghosh et al., 2008; Thodeti et al., 2009). Therefore, we hypothesized that whether TRPV4 may regulate CF differentiation through regulating Rho/Rho kinase pathway.

2a. Absence of TRPV4 attenuates TGF-1-induced CF differentiation: Cardiac fibroblasts were isolated from TRPV4KO mice to confirm TRPV4 is required for

TGF-1-induced CF differentiation. Next, to find whether TRPV4 activation induces pro-fibrotic gene expression, promoter activity assays for -SMA and col11 were performed following stimulation with TGF-1 or TRPV4 agonist (GSK 1016790A) in the presence or absence of TRPV4 antagonist AB159908.

2b. TRPV4 regulates CF differentiation through Rho/MRTF-A pathway: To delineate the molecular mechanism downstream of TRPV4, first, we measured activation of Rho kinase and MRTF-A in response to TRPV4 activator and TGF-

1. We then confirmed the participation of Rho kinase and MRTF-A in CF differentiation by employing pharmacological inhibitors and siRNA knockdown.

Specific Aim 3. To ascertain the functional role of TRPV4 in cardio protection following Myocardial Infarction (MI)

Although TRPV4 is implicated in CF differentiation and angiogenesis, it is not known whether and how TRPV4 protects heart from ischemia induced by myocardial infarction. Therefore, in this aim, we investigated if TRPV4 deletion offers cardioprotection following MI by assessing cardiac function, cardiomyocyte hypertrophy, cardiomyocyte apoptosis, angiogenesis and cardiac fibrosis.

3a. TRPV4 deletion preserves cardiac structure and function following acute MI:

WT and TRPV4KO mice were subjected to acute MI via left anterior descending artery ligation. Cardiac function and structure were analyzed using 2D echocardiography, immunohistochemistry and PSR staining.

3b. Deletion of TRPV4 offers cardioprotection via decreased cardiomyocyte apoptosis and enhanced angiogenesis: To determine the molecular mechanism in cardioprotection offered by TRPV4 deletion, we measured cardiomyocyte apoptosis (TUNEL assay) and angiogenesis (CD31 immunostaining) in heart sections from sham and MI, WT and TRPV4KO mice.

CHAPTER II

Materials & Methods

Animals

WT and TRPV4 knockout (TRPV4KO) male mice in the C57BL/6 background at the age of 4-6 months and 12-16 weeks were used for Transverse aortic constriction (TAC) and Myocardial infarction (MI) surgeries, respectively. All mice were acclimatized in the conventional facility to standard laboratory environment and kept on a 12:12 h day/night cycles and accessed to standard chow and tap water ad libitum. All the studies were approved by the Institutional Animal Care and Use Committee (IACUC) at the Northeast Ohio Medical University (NEOMED),

Rootstown, OH (Protocol# 17-06-117; 15-029; 13-010; 10-007)

Surgical Procedures

Transverse Aortic Constriction (TAC)

TAC was attained via ligating the aorta in between branchio-cephalic and carotid artery (Figure 10A). Mice were anesthetized with 3% isoflurane and secured on a surgical pad (Indus instruments) on its supine position. Hair was removed from the

chest to neck using depilated cream and then disinfected the skin with an iodine solution. An incision was made from neck to sternum till 4th or 5th rib to separate the muscle around the trachea. Aorta was exposed by making an incision on the

1st rib of the sternum separating with retractors. Aortic constriction was made by passing the 5-0 nylon suture around the aorta along with the 27 gauge blunted needle. The sternum and skin were closed using 5-0 vicryl suture. Then anesthesia was turned off, Mice were then administered with Buprenex (0.03mg/kg) as an analgesic and monitored until moved to facility. TAC was further confirmed by measuring the flow velocities using, Doppler 2D echocardiography (Figure 10B)

Myocardial Infarction (MI)

Myocardial infarction was induced in WT and TRPV4KO male mice by ligating left anterior descending (LAD) artery then allowed to recover and undergo a post-MI assessment at various time points. To perform surgery, mice were first intra peritoneally injected with atropine sulfate (0.04 mg/kg body wt, i.m.; Phoenix

Scientific, St. Joseph, MO, USA) as a pre-anesthetic to prevent respiratory secretions that may obstruct airways during surgery. Then mice were intubated with 20 gauge angio-catheter through visualizing the trachea with the help of Biolite illuminator (Harvard apparatus) under anesthesia. The intubation catheter was connected to rodent ventilator (Minivent type 845; Hugo Sachs Electronik, Harvard

Apparatus, Germany) and secured with a knot to its mouth. Then, mice were protected to surgical pad (Indus instruments) in supine position. The mouse

Figure 10. Transverse Aortic Constriction (TAC). A) Diagrammatic representation of transverse constriction of aorta. B) Doppler pulsed wave representation of flow velocities at constriction site. The increase in flow velocity confirms aortic constriction.

was continuously anesthetized with 3% isoflurane through the ventilator and tidal volume and frequency were calculated from the following equation.

Vt = 6.2 x Mb

Where Vt is tidal volume (mL) and Mb is animal body mass (in kg)

BPM=53.5 x Mb

Where BPM = ventilation rate (breaths/minute) (Tarnavski et al., 2004)

Hair was removed, skin was disinfected with iodine solution, and an incision was made along the sternum towards neck to separate the pectoral muscle. A small incision made in between 4th and 5th ribs with blunt scissors exposing the anterior wall and apex of the left ventricle. Left anterior descending artery was located which origins from the root and followed down to mid papillary muscles where ligation was made with 8-0 silk suture. The induction of infarction was confirmed with ST elevation in EKG apparatus (Mouse monitor, Indus instruments)

(Figure 11). The thoracic cavity was closed with 5-0 vicryl suture and the intrathoracic negative pressure was generated through 24 guage angio-catheter.

The skin was closed using 5-0 vicryl suture and the lungs were inflated parting obstructing outflow of the ventilator. Mice were then administered with analgesics

Buprenex (0.03mg/Kg) and anesthesia was stopped. The mouse was monitored until it recovered from anesthesia and surgery and have normal behavior then transferred to animal facility.

Figure 11. Electrocardiogram showing myocardial Infarction: EKG showing the ST elevation (Red arrow) and reciprocal ST depression (green arrow) on the opposite wall following left anterior descending artery ligation (LAD) to induce myocardial infarction in mice.

2D-echocardiography

The murine cardiac function was measured using a Vevo 770 system (Visual

Sonics.Inc, Toronto Canada) with a 710B-075 transducer (20-30MHz) with a frame rate of 40-60 Hz. Briefly, animals were anesthetized using 3% isoflurane (1.0 ml/min) through a nose cone and secured in its supine position on an adjustable platform equipped with EKG to monitor heart rate and respiration rate. 2D and pulsed Doppler wave (PW) images were acquired in parasternal short, long axis and apical four chamber views followed by PW inflows were obtained at mid-papillary level. All measurements were calculated offline by blinded reviewer using Vevo 770/3.0 software (Figure 12).

Histology

Animals were euthanized 28 days after TAC and 8 weeks of MI, respectively.

Hearts were excised and fixed in 4% paraformaldehyde (PFA). Prior to sectioning, fixed tissues were processed and embedded in paraffin and sectioned with a microtome (Leica Biosystems) at 7-10 m thickness laid on to super frosted slides.

For cryosections, tissues were rinsed in PBS and then embedded in OCT (Optical controlling temperature) solution and then sectioned using Leica 2000 system.

Cardiac hypertrophy

Paraffin sections of heart were deparaffinized with a series of washes of xylene, ethanol (100, 90 and 70%) and water. Then round marks were made around the section with aqua repellent marker and incubated with 1:500 Alexa Flour-594 wheat germ agglutinin (Invitrogen) for 1 h. After incubation, slides were washed

Figure 12. Echocardiographic analysis of cardiac function. A) Basal long axis view of left ventricle depicting aorta, LV, LV anterior and posterior walls, and atrial appendages. B) M-mode image at mid papillary level demonstrating LV size, LV wall thickness during systolic and diastolic rhythms. LV- Left ventricle; RA-Right

Atria; LA-Left Atria; AWT- Anterior wall thickness; LVID – Left ventricular internal diameter; PWT – Posterior wall thickness; d- diastolic; s- systolic.

in PBS and then mounted with DAPI containing Vecta shield mounting media

(Vector Laboratories). Images were acquired randomly around the heart using

Olympus IX80 fluorescence microscope and myocyte cross-sectional area was measured using Image J software.

Cardiac fibrosis

Cardiac fibrosis was measured by staining for collagen. Paraffin sections from post-MI, post-TAC and sham hearts were stained with Picrosirius Red (PSR) and

Masson’s Trichrome. Images were acquired, and total collagen deposition was measured using ImageJ software. Additionally, individual collagen types were observed using polarized light microscopy on PSR stained sections (Whittaker,

Boughner, & Kloner, 1989). Briefly, the presence of red to orange birefringence indicates collagen I whereas green to yellowish indicates collagen III, under polarized light. The percent of collagen was calculated from 2X images of the heart

(n>4 hearts per group).

TUNEL assay

Myocyte apoptosis was analyzed by TUNEL assay of heart sections from 7 days post-MI. Heart cryosections were fixed with paraformaldehyde following manufacturer’s protocol (Takara Bio Inc, Japan). Briefly, heart sections were permeabilized with 0.2% TritonX-100 and then incubated with terminal deoxy transferase (TdT). Following that, sections were labeled with a primary antibody against TdT enzyme. After washing, sections were incubated with a secondary antibody conjugated to Alexa Flour-488. Images were acquired using Olympus

IX80 fluorescence microscope and the apoptotic cells were quantified.

Capillary Density

Post-MI heart sections were fixed with cold acetone and then washed with water and PBS. Slides were blocked with 10% normal donkey serum and incubated with anti-rat mouse CD31 (1:50) antibody for overnight. Sections were then incubated with Alexa Flour-594 secondary antibody (1:500) and images were acquired at 20X and 60X using Olympus IX80 fluorescence microscope. Capillary density was quantified by measuring the CD31 positive vessels with ImageJ software and represented as capillary density per field.

Isolation of rodent cardiac fibroblasts

Cardiac fibroblasts were isolated from 12-15-week-old mice or rat. Briefly, animals were euthanized, hearts (only ventricles in case of rat) were minced in

ADS buffer (HEPES-18.3 mM; MgSO40.8mM; NaH2PO4-1mM; NaCl-116.4 mM; D-

Glucose-5.5 mM; KCl-5.5 mM; Phenol Red-0.02 g) and tissues were digested with collagenase 2 (0.2mg/mL) and trypsin (0.75 mg/mL). The cell suspension was centrifuged, and the pellet was resuspended in growth media (DMEM low glucose,

10% FBS, 1% Penicillin and streptomycin, 1.5 ug of FGF, 20 ng of VEGF, and 50 mg of Heparin) then plated on 60 mm dish. After 24 h, cells were washed with PBS and then supplemented with growth media. mCF (mouse CF) were used from 0-2 passages and rCF (rat CF) were used up to 3 passages. Importantly, at these early passages, both mCF and rCF showed low expression of alpha-smooth muscle actin (-SMA) in the cytoplasm but it is not incorporated into stress fibers. This phenotype is often characterized as proto-myofibroblasts but for brevity we referred them as CF.

CF differentiation

To differentiate the cardiac fibroblasts into myofibroblasts, cells were serum starved and then incubated with TGF-1 (10ng/mL) for 24 h. CF differentiation was determined by -SMA incorporation in to stress fibers. In indicated experiments, cells were either pre-treated with the TRPV4 antagonist (AB159908) or Rho kinase inhibitor (Y27632) or MRTF-A inhibitor (CCG1423).

Calcium Imaging

CF cultured on MatTek glass bottom dishes were loaded with Fluo-4/AM (3M) for

20min, washed and kept in calcium medium on an inverted Olympus IX70 confocal microscope. Images were acquired every 3 seconds before and after stimulation with TRPV4 agonist GSK1016790A (100 nM). The change in Fluo-4 flourescence intesity was calculated as F/F0 (ratio of normalized intensity relative to 0 time) using Microsoft Excel. In some of the experiments CF were incubated with TRPV4 antagonist AB159908 or GSK2193874 for 20 min prior to stimulating with

GSK1016790A.

Immunofluorescence

After appropriate stimulation, cells were fixed in 4% paraformaldehyde then permeabilized with 0.25% TritonX-100 and blocked with 10% FBS (Fetal Bovine

Serum). Then, cells were incubated with primary antibodies such as - SMA

(1:250; Sigma) and MRTF-A (1:100 Santa Cruz) for 1h. Cells were then washed

with PBS (X 3) and incubated with respective Alexa Fluor secondary antibodies.

After washing with PBS (X 3), cells were mounted with DAPI contained mounting media. Images were acquired using Olympus IX80 fluorescence microscope and analyzed using ImageJ.

Western blotting

Cells were lysed with 1% Triton X-100 containing protease and phosphatase inhibitors, protein concentration was measured using BCA protein assay (Thermo

Scientific). Lysates were subjected to SDS-PAGE gel electrophoresis and then transferred onto PVDF membrane, PVDF membrane was blocked with 5% milk and then incubated overnight with primary antibodies (MRTF-A; 1:500; Tubulin;

1:2000). Membranes were then washed with TBST (X 3) and incubated with HRP conjugated secondary antibodies (1:20,000) for 1h at room temperature. After incubation, membranes were washed with TBST (x 3), incubated with chemiluminescence reagent and developed using Protein Simple. The intensity of protein bands was quantified using the ImageJ. qPCR

Cells were lysed, and RNA was isolated using RNeasy Mini Kit (Qiagen). RNA concentration was measured with the NanoDrop 2000 UV-Vis Spectrophotometer. cDNA was synthesized using qScript cDNA synthesis kit (Quanta Biosciences) and qPCR was carried out with the Fast SYBR green master mix (Applied Biosystems) using the Fast-Real-Time PCR system (Applied Biosystems). Real-time primers for GAPDH, MRTF-A, and TRPV4 were purchased from IDT technologies. Gene

expression was analyzed relative to GAPDH values and the ΔΔCT values are expressed as a fold change.

Promoter activity Assay rCFs were transfected with pGL3--SMA or pGL3-col11 along with p- galactosidase plasmids using lipofectamine 2000 and media was changed after 6 h. Cells were stimulated with TGF-1 or GSK in the presence or absence of TRPV4 antagonist (AB159908) or MRTF-A inhibitor (CCG1423) and after 24 h, luciferase and -galactosidase assay were carried out individually. Luciferase assay was performed using dual Glo luciferase assay kit from Promega following manufacture’s protocol. Luminescence was detected using Glomax multi detection system. Meanwhile, -galactosidase activity was assayed in lysates after incubation with 2X assay buffer and absorbance was measured at 420 nm.

Rho-kinase activity assay

Rho kinase activity was measured using Cyclex Rho kinase activity assay kit (MBL int) following the manufacturer’s protocol. Briefly, mCFs were serum starved and stimulated with TGF-1 in the presence or absence of TRPV4 antagonist

AB159908 or CCG1423 and incubated for 1h. Cell lysates were mixed with activation buffer and then added to the pre-coated 96 well plate and then incubated for 2 h at 40C. After incubation, the plate was rinsed, incubated with HRP- conjugated antibody. Color was developed by adding substrate and the absorbance was measured at 450 nm, after stopping the reaction.

siRNA

WT-mCFs cultured in 6 well plate (70% confluency) were transfected with 10 nM of control (non-target smart pool siRNA) and MRTF-A specific siRNAs

(Dharmacon) using siLentiFect (Biorad) in Opti-MEM. Six hours after transfection,

Opti-MEM was replaced with complete growth media. After 48 h, cells were lysed and MRTF-A knockdown was confirmed by western blotting and qPCR.

Statistical analysis

The data was presented as mean ± SEM from at least three independent experiments. The significance was determined using Student’s t- test, analysis of variance (ANOVA) and Tukey’s post-hoc analysis with significance set at p ≤ 0.05.

Table 1. List of primers

Forward Reverse

TRPV4 5’ CCT GCA CAT TGCCAT CGAAC 3’ 5’ ATC CTT GGG CTG GAA GC 3’

MRTF-A 5’CCC TGG TTA AGA GAC TGT G 3’ 5’GCT GAA ATC TCT CCA CTC TGA A 3’

GAPDH 5’ GGG TCC CAGCTT GGTTCATC 3’ 5’ ATC CGT TCA CAC CGA CCT TC 3’

CHAPTER III

Results

3.1. Specific Aim 1. Determine the physiological significance of TRPV4 in cardiac remodeling following pressure overload-induced cardiac hypertrophy

3.1.1. Absence of TRPV4 preserves cardiac structure and function post- transverse aortic constriction surgeries

Heart failure due to pressure and/or volume overload is characterized with cardiac hypertrophy and increased interstitial fibrosis which can lead to diastolic and systolic dysfunction. To determine the physiological significance of TRPV4 in in vivo, WT and TRPV4KO mice were subjected to pressure overload by TAC and cardiac function and structure was measured before and 28 days after TAC.

First, we confirmed the sucess of TAC surgeries by measuring flow velocities.

We found a 75 + 5 % increase in flow velocity distal to the constriction area in

post-TAC mice (7 days) compared to sham animals confirming that TAC surgeries were successful. Cardiac hypertrophy was assessed by staining the heart sections with wheat germ agglutinin (WGA) and by measuring myocyte cross-sectional area (Figure 13 A, B) as well as mass of the left ventricles

(Figure 13C). WGA staining showed a significant increase in myocyte cross- sectional area in WT-TAC animals compared to WT-sham animals (934 ± 51 vs

312± 24) 28 days post-TAC (Figure 13 A, B). Importantly, we found that there is a non-significant increase in myocardial cross-sectional area in TRPV4KO-

TAC mice compared to TRPV4KO-sham mice. Interestingly, WT-TAC mice exhibited significantly higher myocyte cross- sectional area than TRPV4-KO mice. Next, M-mode images from echocardiography revealed that LV mass was enhanced in WT-TAC mice compared to TRPV4KO-TAC mice (Figure 13C). These findings indicate that absence of TRPV4 alleviates the effects of the pressure overload on cardiomyocyte hypertrophy and preserves structural integrity.

Figure 13. TRPV4 deletion maintains myocardial integrity following TAC. A)

Representative images (20X) of WGA immunostaining and B) quantification of myocardial cross-sectional area in WT and TRPV4KO hearts, 28 days after TAC.

C) Quantification of LV mass corrected to body weight in WT and TRPV4KO mice,

28 days post-TAC (n=6-9; significance *p≤0.05)

Next, we found that significant akinesis of posterior wall in WT-TAC hearts compared to WT-sham hearts suggesting that pressure-overload diminished cardiac function. However, TRPV4KO-TAC hearts exhibited normal posterior wall movement (Figure 14A). Further, cardiac function (ejection fraction and fractional shortening) analysis using 2D echocardiography found that both ejection fraction and fractional shortening were significantly decreased in WT-

TAC mice compared to WT-sham (%EF: 26.84± 4.14 vs 62.4 ± 3.71). Notably, we found that both ejection fraction and fractional shortening were improved in

TRPV4KO-TAC mice compared to WT-TAC mice (Figure 14B). Taken together, these findings indicate that the absence of TRPV4 preserves the myocardial structure and function when subjected to pressure overload-via

TAC.

Figure 14. Absence of TRPV4 preserves cardiac function after TAC- induced pressure overload. A) M-mode images representing the posterior wall akinesis in WT animals after TAC compared to Sham. In contrast, posterior wall kinesis is intact in TRPV4KO mice post- TAC. B) Quantitative analysis of cardiac function (% ejection fraction and % fractional shortening) in WT,

TRPV4KO mice, 28 days after TAC (n=6-9; significance at *p<0.05).

3.1.2. TRPV4 deletion attenuates cardiac fibrosis induced by TAC.

To determine if TRPV4 deletion preserves cardiac structure and function by inhibiting cardiac fibrosis, we stained heart tissues for collagen with PSR and

Masson’s Trichrome (Figure 15A, B). PSR staining revealed that WT-TAC hearts exhibited increased interstitial fibrosis compared to sham following 28 days of TAC. Conversely, TRPV4KO-TAC hearts exhibited significantly less fibrosis (Figure 15A). The unique property of the collagens under polarized light after staining with PSR helps to discriminate between different collagens based on their color patterns. More mature type I collagen will appear red to orange while collagen III will appear as green to yellow. When we observed PSR staining in polarized light, WT-TAC hearts showed dense red pool of staining

(indicative of mature Collagen-I fibers) with less dense green pockets, whereas

TRPV4KO-TAC hearts displayed minimal color patterns or light yellow (Figure

15A). Similarly, staining with Masson's Trichrome demonstrated robust blue staining indicating high collagen content (fibrosis) in WT-TAC hearts compared to TRPV4KO-TAC hearts (Figure 15B). Quantification of fibrosis (collagen content) revealed that TAC-induced significant amount of fibrosis in the hearts of WT-TAC compared to sham and TRPV4KO-TAC mice. Notably, TRPV4KO-

TAC hearts show very little fibrosis which is comparable to sham mice (Figure

15C). These data suggest that TRPV4 deletion preserved cardiac function by maintaining myocyte structural integrity and reducing collagen deposition following TAC.

Figure 15. TRPV4 deletion reduces cardiac fibrosis post-TAC. A)

Representative images (10X) of post-TAC WT and TRPV4KO hearts stained with

Picrosirus red (PSR), and polarized microscopy revealed increased deposition of matured collagen post-TAC. B) Representative images (10X) stained with

Masson's Trichrome showing cardiac fibrosis (ECM) in WT and TRPV4KO hearts post-TAC. C) Quantification of percent collagen deposition to LV area in stained

(PSR) sections of WT and TRPV4KO hearts 28days after TAC (n=5-9; significance at *p<0.05).

3.2. Specific Aim 2. To delineate the molecular mechanisms by which TRPV4 regulate cardiac fibroblasts differentiation into myofibroblasts.

3.2.1 TRPV4 regulates profibrotic gene expression in cardiac fibroblast

Cardiac fibroblast differentiation into myofibroblast is the key event in the cardiac remodeling process. Previously, we have found that TRPV4 plays a crucial role in CF differentiation via integrating soluble and mechanical signaling in rat cardiac fibroblasts (rCF) (Adapala et al., 2013). Importantly, findings from Aim1 indicate that absence of TRPV4 attenuates cardiac fibrosis following TAC.

Therefore, in this aim, we investigated the underlying molecular mechanisms by which TRPV4 regulate TGF-1 induced CF differentiation.

To confirm the role of TRPV4 in mouse CF differentiation, first we have isolated CF from WT and TRPV4KO mice as described in methods and mCF at 1-

2 passages were used in all the experiments. Functional deletion of TRPV4 was confirmed with qPCR and calcium influx assays. qPCR analysis revealed robust expression of TRPV4 in WT mCFs which is absent in TRPV4KO mCF (Figure

16A). Next, WT-mCFs exhibited robust calcium influx following stimulation with

TRPV4 agonist, GSK1016790A (GSK1). Further, GSK1-induced calcium influx was attenuated in the presence of TRPV4 antagonist AB159908. Furthermore,

TRPV4KO mCF didn't show any calcium influx after GSK1016790A treatment confirming the functional deletion of TRPV4 in these cells (Figure 16 B-D). Next, we evaluated mCF differentiation by TGF-1 as evidenced by -SMA incorporation into stress fibers. We found that WT mCF displayed increased

Figure 16. Functional characterization of TRPV4 in mouse CF. A) qPCR analysis of TRPV4 expression in isolated mCF from WT and TRPV4KO mice. B and C) Representative ratio images (20X) and traces showing relative changes in cytosolic calcium in response to a selective TRPV4 agonist, GSK1016790A (100 nM) in Fluo-4 loaded WT and TRPV4KO mCF (n=300). Arrow indicates the time at which TRPV4 agonist added to cells. D) Quantitative analysis of cytosolic calcium influx induced by GSK1016790A in mCF. (F/F0 = ratio of fluorescence intensity relative to time 0). The results shown are mean ± SEM from 3 independent experiments. Significance was set at p≤0.05.

-SMA expression and incorporation into stress fibers following TGF-1 treatment compared to cells treated with controls (Figure 17A). However, TGF-1-induced

CF differentiation was completely attenuated in TRPV4KO mCF. Quantitative analysis revealed that TGF-1 induced robust differentiation (percent of myofibroblasts) of WT-mCF which was completely attenuated in TRPV4KO-mCF

(80.83±6.11 Vs 19.71±1.25). In fact, the myofibroblast percentage in TRPV4KO- mCF were shown to be at the level of unstimulated cells (Figure 17A). Next, to find out whether TRPV4 activation affects fibrotic gene expression, we performed pro-fibrotic gene promoter activity assays. rCFs were co-transfected with pGL3Col11 / pGL3--SMA along p-galactosidase then stimulated with TGF-1 in presence or absence of TRPV4 antagonist AB159908. As a control, we also stimulated CF with TRPV4 agonist, GSK1016790A. As shown in Figure 17B, the promoter activity of Col1β1 and -SMA was increased after TGF-1 treatment, which is significantly inhibited in presence of AB159908. Notably, TRPV4 activation with GSK1016790A also enhanced -SMA and col11 promoter activity compared to control or in the presence of AB159908. These findings indicate that

TRPV4 channels mediate CF differentiation via activation of pro-fibrotic gene expression.

Figure 17. TRPV4 is required for fibrotic gene expression in mouse cardiac fibroblast differentiation into myofibroblast. A) Fluorescence images (20X) showing -SMA in WT and TRPV4KO mCF following TGF-1 stimulation.

Quantitative analysis of fibroblasts differentiation (-SMA positive cells). Note that complete attenuation of TFG-1-induced differentiation in TRPV4KO mCF. B) rCFs (Rat cardiac fibroblasts) were transfected with pGL3--SMA or pGL3-Col11 along with p-galactosidase plasmids and TRPV4 (GSK)-induced promoter

(luciferase) activity was measured in the presence or absence of TRPV4 antagonist, AB1. The data presented are mean + SEM of three separate experiments. Significance was set at p≤0.05.

3.2.2. TRPV4 mediates CF differentiation through Rho/Rhokinase/MRTF-A pathway

Previous studies from our lab demonstrated that TRPV4 regulates endothelial cell reorientation through modulating Rho/ROCK signaling (Ghosh et al., 2008; Thodeti et al., 2009). Therefore, to investigate the molecular mechanism by which TRPV4 regulates CF differentiation, we focused on mechanosensitive

Rho/MRTF-A signaling pathway. First, we measured Rho kinase activity in WT and

TRPV4KO mCFs in response to TGF-1 in the presence of TRPV4 antagonist.

TGF-1 induced robust Rho kinase activity in WT-mCF which was reduced in the presence of TRPV4 antagonist AB159908 (0.77+0.014 Vs. 0.25+0.01) (Figure

18A). Importantly, TRPV4KO mCF exhibited reduced low levels of Rho kinase activity in response to TGF-1 (Figure 18B). Next, we measured TGF-1-induced differentiation in WT-mCF in the presence of ROCK inhibitor, Y27632. We found that pre-treatment of mCF with Y-27632 significantly decreased TGF-1-induced mCF differentiation compared to untreated WT mCF (Figure 18C, D).

Figure 18. TRPV4 mediates CF differentiation via Rho/Rho kinase pathway.

A) TGF-1 induces Rho kinase activation in WT mCFs which is attenuated by

TRPV4 antagonist AB159908. B) TGF-1-induced Rho kinase activation is suppressed in TRPV4KO mCF. C) Representative images (20X) showing TGF-1- induced differentiation (as indicated by -SMA staining in stress fibers) in the presence or absence of Y27632 (Y27) in WT mCFs. D) Quantitative analysis of mCF differentiation. The data presented are mean + SEM of three separate experiments. Significance was set at p<0.05.

Since Rho/Rho kinase- activates a mechanosensitive transcription factor,

MRTF-A in pulmonary fibroblasts, (Gombedza et al., 2017) we asked if MRTF-A is involved in TRPV4/TGF- mediated CF differentiation. To explore this, first we measured MRTF-A activation by assessing its nuclear translocation. WT-mCFs were stimulated with either TGF-1 or GSK in the presence or absence of

AB159908 and stained with specific antibodies against, MRTF-A. We found that both TGF-1 and GSK1 increased the translocation of MRTF-A into nucleus as evidenced by colocalization of MRTF-A and DAPI (Figure 19). Further, this TGF-

1/ GSK1-induced MRTF-A nuclear translocation was significantly inhibited by

AB159908. Importantly, AB1 treatment alone did not inhibit the MRTF-A nuclear translocation. Next, to find out, if MRTF-A mediates TGF-1/TRPV4-induced CF differentiation, we treated mCF with MRTF-A inhibitor, CCG004 and measured CF differentiation. As expected, we found robust differentiation of WT-mCF in response to TGF-1 as evidenced incorporation of -SMA into stress fibers. In contrast, CCG1423 treatment significantly attenuated TGF-1-induced mCF differentiation (not shown). To confirm this, we knocked down MRTF-A in WT-mCF using specific siRNAs. First, we standardized the siRNA concentration and found that 10nM concentration of siRNA induced 70% reduction in MRTF-A expression as measured by Western blot and qPCR (Figure 20). Next, we found that both

TGF-1 and GSK-induced differentiation was significantly inhibited in MRTF-A knocked down WT- mCFs compared to control siRNA-treated mCF (Figure 21A).

Next, we asked if MRTF-A is required for TRPV4/TGF-1-induced fibrotic gene expression. We found that

Figure 19. TRPV4 mediates TGF-1-induced MRTF-A activation. A)

Representative images (20X) of MRTF-A (green) nuclear translocation in WT-mCF in response to TGF-1 (10 g/ml) or GSK (100 nM) in the presence or absence of

AB159908. Nuclei were stained with DAPI (blue). B) Quantitative analysis of percent of cells that exhibited MRTF-A nuclear translocation (colocalization of

DAPI and MRTF-A). The data presented are mean + SEM of three separate experiments. Significance was set at p<0.05.

Figure 20. Silencing of MRTF-A using MRTF-A specific siRNAs. WT-mCFs were transfected with control and MRTF-A specific siRNAs using siLentiFect and cells were lysed 24-48 h after transfection for the measurement of mRNA and protein expression. A) qPCR and B) Western blot analysis of MRTF-A expression in control and MRTF-A knocked down cells. The data presented are mean + SEM of two separate experiments. Significance was set at p<0.05.

Figure 21. TRPV4 mediates mCF differentiation through MRTF-A pathway. A)

Immunofluorescence images (20X) showing TGF-1/GSK-induced differentiation of CF to MF (-SMA (red) incorporation into stress fibers) in control siRNA and

MRTF-A siRNA treated mCF. Note that siRNA knowndown of MRTF-A attenuated

CF differentiation. B-C) Pharmacological inhibition of MRTF-A with CCG1423 significantly inhibited TGF-1/GSK1- induced fibrotic gene (-SMA) promoter activity. The data presented are mean + SEM of three separate experiments.

Significance was set at p<0.05.

TGF-1/GSK-induced fibrotic promoter activities (col11 and -SMA, respectively) were significantly inhibited in the presence of MRTF-A inhibitor, CCG1423 (Figure

21B, C).

Finally, we asked if Rho/Rho kinase act upstream of MRTF-A during TGF-

1/TRPV4-induced CF differentiation. Indeed, pre-treatment with CCG1423 did not change TGF-1/GSK-induced activation of Rho kinase activity indicating that

Rho kinase acts upstream of MRTF-A in TGF1/GSK1-induced CF differentiation

(Figure 22).

Taken together, these findings confirmed that TRPV4 mediates TGF-1 induced CF differentiation by regulating profibrotic gene expression through modulation of Rho/Rhokinase/MRTF-A signaling pathway.

Figure 22. MRTF-A acts downstream of Rho kinase during CF differentiation.

WT mCFs were stimulated with TGF-1 in the presence or absence of MRTF-A inhibitor CCG1423 (CCG) for 1h and Rho kinase activity was measured. Note that

CCG1423 treatment failed to inhibit TGF-1-induced Rho kinase activity. The data presented are mean + SEM of three separate experiments. Significance was set at *p<0.05.

3.3. Specific Aim 3. Ascertain the functional role of TRPV4 in cardioprotection following myocardial infarction.

3.3.1. TRPV4 deletion protects myocardium from ischemia-induced pathological remodeling following acute MI

Ischemic heart diseases are more predominant cause of mortality and morbidity in the world. Although, recent sophisticated interventional therapies increase patient survival from myocardial infarction, MI often leads to chronic complications such as cardiac hypertrophy, cardiac fibrosis eventually leading to heart failure. In Aims 1 and 2, we determined that TRPV4 deletion preserves cardiac structure following pressure overload by attenuating cardiac fibrosis via modulation of CF differentiation and underlying molecular mechanisms. In this aim, we investigated if TRPV4 deletion also offers cardioprotection from ischemia i.e. following myocardial infarction.

To investigate this, we have subjected WT and TRPV4KO mice to MI by ligating the left anterior descending artery (LAD) and observed cardiac function for

8 weeks. Serial 2D echocardiographic analysis of cardiac function (% ejection fraction-%EF and fractional shortening-%FS) revealed that both EF and FS were reduced in WT-MI mice compared to WT-sham animals within 7 days post-MI which was further declined after 8 weeks (19.87±2.32 Vs 59.14±1.47) (Figure

23A). In contrast, TRPV4KO-MI mice exhibited slight decrease in EF and FS at

7days but that was significantly higher compared to WT-MI (39.93 ±5.42 Vs 19.87±

2.32). Further, cardiac function was preserved in TRPV4KO mice up to 8

Figure 23. TRPV4 deletion preserves myocardial function post-MI. A)

Echocardiographic analysis of cardiac function showed that deletion of TRPV4 significantly preserved the cardiac function compared to WT-MI mice, 8 weeks post-MI.

weeks post-MI and was significantly higher (10 + 2 %) compared to WT-MI mice

(Figure 23A). In addition, M-mode images from 2D echocardiography revealed that WT-MI exhibited anterior wall akiness compared to TRPV4KO-MI 8 weeks, post-MI.

Anatomical examination of the hearts 8 weeks after MI revealed that WT-

MI hearts illustrated damaged myocardium and anterior wall below the ligation site to apex region compared to WT-Sham hearts, whereas, TRPV4KO hearts showed less damaged myocardium following MI (Figure 24A). Further, to find out if TRPV4 deletion preserve the myocyte integrity post MI, we stained heart sections with

WGA. WGA staining revealed that WT-MI hearts exhibit increased myocyte cross sectional area at normal zone of myocardium compared to WT-sham animals

(Figure 24B). However, TRPV4KO-MI hearts demonstrated significantly reduced myocyte cross sectional area compared to WT-MI animals. Next, we asked if

TRPV4 deletion also reduces cardiac fibrosis (similar to TAC) and scar formation in response to MI. To determine this, we have stained 8 weeks post-MI heart sections from WT and TRPV4KO mice with Masson’s Trichrome and PSR.

Masson’s Trichrome and PSR staining revealed that WT-MI hearts have increased fibrosis both at infarct (scar) and remote regions of the myocardium. In contrast,

TRPV4KO-MI heart sections showed less fibrosis at infarct (scar) and no fibrosis at remote zone. Notably, TRPV4KO-MI hearts exhibited viable tissue at the infarcted areas which indicate that absence of TRPV4 preserved myocardium and attenuated deposition and organization of ECM at the infarct zone. Quantitative analysis revealed that collagen volume in

Figure 24. Absence of TRPV4 preserves myocardial structure and integrity following MI. A) Representative images of whole hearts from WT and TRPV4KO mice, 8 weeks post-MI showing infarcts. B) Histological analysis of heart sections stained with WGA showing (60X) that myocardial cross-sectional area in WT and

TRPV4KO mice, 8 weeks post-MI. C) Quantitative analysis revealed that a significant increase in myocardial cross-sectional area in WT mice 8 weeks after

MI compared to sham. However, myocardial cross-sectional area is significantly lower in TRPV4KO mice compared to WT mice, post-MI. The data presented are from n> 6. Significance was set at *p<0.05.

MI MI WT

Note Note that

zones of WT hearts

Sections from post

led significantly higher collagen collagen higher significantly led

Heart

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)

Significance was set at set *p<0.05. was Significance

post post MI

TRPV4KO TRPV4KO mice 8 weeks post MI.

MI. n

week

) to reveal cardiac fibrosis (collagen content).

WT WT and

d of any living tissue) and remote

PSR

collagen deposition collagen

(red)

irius irius red (

fibrosis ( fibrosis

fibrosis

icro

Quantification of Quantification

stained with

KO hearts exhibit reduced cardiac fibrosis

TRPV4

Figure and TRPV4KO mice were Representative images (4X) showing cardiac increased fibrosis (collagen content) in infarct (with scar devoi compared to sham hearts. In contrast, TRPV4KO hearts show reduced fibrosis in infarct (few areas of fibrosis with zones. remote and tissue) intact hearts, 8 to post weeks TRPV4KO compared content WT hearts in

TRPV4KO hearts was reduced compared to WT mice following MI (Figure 25).

Further, polarized microscopy images from PSR stained heart sections from WT-

MI mice revealed densely red scars made up of matured type I collagen throughout entire scar volume, suggesting near total loss of viable tissue in the infarcted region as well as at remote zone areas during remodeling process following MI. However, images from TRPV4KO mice demonstrated a sparse pattern of red birefringence indicating reduced deposition of matured type I collagen at the infarcted zone with viable tissue (Figure 25). These findings indicate that deletion of TRPV4 preserved cardiac function by reducing cardiac fibrosis and preserving myocyte and myocardial integrity, 8 weeks post-MI.

3.3.2. Absence of TRPV4 reduces myocyte apoptosis and enhances coronary angiogenesis, post-MI

To determine the molecular mechanism by which TRPV4 deletion offers cardio-protection, we measured myocyte apoptosis and angiogenesis. Images from TUNEL assay showed that increased TUNEL positive cells in heart sections from WT-mice compared to TRPV4KO-mice, 7 days post-MI (Figure 26A).

Quantification of TUNEL positive cells revealed that TRPV4 deletion significantly reduced the number of TUNEL positive cells compared to WT-MI animals indicating TRPV4 absence diminished myocytes apoptosis post-MI (Figure 26B).

In addition to myocyte apoptosis, revascularization is a crucial phase to attenuate the progression of myocardial infarction. Recent studies demonstrate the importance of angiogenesis, revealing that angiogenesis is established within three days of post-MI (Kobayashi et al., 2017). Previously, our lab has shown that

TRPV4 negatively regulates angiogenesis and TRPV4KO mice exhibit increased tumor angiogenesis (Adapala et al., 2016; Thoppil et al., 2015; Thoppil et al.,

2016). Therefore, we have measured capillary density in 7 days post- MI heart sections by immunostaining for CD31 (endothelial cell marker). In general, remote

(normal) zones showed significantly high capillary density in WT hearts which was almost double in TRPV4KO hearts. In contrast, we found that significantly less capillaries in infarct/boarder regions from WT and TRPV4KO hearts 7 days, post-

MI. However, quantification of capillaries in infarct/boarder zones revealed that

TRPV4KO hearts showed significantly higher capillary

reduced reduced

KO hearts showed

following following 7 days post MI

: green; nuclei: DAPI) in heart

TRPV4

TUNEL

wever,

Quantification of TUNEL positive cells per field

MI. Ho

Significance was set at *p<0.05. wasat set Significance

7days 7days following MI.

mice

is significantly reduced in TRPVKO hearts

compared to WT compared to

photo photo micrographs (20X) showing myocyte apoptosis (

WT WT and TRPV4KO

yocyte yocyte apoptosis

increased myocyte apoptosis in WT hearts, post

Figure Representative sections from revealed myocyte apoptosis

Figure 27. TRPV4KO hearts exhibit increased angiogenesis, 7 days post MI.

A) Representative images (20X) of heart sections from WT and TRPV4KO mice following 7 days post MI stained with CD31 to visualize capillaries (red). Images were acquired at normal and infarcted areas. B) Quantification of capillary density at infarcted area shows significantly higher capillaries (angiogenesis) in TRPV4KO hearts compared to WT mice, 7 days following MI. n>3 Significance was set at

*p<0.05.

density compared to WT hearts (53.14 + 6.38 Vs 27.9+3.85) (Figure 27).

Taken together, these results indicate that absence TRPV4 reduces myocyte apoptosis and fibrosis probably by increasing coronary angiogenesis, which aids in preserving cardiac structure and function following myocardial infarction.

CHAPTER IV

Discussion

The data presented in this dissertation demonstrate that TRPV4 plays a crucial role during cardiac remodeling following myocardial injury or insult. We conclude this based on our findings that 1) absence of TRPV4 preserved cardiac function and structure, with minimal interstitial fibrosis, following pressure overload-induced cardiac stress, 2) TRPV4 mediated CF differentiation into MF by modulating the novel mechanosensitive transcriptional Rho/Rhokinase/MRTF-A pathway, and 3) TRPV4 deletion offered cardioprotection from ischemia by preserving myocardial integrity and cardiac function, by preventing myocyte apoptosis and enhancing angiogenesis.

Heart failure is a clinical condition in which the heart is unable to pump enough blood to meet the metabolic requirements of the body. Continuous exposure to risk factors, such as smoking, hypertension, obesity, and diabetes,

can contribute to cardiomyopathies, including cardiac hypertrophy, myocardial infarction, and aortic stenosis (Benjamin et al., 2017; Go et al., 2013; Mozaffarian et al., 2015). The progression of heart disease is associated with extensive structural changes in chamber geometry and myocardial architecture. This can affect the mechanical performance of the heart and potentially trigger the remodeling process (Mann & Bristow, 2005). The goal of the remodeling process is to compensate cardiac injury or insult and aid in the recovery of the myocardium by depositing ECM. However, uncontrolled remodeling can result in excessive deposition of ECM which can disrupt the coordination of myocardial excitation and contraction coupling during systole and diastole. Both soluble and mechanical factors can be involved in abnormal remodeling. The development of soluble factor-based therapeutic agents is active at the preclinical level and aims to target cardiac fibrosis in chronic and acute cardiomyopathies (Z. Fan & Guan, 2016;

Fraccarollo et al., 2012). However, these agents have modest efficacy at the clinical level, which require investigation into alternative therapeutic pathways.

Focusing on novel mechanosensors and targets in mechanotransduction has become popular in recent years for the treatment of various cardiovascular diseases.

Among the studied mechanical factors, TRPV4 has received greater attention in various physiological and pathological conditions, including cancer and cardiovascular diseases. TRPV4 is a mechanosensitive ion channel, activated by mechanical stimuli such as shear stress, ECM stiffness, and cyclic strain, involved in both physiological and pathological conditions (Adapala et al., 2013; Matthews

et al., 2010; O'Neil & Heller, 2005; Thodeti et al., 2009). Previously, we have demonstrated that TRPV4 pharmacological inhibition, or deletion with siRNA, attenuated TGF1-induced CF differentiation (Adapala et al., 2013). Further, we found that TRPV4 mediates CF differentiation by integrating soluble (TGF1) and mechanical factor signaling. However, the functional role of TRPV4 in cardiac remodeling, post-myocardial injury or insult, is not known.

The physiological significance of TRPV4 in cardiac remodeling following pressure overload-induced cardiac hypertrophy

Irreversible enlargement of the heart is triggered by multiple stimuli including smoking, hypertension, aortic stenosis, myocardial infarction and stress, which alters the biochemical and mechanical properties of the myocardium, leading to pathological remodeling which can results in heart failure. Multiple soluble (TGF1, Ang II) and mechanical factors (mechanical stretch, matrix stiffness) are involved in this process. Recently, many studies have demonstrated the effects of soluble factors in hypertrophic cardiomyopathy. For example, mouse hearts treated with TGF1 neutralizing antibody, as well as TGF2r deletion, reduced fibrosis and exhibited persisted chamber dilation and dysfunction following pressure overload (Koitabashi et al., 2011; Koitabashi & Kass, 2011).

Moreover, Angiotensin II is widely used to study hypertension-induced hypertrophy and heart failure in mice (Crowley et al., 2006). Blockade of Ang II activity with

ACE inhibitors and ARBs was able to lower blood pressure and reduce heart failure

(Booz & Baker, 1996; Saleem, Bharani, & Gauthaman, 2010). The activation of both TGF1 and angiotensin is dependent on the alterations in mechanical forces

and stiffness (Hinz et al., 2007; Yamazaki et al., 1995); however, the mechanosensor and their effects in heart failure remain elusive.

Multiple studies have provided evidence that alterations in Ca2+ handling contributes to heart failure by activating abnormal calcium-dependent signaling

(Yano, Ikeda, & Matsuzaki, 2005). Among many calcium channels, the transient receptor potential ion channel family has received greater attention in regulating the pathophysiology of the heart. TRPC-1, -3, -4,-6, TRPM4, TRPM7, and TRPV-

1, -2, -4 are expressed in the heart and their role has been established in cardiac dysfunction. Mice lacking TRPC1 demonstrated preserved cardiac function and failed to exhibit the maladaptive effects of fibrosis or hypertrophy compared with

WT animals, 4 weeks after transverse aortic banding and angiotensin II infusion

(Seth et al., 2009). Interestingly, double knockdown of TRPC3 and TRPC6 in mice ameliorated the maladaptive changes in systolic and diastolic function, while individual knockdown of TRPC3 or TRPC6 was not protective post-TAC (Onohara et al., 2006; Seo et al., 2014). Several studies demonstrated that TRPC channels modulate the expression of -myosin heavy chain through regulating calcineurin-

NFAT signaling during heart failure (Nakayama, Wilkin, Bodi, & Molkentin, 2006;

Seo et al., 2014). Although TRPC3 and TRPC6 are both activated by mechanical stress, their inhibitors failed to protect the effects of cardiac hypertrophy (Seo et al., 2014). TRPV1 and TRPV2, also involved in cardiac remodeling, are upregulated post-TAC. Absence of TRPV1 reduced cardiac hypertrophy as well as collagen III and apoptotic markers, such as cleaved caspases. Dietary capsaicin, a known TRPV1 activator, attenuated MMP activity and angiotensin II-induced

fibroblast proliferation (Buckley & Stokes, 2011; Q. Wang et al., 2014). TRPV2 deletion demonstrated no effect on cardiac function and progression of heart failure following MI (Koch et al., 2017). TRPM7 and TRPV4 have both demonstrated to regulate CF differentiation in vitro (Du et al., 2010). In vivo, pharmacological inhibition of TRPM7 reduced vascular remodeling in rats, post-

TAC (Li et al., 2014). Although TRP channels are implicated in cardiac remodeling and disease, there are no studies on the role of TRPV4 in chronic heart failure.

In our current study, we provide the physiological significance of TRPV4 in heart failure. TAC induces pressure-overload which is sensed by mechanosensors in the various cells of heart and respond by triggering myocyte hypertrophy as well as interstitial fibrosis. Indeed, we found that WT-TAC animals exhibit increased myocyte-cross sectional area and LV mass. However, we found that the absence of TRPV4 maintained myocyte cross sectional area and left ventricular mass, preserving systolic and diastolic function following pressure overload. Although we do not know the exact mechanism for this reduced myocyte hypertrophy, we speculate that absence of mechanosensor such as

TRPV4, render the cells not to respond to the mechanical signals that trigger hypertrophy. Alternatively, absence of TRPV4 may inhibit CF differentiation to

MF and reduces cardiac fibrosis which will limit the hypertrophy. In fact, histological analysis demonstrated that TRPV4KO hearts exhibited significantly less collagen deposition after TAC when compared with their WT counterparts.

Additionally, in Aim 3, we found that TRPV4KO hearts exhibit increased capillary density which may preserve cardiomyocyte survival and structure by providing

required blood flow and preventing interstitial fibrosis. Irrespective of the exact mechanism by which it preserves cardiac structure and function, targeting

TRPV4 offers novel approach to inhibit cardiac hypertrophy.

TRPV4 regulates cardiac fibroblasts differentiation through modulating

Rho/Rhokinase/MRTFA pathway

Pathological fibrosis after injury plays a dynamic role in ventricular remodeling and heart failure. TGF1 is universally known as a potent stimulator of fibroblast differentiation. TGF- was shown to induce CF differentiation via canonical SMAD pathway and non-canonical p38 MAPK pathway (Leask &

Abraham, 2004; Y. E. Zhang, 2009). Recent evidence suggests that TRP channels also participate in CF differentiation. Du et al. demonstrated that

TRPM7 but not TRPC3, TRPC6 and TRPV4 is required for atrial fibroblast differentiation (Du et al., 2010). However, the molecular mechanism by which

TRPM7 mediates these effects are not known. TRPC3 on the other hand was shown to induce CF proliferation and differentiation and may induce CF differentiation through SRF (Harada et al., 2012; Saliba et al., 2015). In contrast,

TRPC6 was shown to trigger -SMA expression via calcineurin/NFAT pathway

(Onohara et al., 2006; Seth et al., 2009). It is evident that most of the work on soluble factors and TRP channels focused on either CF proliferation or -SMA secretion without considering the contribution of mechanical factors which are required for -SMA incorporation in to stress fibers.

Our present work, therefore, focused on mechanical signaling in CF differentiation with TRPV4 as a candidate mechanosensor. Recent studies have

demonstrated the alterations in ECM stiffness regulate CF differentiation (Galie,

Westfall, & Stegemann, 2011). Cells sense mechanical forces applied to ECM via integrins which in turn transfer cellular contractile forces onto the ECM to modulate the phenotype of the cell (Hinz, 2010; Hinz et al., 2007). However, the role of mechanical factors and mechanotransduction pathways in CF differentiation and cardiac remodeling have not been thoroughly investigated.

We have previously demonstrated that TRPV4 channels integrate TGF1 and stiffness-induced CF differentiation in rat ventricular fibroblasts (ref). To delineate the molecular mechanism, in Aim2, we employed mCFs from both WT and TRPV4KO mice and measured TGF1-induced CF differentiation. To our surprise, contrast to pharmacological inhibition or siRNA downregulation, deletion of TRPV4 completely attenuated TGF-1-induced CF differentiation.

Further, direct activation of TRPV4 triggered pro-fibrotic gene activation in CF suggesting that TRPV4 is a critical component of CF differentiation and may regulate it through a mechano-transcriptional pathway. Indeed, we found that

TGF-1/TRPV4 activates Rho/Rho kinase/MRTF-A pathway upstream of CF differentiation.

Rho kinase is a crucial regulator of actin reorganization and controls many cellular functions, such as motility, adhesion, differentiation, and proliferation.

Inhibition of Rho kinase, with Y27632, demonstrate to reduce SMA expression in smooth muscle cells (Bourgier et al., 2005). Global or fibroblasts-specific deletion of Rho kinase, attenuated fibrosis following Ang II infusion-induced heart failure (Lighthouse & Small, 2016; T. Shimizu et al., 2017). We have previously

demonstrated that TRPV4 regulates mechanical stretch-induced endothelial cell reorientation via activation of Rho/Rho kinase pathway (Ghosh et al., 2008;

Thodeti et al., 2009). In the present study, we found that TGF1 increased Rho kinase activity which was significantly reduced upon TRPV4 inhibition. Further, inhibition of Rho kinase with Y27632 significantly attenuated TGF-1/TRPV4- induced CF differentiation. These findings confirmed that TRPV4 channels regulate CF differentiation by modulating Rho.Rho kinase pathway. One of the downstream effect of Rho kinase is polymerization of monomeric G-actin into F- actin which was shown to activate a mechanosensitive transcription factor,

MRTF-A (Miralles, Posern, Zaromytidou, & Treisman, 2003). When released from monomeric actin MRTF-A translocates to nucleus and induce fibrotic gene expression (Huang et al., 2012). We found that activation of TRPV4 by GSK or

TGF-1 treatment induced MRTF-A nuclear translocation in mCF which is sensitive to TRPV4 inhibition suggesting that MRTF-A is downstream of TRPV4 in CF differentiation. Further, pharmacological inhibition of MRTF-A significantly inhibited TGF-1/GSK-induced fibrotic gene promoter activity as well as CF- differentiation. Importantly, siRNA knockdown of MRTF-A attenuated TGF-1- induced CF differentiation confirming that MRTF-A mediates CF differentiation downstream of TGF-1/TRPV4. By using MRTF-A null mice, Small et al. have previously demonstrated MRTF-A controls myofibroblast differentiation and fibrosis following myocardial infarction (Small et al., 2010) . Further, a small molecule activator of MRTF-A has been shown to increase MF differentiation during wound healing (Velasquez et al., 2013). However, the upstream

mechanosensor or mechanotransduction mechanisms that regulate Rho kinase/MRTF-A during CF differentiation is not known. Our findings demonstrate that MRTF-A is activated by TRPV4 and identifies TRPV4 is a mechanosensor in CF that regulates Rho/Rhokinase/MRTF-A mechano-transcriptional pathway.

Previous studies on the role of soluble factors such as TGF-1 and other

TRP channels in CF differentiation mostly focused on CF proliferation and -SMA expression and identified the molecular mechanism to be either classical

SMAD/SRF pathway or p38/calcineurin/NFAT pathway (Leask & Abraham, 2004;

Y. E. Zhang, 2009). Both pathways were shown to activate fibrotic gene expression including collagen 1 and -SMA. However, the mechanism that regulate -SMA incorporation into stress fiber is not known which requires mechanical forces. Our findings suggest that TRPV4 dependent activation of Rho kinase not only induce -SMA expression via MRTF-A, but also promotes -SMA incorporation into stress fibers. We speculate that TRPV4 activation induces activation of additional integrins which will increase mechanical stiffness of ECM on one hand and TGF- activation on the other hand. Once activated TGF-1 can activate TRPV4-dependent CF differentiation triggering a positive feedback mechanism (Figure 28). Recently it was shown that pulling of a single integrin can modulate the conformational changes in latent TGF1 to release of active

TGF1 from the ECM supporting such possibility (Buscemi et al., 2011). In conclusion, we demonstrate that TRPV4 act as an upstream mechanosensor that not only activates-SMA synthesis but also its incorporation into stress fibers

during CF differentiation. Further, this pathway may trigger a positive feedback signaling involving integrins and TGF-1 that sustains CF differentiation to MF.

Figure 28. Schematic representation of molecular mechanism by which

TRPV4 mediate TGF-1 induced CF differentiation into MF A) TRPV4 integrates TGF-1 (soluble) and mechanical (ECM stiffness/force) during CF differentiation. Upon stimulation, TRPV4 activates Rho/Rho kinase activity, which in turn induces MRTF-A release from G-actin and translocation to nucleus to trigger pro-fibrotic gene expression (-SMA). Additionally, TRPV4/Rho kinase pathway induces -SMA incorporation into stress fibers. Increased mechanical tension due to stress fiber contraction could activate/pull integrins which in turn release TGF-1 from latent TGF-complex. TGF-then induces TRPV4 expression and activation and sustains CF differentiation to MF in this positive feed-back mechanism.

The functional role of TRPV4 in cardiac remodeling post myocardial infarction

Ischemic heart diseases are the leading cause of mortality and current interventional therapies are mainly focused on rescuing the damaged myocardium.

However, an increase in the recurrent incidences of myocardial infarction and patient survival rate enhanced burden of long term care as these patients undergo adverse cardiac remodeling leading to cardiac fibrosis and heart failure. Hence, there is an urgent need to find alternative therapies for cardiac fibrosis (Mozaffarian et al., 2015). Therefore, we focused on mechanotransduction mechanisms and found that TRPV4 is a mechanosensor that regulate CF differentiation and absence of TRPV4 preserved cardiac structure and function in response to pressure-overload. We found that deletion of TRPV4 offered cardioprotection from

MI-induced ischemia. Importantly, we found that TRPV4KO hearts exhibited decreased scar formation at the infarcted region compared to the WT-MI animals.

Further, we found that the myocardium was preserved at the infarcted region even after 8 weeks post MI, indicating that targeting TRPV4 not only inhibits fibrosis but also preserves cardiac muscle in the infarcted region. In order to find out the molecular mechanisms in cardio-protection offered by deletion of TRPV4, we found that absence of TRPV4 decreased myocyte apoptosis and increased coronary angiogenesis. Although cell death following acute injury mainly occurs through necrosis, previous studies demonstrated that apoptosis is also involved in myocyte loss in the later stages of MI (Anversa et al., 1998). TRPV4 has recently been implicated in apoptosis. Intra-cerebrovascular injection of a TRPV4 agonist

was shown to increase apoptosis in the hippocampi whereas TRPV4 inhibition reduced apoptosis after middle cerebral artery occlusion. In addition, activation of

TRPV4 revealed enhanced apoptosis by downregulating the PI3K/Akt pathway and increasing p38MAPK signing (Jie et al., 2015). In cardiomyocytes, hypoxia/reoxygenation-induced cell injury was reversed by TRPV4 antagonist, which increased intracellular calcium and reactive oxygen species (Wu et al.,

2017). We have previously shown that TRPV4-deficient tumor endothelial cells exhibit increased proliferation and TRPV4 activation inhibits this proliferation selectively i.e. without changing the proliferation of normal endothelial cells (EC)

(Thoppil et al., 2015). Based on these findings, we believe that the survival of the myocardium in post-MI TRPV4KO hearts is, at least in part, due to reduced myocyte apoptosis. However, the molecular mechanisms by which TRPV4 regulates cardiomyocyte survival or apoptosis is not known. However, we have previously demonstrated that TRPV4KO EC and TRPV4-deficient tumor EC both exhibit a high basal ERK1/2 activity (Thoppil et al., 2015). These findings suggest that in normal cells TRPV4 negatively regulate ERK1/2 activity and cell proliferation and deletion of TRPV4 may activate these pathways to enhance cell proliferation and survival.

Angiogenesis/revascularization is a crucial phase in the remodeling process following MI, as the damaged existing vasculature cannot meet the increased demand in oxygen for viable myocardium. Loss of vasculature can result in the progression of myocyte damage and expand scar. The enhanced expression of

VEGFR2 at regions of hypoxia in the infarct zone can initiates angiogenesis from

existing vasculature in the endocardium. We have previously demonstrated that

TRPV4 negatively regulates angiogenesis as evidenced by increased tube/sprout formation in vitro and enhanced capillary density in subcutaneous Matrigels and/or tumors implanted in TRPV4KO mice (Thoppil et al., 2016). Findings from the present study demonstrate that angiogenesis, via detection of CD31, is enhanced at infarct/border zones in TRPV4KO hearts compared to WT hearts, post-MI. In addition, TRPV4KO hearts show increased basal capillary density. We believe that this increased capillary density may allow for better blood flow which reduces myocyte apoptosis and cardiac fibrosis leading to the cardioprotection observed in

TRPV4KO hearts following MI.

Although an orally active TRPV4 blocker was shown to prevent pulmonary edema induced by chronic heart failure, a direct role for TRPV4 in heart failure has not been studied (Thorneloe et al., 2012). While our work is in progress Dong et al showed that TRPV4 inhibition or deletion offered protection against acute ischemia/reperfusion injury (Dong et al., 2017). However, these studies did not address a direct role of TRPV4 in cardiac remodeling during cardiac hypertrophy or myocardial infarction. In the present study, by employing well known models of cardiac hypertrophy and myocardial infarction in TRPV4KO mice, we found that the absence of TRPV4 preserved cardiac structure and function post-MI and TAC.

Further, we delineated the molecular mechanism and identified a novel TRPV4 dependent mechano-transcriptional Rho-Rho kinase/MTRF-A pathway that mediates CF differentiation to MF.

Altogether, our results demonstrate a crucial role for TRPV4 in cardiac remodeling following chronic, as well as acute, injury/insult and identifies TRPV4 as a novel soluble factor-independent target for the treatment of cardiac fibrosis, hypertrophy and myocardial infarction.

Conclusions

The primary goal of this dissertation was to investigate the hypothesis that targeting mechanosensitive ion channel TRPV4 protects heart from cardiac hypertrophy and myocardial ischemia by preventing adverse cardiac remodeling.

The aims were 1) to determine the physiological significance of TRPV4 in cardiac remodeling following pressure overload-induced cardiac hypertrophy 2) to delineate the molecular mechanisms by which TRPV4 regulate cardiac fibroblasts differentiation into myofibroblasts and 3) to ascertain the functional role of TRPV4 in cardio protection following myocardial infarction.

Overall, we found that TRPV4 is a mechanosensor and deletion of TRPV4 offers cardioprotection in response to pressure overload-induced hypertrophy and myocardial infarction-induced ischemia. Specifically, we found that the absence of

TRPV4 preserved cardiac structure and function in response to TAC and MI probably through the inhibition of cardiac fibroblast differentiation to myofibroblasts and dependent fibrosis. We further dissected the molecular mechanism and identified that TRPV4 mediates CF differentiation via a novel mechanotranscriptional Rho/Rhokinase/MRTF-A pathway. Finally, we found that the absence of TRPV4 preserved myocardial structure in response to MI via reduced myocyte apoptosis and increased coronary angiogenesis.

These findings are clinically important as the traditional anti-fibrotic strategies which are mostly focused on targeting soluble factor (TGF-) signaling have shown only modest success in the clinical trials. Further, most of the attention on revascularization of ischemic hearts is concentrated on VEGF-treatment and

stem cell therapy which are not very successful in clinics. Our findings from this study, thus, will provide a mechanotransduction based-therapeutic means to manipulate cardiac fibrosis and coronary angiogenesis in situations where chemical treatments using growth factor or antagonizing growth factor signaling have proven ineffective.

Future directions

 Direct connection between TRPV4 and TGF-1: focusing on p38 MAPK and

PI3-Kinase.

 Direct connection between TRPV4 and Rho/Rho kinase.

 Generation of :

-Fibroblast specific TRPV4KO mice: Tcf-21-Cre (CF) and Periostin-Cre

(MF)

-Endothelial specific TRPV4KO mice: Tie-2-Cre and VE-Cadherin-Cre

-Cardiomyocte specific TRPV4KO mice: Myh6-Cre

 Translational: Effect of TRPV4 antagonists on hypertrophy and MI.

 Other fibrosis models: Angiotensin II infusion.

 Other ischemia models: Ischemia/Reperfusion injury, hypoxia- in vitro.

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