Grk5 Is a Novel Regulator of Fibroblast Activation and Cardiac Fibrosis

GRK5 IS A NOVEL REGULATOR OF FIBROBLAST ACTIVATION AND CARDIAC FIBROSIS

A Dissertation Submitted to the Temple University Graduate Board

In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY

by Akito Eguchi May 2020

Examining Committee Members:

Walter J. Koch PhD, Advisory Chair, Department of Pharmacology Joseph Y. Cheung MD, PhD, Department of Medicine Konstantinos Drosatos PhD, Department of Pharmacology John Elrod PhD, Department of Pharmacology Douglas Tilley PhD, Department of Pharmacology Laurel Grisanti PhD, External Member, Department of Biomedical Sciences, University of Missouri

ABSTRACT

Rationale: Pathological remodeling of the heart is a hallmark of chronic heart failure (HF) and these structural changes further perpetuate the disease. Cardiac fibroblasts are the critical cell type that is responsible for maintaining the structural integrity of the heart.

Stress conditions, such as a myocardial infarction (MI), can activate quiescent fibroblasts into synthetic and contractile myofibroblasts. G protein-coupled receptor (GPCR) kinase

(GRK) 5 is an important mediator of cardiovascular homeostasis through dampening of

GPCR signaling, and is expressed in the heart and upregulated in human HF. Of note,

GRK5 has been demonstrated to translocate to the nucleus in cardiomyocytes in a calcium- calmodulin (Ca2+-CAM)-dependent manner, promoting hypertrophic gene transcription through activation of NFAT. Interestingly, NFAT is also involved in fibroblast activation.

GRK5 is highly expressed and active in cardiac fibroblasts (CFs), however its pathophysiological role in these crucial cardiac cells is unknown.

Objective: The aim of this study is to elucidate the role of GRK5 in the activation of cardiac fibroblasts in vitro and cardiac fibrosis after injury in vivo.

Methods and Results: We demonstrate using adult cardiac fibroblasts that genetic deletion of GRK5 inhibits Angiotensin II (AngII) mediated fibroblast activation. Fibroblast-specific deletion of GRK5 in mice decreased fibrosis and cardiac hypertrophy after chronic AngII infusion compared to non-transgenic littermate controls (NLCs). Fibroblast-specific deletion of GRK5 was also protective in mice after ischemic injury as they presented with preserved systolic function, decreased fibrosis, and decreased hypertrophy compared to

NLCs. Mechanistically, we show that nuclear translocation of GRK5 is involved in fibroblast activation.

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Conclusions: We present novel data demonstrating that GRK5 is a regulator of fibroblast activation in vitro and cardiac fibrosis in vivo. This adds to previously published data which demonstrates the potential beneficial effects of GRK5 inhibition in the context of cardiac disease.

For Romeo,

The best pupper that ever lived.

FUNDING

Akito Eguchi was partially supported by three National Institutes of Health/National

Heart, Lung, and Blood Institute (NIH/NHLBI) grants: P01 HL091799 (to Walter Koch),

P01 HL075443 (to Walter Koch), and F30 HL143953 (to Akito Eguchi).

ACKNOWLEDGMENTS

First and foremost, I need to thank Wally Koch for being an exceptional thesis advisor. I initially joined your lab because of the amazing science that you and your lab had conducted. However, knowing what I know now, I would have joined your lab because of your generosity, your mentorship, and your personality. It was truly an honor and a privilege to have studied under you. Thank you for your patience, your guidance, and your encouragement when experiments were not going well. You always listened to my ideas and allowed me an incredible amount of freedom in developing my project, which was crucial for my development as an independent thinker and scientist. I am extremely grateful and could not have asked for a better mentor. I will look back on these years with pride and fond memories.

I would like to thank the members of my thesis advisory committee, Joseph

Cheung, Konstantinos Drosatos, John Elrod, and Douglas Tilley, for your support, your feedback, and your genuine interest in my research. Without your contributions, my project would not have been as complete as it is, and for that I am truly grateful. I would also like to acknowledge Laurel Grisanti, my external examiner. We first met when I rotated in Dr. Tilley’s lab and you have been nothing but encouraging and supportive, both personally and professionally. Thank you for your time and effort in supporting my dissertation.

I would like to thank the Center for Translational Medicine in general. The collaborative atmosphere that this center has encouraged has made the past four years incredibly rewarding. Everyone is willing to help one another whether it be lending supplies, providing feedback, or helping with techniques. I would also like to

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acknowledge the MD/PhD program, and specifically Dr. Dianne Soprano for giving me this amazing opportunity to join this program.

A huge thank you goes to the members of the Koch lab, both past and present.

There has been a lot of turnaround in terms of members, but all of you have been more than willing to help in regards to experimental design/techniques and providing advice.

Above all else, I am so grateful for all of the friendships I have developed. You are the reason that I looked forward to coming into work, even when experiments weren’t working well.

Special thanks needs to go to Ken Gresham, whose name I will always misspell.

You started off being the new generic looking guy in the Koch lab that wore cowboy boots to work. I’m glad that you stopped wearing them, so that we could become close friends. I will very much miss our overly loud, strange, and vulgar conversations that we had in the lab and I hope to continue to have those conversations outside of work.

Another special thanks goes to Ryan Coleman. You are one smart grad student and I wish

I had befriended you earlier because you are one of the most interesting people that I have ever met.

I would like to thank the lunch group, both past and present, both for entertainment purposes as well as some serious conversations. Alana Vagnozzi, Krista

Connelly, Toby Thomas, Cassandra Wolsh, Mike Lee, Ken, and Ryan, thanks for the good times.

I would not be where I am today without the support of my family. My father is the one who got me interested in both medicine and science, and without your support

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and encouragement, I would not be pursuing both. I also cannot thank my mother enough, who has been a constant source of love and encouragement.

Finally, I would not have been able to finish this degree without the amazing patience, strength, and humor of my girlfriend, Danielle. Thank you for prioritizing my education and being incredibly loving and supportive. I hope that you now understand what you got yourself into.

TABLE OF CONTENTS

Page

ABSTRACT ...... iii

DEDICATION ...... v

FUNDING...... vi

ACKNOWLEDGMENTS ...... vii

LIST OF TABLES AND FIGURES ...... xiv

LIST OF ABBREVIATIONS ...... xvi

CHAPTER

1. HEART FAILURE AND CARDIAC FIBROSIS ...... 1

Heart Failure and Cardiac Remodeling ...... 1

Fibroblasts ...... 3

Myofibroblasts ...... 6

Cardiac Fibrosis ...... 9

Resident Cardiac Fibroblasts ...... 9

Cardiac Myofibroblasts ...... 10

Signaling Pathways Associated with Fibroblast Activation ...... 13

Transforming Growth Factor β (TGFβ) Signaling ...... 13

Angiotensin II (AngII) Signaling ...... 15

Endothelin 1 (ET-1) Signaling ...... 17

Calcium (Ca2+) Dependent Signaling ...... 18

2. G PROTEIN-COUPLED RECEPTOR KINASE 5 IN CARDIAC

PATHOPHYSIOLOGY ...... 22

G Protein-Coupled Receptor (GPCR) Signaling Overview ...... 22

GPCR Structure ...... 23

GPCR Function and G Protein Activation ...... 24

GPCR Regulation ...... 25

G Protein-Coupled Receptor Kinases (GRKs) ...... 28

GRK Tissue and Subcellular Localization ...... 30

GRK Regulation ...... 31

G Protein-Coupled Receptor Kinases 5 (GRK5) ...... 31

Pathophysiological Roles of GRK5 ...... 31

GRK5 in the Cardiovascular System ...... 33

3. GRK5 IS A NOVEL REGULATOR OF FIBROBLAST ACTIVATION

AND CARDIAC FIBROSIS ...... 37

Introduction ...... 37

Methods...... 39

Experimental Animals ...... 39

Isolation of Adult and Neonatal Mouse Cardiac Fibroblasts and

Myocytes ...... 40

Real Time PCR ...... 42

Surface Sensing of Translation (SUnSET) Assay ...... 43

Calmodulin Capture Assay ...... 43

Immunoblotting...... 43

In Vivo AngII Infusion and Model of Myocardial Infarction ...... 44

Transthoracic Echocardiographic Analysis ...... 44

Assessment of Myocardial Fibrosis and Hypertrophy ...... 44

Immunofluorescence ...... 45

Drug Treatments ...... 45

NFAT Luciferase Reporter and Luciferase Assay ...... 46

Collagen Gel Contraction Assay ...... 46

Statistical Analyses ...... 46

Results ...... 47

GRK5 is Required for Fibroblast to Myofibroblast

Transdifferentiation ...... 47

Decreased Levels of GRK5 in Fibroblasts Are Beneficial to AngII

Infused Myocardium ...... 49

Fibroblast Specific Loss of GRK5 Protects Against Cardiac

Ischemic Injury ...... 52

Non-canonical GRK5 Activity is Responsible for Fibroblast

Activation ...... 54

Discussion ...... 57

4. CONCLUSIONS AND SIGNIFICANCE ...... 69

Extended Discussion and Future Directions ...... 69

Conclusions ...... 74

REFERENCES CITED ...... 75

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LIST OF TABLES AND FIGURES

Figure ID Page

Table 1. Published markers for cardiac fibroblasts ...... 4

Figure 1. Mechanisms of smooth muscle and myofibroblast contraction ...... 8

Figure 2. Signaling pathways involved in fibroblast activation ...... 12

Figure 3. GPCR activation and desensitization ...... 27

Figure 4. GRK5 is necessary for fibroblast transactivation into myofibroblasts ...... 48

Figure 5. GRK5 levels in fibroblasts isolated from GRK5KO mice ...... 50

Figure 6. GRK5 fibroKO are protected against AngII mediated cardiac hypertrophy and

fibrosis...... 51

Figure 7. GRK5 fibroKO are protected against MI mediated cardiac dysfunction and

fibrosis...... 53

Figure 8. Non-canonical GRK5 activity promotes fibroblast activation ...... 56

Supplemental Figure 1. Immunoblot probing for GRK5 in MACFs isolated from WT and

GRK5KO mice ...... 63

Supplemental Figure 2. GRK5KO MACFs are resistant to TGFβ mediated α-SMA

expression ...... 64

Supplemental Figure 3. GRK5KO MACFs demonstrate decreased wound closure by in

vitro scratch assay ...... 65

Supplemental Figure 4. GRK5KO MACFs do not demonstrate changes in proliferation

...... 66

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Supplemental Figure 5. GRK5KO MACFs have decreased protein translation rates after

AngII stimulation compared to WT MACFs ...... 67

Supplemental Figure 6. GRK5 nuclear effects mediate activation ...... 68

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LIST OF ABBREVIATIONS

ACE angiotensin converting enzyme

AngII Angiotensin II

ANOVA analysis of variance

α-SMA α-smooth muscle actin

AT1R angiotensin type I receptor

β-AR β-adrenergic receptor

BK12 GRK2-overexpressing mice

Ca2+ calcium

CaM calmodulin cAMP cyclic adenosine monophosphate cDNA complementary Deoxyribonucleic acid

CF cardiac fibroblast

Cn calcineurin

CVD cardiovascular disease

ECM extracellular matrix

EF ejection fraction

EMT epithelial to mesenchymal transition

ET1 endothelin 1

FS fractional shortening

GDP guanosine diphosphate

GTP guanosine triphosphate

GEF guanine nucleotide exchange factor

GPCR G protein-coupled receptor

GRK G protein-coupled receptor kinase

GRK5 fibroKO collagen1α2-CreER(T)/GRKfl/fl mice

HF heart failure

HFpEF heart failure with preserved ejection fraction

HFrEF heart failure with reduced ejection fraction

IP3 inositol triphosphate

IP3R IP3 receptor

JNK c-Jun N-terminal kinase

KO knockout

LV left ventricle

MACF mouse adult cardiac fibroblast

Malb malbrancheamide

MAPK mitogen-activated protein kinase

MCU mitochondrial calcium uniporter

MEF mouse embryonic fibroblast

MI myocardial infarction

MLC myosin light chain mtCU mitochondrial calcium uniporter channel complex

NFAT nuclear factor of activated T-cells

NIH National Institutes of Health

NLC non-transgenic littermate controls

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NLS nuclear localization signal

NRCF neonatal rat cardiac fibroblast

PIP2 phosphatidylinositol 4,5-bisphosphate

PKC protein kinase C

PLC phospholipase C qRT-PCR quantitative real time polymerase chain reaction

RAAS renin-angiotensin-aldosterone system

SUnSET surface sensing of translation

TAB TAK1-binding protein

TAK1 TGFβ-activated kinase 1

Tcf21 transcription factor 21

TGFβ transforming growth factor β

TGFβR transforming growth factor β receptor

TK45 GRK5 overexpressing mice

TRPC transient receptor potential channel

WT wild type

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CHAPTER 1

HEART FAILURE AND CARDIAC FIBROSIS

Heart Failure and Cardiac Remodeling

Cardiovascular disease (CVD) is the leading cause of mortality in the United

States as well as globally (Benjamin et al., 2019; Mensah et al., 2019). CVDs include coronary heart disease, heart failure (HF), stroke, and hypertension. In the US, HF is a major public health concern, where there are currently 6.2 million Americans suffering from HF and this number is expected to rise to over 8 million by 2030 (Benjamin et al.,

2019). This syndrome also has a significant impact on the economy with the cost of care for the treatment of HF in the United States being $35.4 billion in 2010 and this is expected to rise to $69.8 billion by 2030. In this scope, improvements in therapeutics for the treatment of HF can reap enormous benefits.

HF is a clinical syndrome characterized by the heart’s inability to pump blood sufficiently to meet the metabolic demands of the body. It is a complex disease, which is the end result of a multitude of etiologies ranging from ischemic injuries caused by coronary heart disease, genetic causes, such as hypertrophic cardiomyopathy, or chronic injuries, such as diabetes and hypertension. No matter the causative injury, the heart fails when there is a disruption in its pumping capacity or its ability to fill. Therefore, the disease has been stratified into HF with reduced ejection fraction (EF) (HFrEF) or HF with preserved EF (HFpEF). The incidence of each is roughly equal in the US.

The pathophysiology of HF is as complex as its etiology. However, a major contributing factor to the pathogenesis of HF is a structural change in the heart termed cardiac remodeling. Because the heart is a terminally differentiated organ that can only

replace cardiomyocytes at a rate of <1% per year, the heart relies on 2 major reparative approaches after injury: cardiomyocyte hypertrophy and cardiac fibrosis (Bergmann et al., 2009). After injury, physical stress and neurohormonal signals promote cardiomyocyte growth, leading to a temporary preservation of cardiac function (van

Berlo et al., 2013). This hypertrophic response leads to ventricular thickening and stiffening. The Laplace law states that ventricular wall stress is proportional to ventricular pressure and radius and is inversely proportional to wall thickness (Grossman et al.,

1975). By inducing ventricular hypertrophy, the heart is able to reduce wall stress, thereby reducing oxygen demand as an adaptive response to injury. However, persistent stress eventually causes a transition from adaptive hypertrophy to maladaptive HF and the molecular mechanism that underlies this switch remains incomplete (Burchfield et al.,

2013).

Another hallmark feature of cardiac remodeling after injury involves excessive deposition of extracellular matrix (ECM) proteins. These proteins make up both the scar as a replacement fibrotic response after injury such as a myocardial infarction or reactive fibrosis (interstitial or perivascular fibrosis) after more chronic insults. The end result of the fibrotic response is an decrease in compliance, contractile dysfunction, and rhythm disturbances (Spinale, 2007). In fact, the amount of cardiac fibrosis correlates strongly with adverse outcomes in both HFrEF and HFpEF (Assomull et al., 2006; Bergmann et al., 2009; Schelbert et al., 2017; Wu et al., 2008; Yan et al., 2006). Therefore, targeting the fibrotic response is an attractive therapeutic strategy for the treatment of HF.

Fibroblasts

ECM deposition and cardiac fibrosis occurs via fibroblast activity. Fibroblasts are mesenchymal in origin, are flat, and have an elongated spindle/stellate shape with various cytoplasmic projections (Souders et al., 2009). Morphologically, fibroblasts differ from other cell types in the heart by an absence of a basement membrane. In the broadest sense, the functional role of a fibroblast is to secrete a variety of ECM proteins, such as collagens and fibronectins, as well as biochemical mediators, such as growth factors and proteases (Kanekar et al., 1998; Souders et al., 2009). Because fibroblasts have comparable functions regardless of their tissue origin (the heart, lungs, skin, etc.), they have been viewed as being a uniform cell type. This view of fibroblast homogeneity has been challenged by data demonstrating vast differences in fibroblast populations between tissues as well as within common tissues. Human lung fibroblasts are heterogeneous in surface marker expression and collagen production while periodontal fibroblasts are also heterogeneous in collagen production (Fries et al., 1994). In addition, human skin fibroblasts isolated from different anatomical sites displayed distinct transcriptional patterns with differential regulation of ECM protein synthesis, metabolism, and signaling pathways that control proliferation and migration (Chang et al., 2002). This immense heterogeneity amongst the fibroblast populations has created unique challenges in determining what truly defines a fibroblast. Although fibroblast functions have been defined in a variety of contexts, no protein markers have been identified that are exclusively expressed by fibroblasts. While a variety of proteins have been shown to be highly expressed in fibroblasts, all of them have been demonstrated to be expressed in other tissues (Table 1) (Ivey and Tallquist, 2016).

Table 1: Published Markers for Cardiac Fibroblasts

CM, cardiomyocytes; CPC, cardiac progenitor cells; EC, endothelial cells; Ep, epicardium; F, fibroblasts; FC, flow cytometry; GT, gene technology; IC, immune cells; IHC, immunohistochemistry; NE, not expressed; P/V, pericytes/vascular smooth muscle cells. * Marker expressed in a subpopulation of fibroblasts.

Adapted from Ivey and Tallquist, 2016.

The ECM strongly influences the function of tissues and the fibroblast population adjacent to it. During development, these epithelial and mesenchymal interactions with the ECM is critical for proper organization and maturation of tissues and organs.

Fibroblasts and the resultant ECM can define the physical properties of tissues, such as in skin, as well as trap critical signaling molecules necessary for development (Iozzo, 2005;

Sorrell and Caplan, 2004). After development, the ECM provides structural support for tissues and organs, form lamina in order to delineate borders separating different cell types that have differing functions, as well as serve as interstitial regions to congregate similar cells with common functions (Kendall and Feghali-Bostwick, 2014). Fibroblasts produce these specialized ECMs by producing and secreting a wide variety of structural proteins in varying amounts. Collagen type I, the most abundant protein in humans and other mammals, provides rigidity due to its triple stranded, rope-like organization. In contrast, elastin and fibrillin combine to form elastic fibers, which allow for stretching of tissues. There are also a variety of other collagen subtypes that provide the ECM unique properties. The relative expression of these structural proteins by fibroblasts allows the tissues to have the proper structural integrity needed for their physiological function. The

ECM also contains adhesive proteins as well as a ground substance comprised of a hydrated gel of proteoglycans. Adhesive proteins, such as fibronectin and laminin, form the connection between cells and the ECM. The ground substance forms a pathway for cell migration of immune cells and fibroblasts, nutrients, and chemical signals.

Fibroblasts produce and secrete all components of the ECM, as well as the proteins to maintain them. Fibroblasts produce lysyl oxidase, which is responsible for collagen maturation by cross-linking collagen fibers (Cox et al., 2013). ECM catabolism is

regulated by matrix degrading enzymes, such as matrix metallopeptidases (MMPs), as well inhibitors of MMPs, tissue inhibitors of MMPs (TIMPs).

Myofibroblasts

During the wound healing process, fibroblasts migrate to the site of injury, proliferate, and become activated. The activation process is induced by a combination of mechanical stress as well as hormones, such as transforming growth factor β (TGFβ), angiotensin II (AngII), and endothelin 1 (ET1) (Tomasek et al., 2002). All of these factors are interconnected and lead to the activation of myofibroblast gene transcription programming (Davis and Molkentin, 2014; Stempien-Otero et al., 2016). The myofibroblast phenotype is characterized by de novo expression of α-smooth muscle actin (α-SMA), an increase in expression and secretion of ECM components, and a resistance to apoptosis. The expression of α-SMA, as seen in smooth muscle cells, allows these cells to contract wounds. α-SMA, along with other intracellular microfilament proteins, such as β-actin, γ-actin, and α-actinin, form stress fibers (Dugina et al., 2001;

Hinz et al., 2003; Kreis and Birchmeier, 1980; Singer, 1979). These stress fibers are connected to extracellular fibronectin fibrils and collagens, allowing myofibroblasts to contract the ECM (Singer et al., 1984; Tomasek et al., 2002). ECM contraction is also dependent on myosin light chain (MLC). In a calcium (Ca2+) dependent pathway, increased intracellular Ca2+ will activate MLC kinase leading to MLC phosphorylation and increased contraction. This is a transient response as the increase in Ca2+ is short- lived and myosin phosphatase inactivates MLC (Katoh et al., 1998). In a Ca2+ independent pathway, RhoA, downstream of an agonist, activates Rho kinase, which can 6

either directly activate MLC by phosphorylation or inactivating myosin phosphatase

(Katoh et al., 2001). The latter RhoA dependent contraction appears to be the predominant pathway that regulates myofibroblast contraction which is in contrast to smooth muscle cells which tends to rely on the Ca2+ dependent pathway (Tomasek et al.,

2002). This is due to their difference in function. Smooth muscle cells contract and relax in response to environmental cues while myofibroblasts need sustained contraction for wound closure (Figure 1).

As mentioned, increased ECM deposition is a major function of myofibroblasts and this is a part of the normal wound healing response. However, chronic injury or dysregulation of the repair process can lead to maladaptive fibrosis. Fibrosis is a scarring process characterized by excess deposition of ECM, leading to organ dysfunction.

Fibrosis plays a significant contributory role in a variety of organ failures in disease, such as in idiopathic pulmonary fibrosis, liver cirrhosis, kidney fibrosis, systemic sclerosis, and cardiac fibrosis. Regardless of disease or organ, maladaptive fibrosis is due to disproportionate myofibroblast activity. Excess ECM and contraction can alter organ structure and disrupt cellular functions, ultimately leading to organ failure (Wynn and

Ramalingam, 2012). Because myofibroblast function and fibrosis contributes to significant morbidity and mortality in a variety of different disease contexts, a better understanding of myofibroblast biology is needed for the development of anti-fibrotic therapies (Wynn and Ramalingam, 2012).

Figure 1. Mechanisms of Smooth Muscle and Myofibroblast Contraction. Smooth muscle contraction is primarily mediated through Ca2+ dependent pathways. Electrical depolarization or activation of Gq pathways leads to an increase in cytosolic Ca2+ through influx via voltage dependent Ca2+ channels or through IP3 mediated Ca2+ release from the sarcoplasmic reticulum (SR). Ca2+ activates MLCK, leading to the phosphorylation of MLC, leading to cross-bridge formation between myosin and actin and subsequent contraction. Myofibroblast contraction is mediated by activation of RhoA, primarily through coupling of G proteins (G12/13). RhoA activated Rho-kinase, which can then either inhibit relaxation by inactivating MLCP or activating MLCK leading to contraction. Adapted from Klabunde, 2011.

Cardiac Fibrosis

Resident Cardiac Fibroblasts

Recent studies have elucidated that cardiac fibroblasts make up roughly 11% of the total cell number of heart (Pinto et al., 2016). These resident cardiac fibroblasts are responsible for maintaining the structural and mechanical integrity of the heart. They are responsible for production and maintenance of the collagen network that is critical for proper conductivity and rhythmicity (Camelliti et al., 2005; Caulfield and Borg, 1979).

The structural components of the heart that are produced by fibroblasts are comprised of collagen types I, III, V, and VI as well as periostin, vimentin, and fibronectin (Snider et al., 2009). However, the presumed basal functions of cardiac fibroblasts as regulators of cardiac ECM are implied. Embryonic loss of fibroblasts leads to hearts with significant reductions in collagens; however, perinatal lethality prevented further investigation into functions of adult cardiac fibroblasts (Acharya et al., 2012; Smith et al., 2011). There is a longstanding dogma that fibroblasts are the exclusive producers of ECM proteins, such as the various collagens. However, this concept has largely been debunked as a variety of other cell types are able to produce and secrete ECM proteins, such as endothelial cells and cardiomyocytes (Borg et al., 1983; Fisher and Periasamy, 1994; Hayashi et al.,

1988). Because of the difficulty of finding fibroblast-specific markers and therefore genetic tools, determining the fundamental roles of fibroblast in cardiac physiology has been a challenge.

Recently, the developmental origin of cardiac fibroblasts has been teased out via various novel lineage tracing studies. In the late 1990’s, developmental biologists demonstrated that the epicardium undergoes epithelial to mesenchymal transition (EMT)

and contributes to the cardiac fibroblast and vascular smooth muscle cell populations

(Dettman et al., 1998; Gittenberger-de Groot et al., 1998; Mikawa and Gourdie, 1996).

Discovery of epicardium specific genes, such as transcription factor 21 (Tcf21), has reconfirmed this finding through lineage tracing (Acharya et al., 2012). In addition, it was demonstrated that resident fibroblasts residing in the right ventricle and the interventricular septum, which make up ~20% of the cardiac fibroblast population, do not originate from the epicardium, but have an endothelial origin (Ali et al., 2014; Moore-

Morris et al., 2014). Furthermore, Tcf21 and platelet-derived growth factor receptor α

(PDGFRα), a receptor tyrosine kinase, are required for proper fibroblast development as deletion of either gene results in a loss of epicardium-derived cardiac fibroblasts and subsequent disruption in the expression of ECM in LV (Acharya et al., 2012; Smith et al.,

2011). Lineage tracing has demonstrated that the fibroblast population involved in fibrotic remodeling after cardiac injury is composed entirely of resident fibroblasts with little to no contributions from circulating fibroblasts or from endothelial, hematopoietic, or epicardial cells (Abe et al., 2001; van Amerongen et al., 2008; Moore-Morris et al.,

2014; Zeisberg et al., 2007; Zhou et al., 2011).

Cardiac Myofibroblasts

Because the heart has a very limited regenerative capacity following injury, the repair process involves the removal of necrotic cardiomyocytes and replacement fibrosis in order to maintain the structural and functional integrity of the heart. After acute cardiac injury, expression of various pro-inflammatory and profibrotic cytokines are upregulated.

This induces “proto-myofibroblast” formation, which is characterized by the formation of 10

cytoplasmic actin stress fibers, allowing migration into the site of injury (Hinz, 2007;

Tomasek et al., 2002). These proto-myofibroblasts begin secreting collagen and fibronectin rich ECM 20-30 hours after injury (Tomasek et al., 2002). High levels of cytokines and mechanical stress leads to the maturation of the proto-myofibroblasts into myofibroblasts (Davis and Molkentin, 2014). These cells have de novo expression of

αSMA that forms a stress fiber network with contractile functions. Myofibroblasts hyper- secrete ECM proteins in order to form a scar as a measure against myocardial rupture. As the scar matures, the tensile strength of collagen increases within the site of injury and myofibroblasts express αSMA, which contributes to pathological remodeling (Travers et al., 2016). In most other tissues, maturation of the scar leads to apoptosis of myofibroblasts; however in the heart, they seem to persist and continue to release profibrotic signals. This leads to activation of other resident fibroblast in remote areas, leading to reactive interstitial fibrosis (Cleutjens et al., 1995; Sun and Weber, 2000).

This further perpetuates the disease as it decreases compliance, ventricular filling, and therefore cardiac output. These myofibroblasts also release pro-inflammatory and pro- hypertrophic signals leading to cardiomyocyte hypertrophy and necrosis, potentiating the pathophysiologic response (Takeda and Manabe, 2011; Turner, 2014).

Fibroblast activation is initiated by a variety of neurohormonal, cytokine, and mechanical signals that intertwine to activate myofibroblast associated gene programming. Figure 2 demonstrates a few of the major signaling pathways demonstrated to induce fibroblast transdifferentiation into myofibroblasts.

Figure 2. Signaling Pathways Involved in Fibroblast Activation TGBβ has canonical (Smad signaling) and non-canonical (MAPK signaling) pathways, which can lead to myofibroblast gene transcription. AngII activated Ca2+ signaling, which leads to activation of NFAT associated gene transcription. Mitochondria regulates intracellular Ca2+, which can activate Ca2+ signaling as well as alter metabolism to promote differentiation.

Signaling Pathways Associated with Fibroblast Activation

Transforming Growth Factor β (TGFβ) Signaling

TGFβ is a family of growth factors, of which TGFβ1 is likely to have the greatest effect, which is known to play a major contributing role in activating the fibrotic response in a variety of organs, including the heart (Leask and Abraham, 2004). After injury,

TGFβ is upregulated and released into the ECM by resident fibroblasts, surrounding mesenchymal cells, and inflammatory cells, such as resident macrophages and monocytes

(Davis and Molkentin, 2014; Stempien-Otero et al., 2016). TGFβ is secreted into the

ECM in an inactive form by being bound to latent TGFβ binding protein and is activated by enzymatic cleavage or by release from the ECM by mechanical tension (Wipff et al.,

2007). After cardiac injury, mechanical stress induces TGFβ release, activating fibroblasts (Hinz, 2007). The myofibroblasts, with their before mentioned α-SMA, are able to further induce TGFβ release through causing additional mechanical tension onto the ECM. This causes a positive feedback loop that maintains TGFβ in its active form allowing for further propagation of this activation response.

TGFβ1 binds to its cell surface receptor a heteromeric receptor composed of

TGFβ receptor 1 (TGFβR1), also known as activin receptor-like kinase 5, and TGFβ receptor 2 (Derynck and Zhang, 2003). Canonical TGFβ signaling involves phosphorylation of Smad2/3 by TGFβR1 leading to complex formation with Smad4. This complex can then translocate to the nucleus and act as a transcription factor leading to transcription of profibrotic genes (Bujak and Frangogiannis, 2007; Bujak et al., 2007).

Currently, it is believed that Smad2 is not a mediator of fibroblast activation (Bonniaud et al., 2004; Hecker et al., 2009). Smad3, on the other hand, is critical in fibroblast activation downstream of TGFβ as it regulates the expression of ECM genes, such as collagens, fibronectin, TIMP-1, and MMPs (Dobaczewski et al., 2010; Verrecchia et al.,

2001). This upregulation can be inhibited by expression of a dominant negative Smad3 or overexpression of inhibitory Smad7 (Verrecchia et al., 2001). Furthermore, fibroblasts isolated from Smad3 knockout (KO) mice are resistant to TGFβ mediated expression of

ECM proteins (Yang et al., 2003). Smad3, however, appears to only be involved in the transcription of ECM proteins and not α-SMA. While the α-SMA promoter has been demonstrated to contain Smad3 binding sites in vitro and Smad3KO fibroblasts have impaired α-SMA expression, Smad3KO mice fail to show a decrease in α-SMA positive cells after MI (Dobaczewski et al., 2011). In addition, overexpression of Smad7, which is a specific inhibitor of canonical Smad2/3 signaling, failed to block TGFβ mediated α-

SMA expression in isolated cardiac fibroblasts (Davis et al., 2012). Therefore, it appears that canonical TGFβ signaling may only underlie select aspects of fibroblast activation.

Non-canonical TGFβ signaling through activated TGFβR2 has also been implicated in fibroblast activation. This leads to the recruitment of TGFβ activated kinase

(TAK1) and TAK1 binding protein (TAB), which then leads to the activation of mitogen activated protein kinase (MAPK) signaling cascades, including c-Jun N-terminal kinase

(JNK) and p38 kinase (Davis and Molkentin, 2014; Davis et al., 2012). Previous literature focused on the canonical TGFβ signaling as the primary source of fibroblast activation, but there is mounting evidence suggesting that this non-canonical pathway

may play a significant role. Inhibition of p38 signaling can inhibit TGFβ induced expression of αSMA, col1a, fibronectin in various fibroblast population, such as mouse cardiac, mouse embryonic (MEFs), and human ocular fibroblasts (Davis et al., 2012;

Meyer-Ter-Vehn et al., 2006). In addition, fibroblast (TCF21 cre) and myofibroblast specific (periostin cre) KO of p38 was protective in two models of cardiac injury while overexpression of the direct activator of p38, MAPK kinase 6 (MKK6), propagated the fibrotic response (Molkentin et al., 2017). Pharmacological inhibition of p38 in vivo also protected against cardiac fibrosis and remodeling after MI in mice and rats (Kompa et al.,

2008; See et al., 2004). These observations support the notion that non-canonical TGFβ signaling also potently regulates cardiac fibroblast activation.

Angiotensin II (AngII) Signaling

The renin-angiotensin-aldosterone system (RAAS), primarily through the activity of AngII, leads to many pathophysiological functions, including activation of fibroblasts and cardiac fibrosis (Forrester et al., 2018; Haudek et al., 2010; Schnee and Hsueh, 2000;

Zhang et al., 2010). Classically, production of angiotensin peptides is initiated by the synthesis and processing of preprorenin in juxtaglomerular cells into renin, which is secreted into the circulation. Renin cleaves angiotensinogen, which is produced by the liver into angiotensin I, which is then processed by angiotensin converting enzyme

(ACE) into AngII (Karnik et al., 2015). There is evidence that all of the components of the RAAS are expressed in the heart and the heart has an organ specific RAAS (De Mello and Frohlich, 2014; Forrester et al., 2018).

AngII binds to its receptor AngII type 1 receptor (AT1R), a G protein-coupled receptor (GPCR), and mediates various functions in fibroblasts, including activation, migration, and proliferation (Villarreal et al., 1993). AT1R is coupled to the heterotrimeric G protein, Gq, which when stimulated leads to the activation of phospholipase C (PLC) through the GTP-bound Gq subunit. PLC catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into two second messengers: inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 diffuses through the cytoplasm and interacts with IP3 receptors on the endoplasmic reticulum causing release of Ca2+. DAG stays localized in the plasma membrane and acts as an activator of protein kinase C

(PKC).

The actions of AngII is believed to be, in part, due to its effects on TGFβ production and subsequent signaling (Leask and Abraham, 2004). Cardiac fibroblasts stimulated with AngII induces TGFβ expression and downstream profibrotic genes, both of which were blocked by losartan, an angiotensin receptor blocker (ARB) (Campbell and Katwa, 1997; Gray et al., 1998; Lee et al., 1995). AngII stimulation of cardiac fibroblasts also activates Smad2/3 and MAPKs (Gao et al., 2009). While TGFβ appears to be downstream of AngII signaling to induce fibroblast activation, AngII is also able induce myofibroblast transdifferentiation independent of TGFβ. AngII treated cardiac fibroblasts express α-SMA in the absence of TGFβR1 while inhibition of p38 inhibited this response. This suggests that p38 signaling acts as a nodal point between the two signaling cascades (Davis et al., 2012).

Inhibition of AngII signaling is currently a mainstay of HF therapy. In fact, ACE inhibitors and AT1R blockers (ARBs) are first line drugs in the treatment of patients with 16

HFrEF (Meyer, 2020). They have been demonstrated to reduce morbidity and mortality in numerous clinical trials, demonstrating the beneficial effects of RAAS blockade (Cohn et al., 1991; CONSENSUS Trial Study Group, 1987; Flather et al., 2000, 2000; Heran et al., 2012). While not approved for the treatment of cardiac fibrosis directly, ARBs have shown to reduce cardiac fibrosis in both humans and animal models (Díez et al., 2002;

Pfeffer et al., 1995; Yang et al., 2009). In vitro treatment of cardiac fibroblasts with losartan attenuated AngII-mediated collagen synthesis and α-SMA expression (Galie et al., 2012). These data present a compelling argument that AngII is a potent activator of fibroblasts and that the protective effects of AngII blockade may be due to its effects on fibroblast function.

Endothelin 1 (ET1) Signaling

ET1 is a vasoactive peptide classically believed to be released by endothelial cells to increase blood pressure. However, recently it has been appreciated that it is also released by inflammatory cells as well as fibroblasts and can promote tissue fibrosis (Shi-

Wen et al., 2006). There are two known ET1 receptors in the heart: the Endothelin-A

(ETA) and Endothelin-B (ETB) receptors. These receptors are expressed primarily in endothelial cells; although there are reports of its expression in both cardiomyocytes and cardiac fibroblasts (Chen et al., 2000; Dashwood and Abraham, 2011). ET1 has been demonstrated to induce fibroblast activation and scar formation in the skin and lung

(Appleton et al., 1992; Guidry and Hook, 1991; Shi-Wen et al., 2004). In cardiac fibroblasts, ET1 causes increased collagen production and myofibroblast formation in cultured cardiac fibroblasts (Hafizi et al., 2004; Nishida et al., 2007). Furthermore,

myofibroblasts isolated from the scar post-MI have increased ET1 expression (Katwa,

2003). The role of ET1 in cardiac fibrosis in vivo is not well characterized (Davis and

Molkentin, 2014).

Currently, there is evidence suggesting that ET1 works synergistically with other profibrotic factors to promote persistence of the myofibroblast phenotype (Leask, 2007,

2010). While ET1 is able to activate fibroblast on its own, it also acts as a downstream mediator of both AngII and TGFβ signaling (Leask, 2010; Shi-Wen et al., 2006; Shi-wen et al., 2007). AngII has been shown to induce ET1 expression in cardiac fibroblasts and this effect was abrogated with losartan treatment (Cheng et al., 2003). ET1 is also regulated by non-canonical TGFβ signaling in pulmonary fibroblasts (Shi-Wen et al.,

2006). Treatment with non-specific endothelin receptor antagonist bosentan was able to partially inhibit TGFβ mediated fibroblast activation (Shi-wen et al., 2007).

Calcium (Ca2+) Dependent Signaling

Recently, the role of sustained cytosolic Ca2+ has been shown to promote the myofibroblast transdifferentiation. Ca2+ likely serves as a nodal second messenger in signaling cascades for a variety of profibrotic factors. Indeed, TGFβ, AngII, and ET1 have all been shown to increase cytosolic Ca2+ concentrations (Alevizopoulos et al.,

1997; Furuya et al., 2005; McGowan et al., 2002; Muldoon et al., 1988; Ostrom et al.,

2003). There are a variety of Ca2+ influx mechanisms in different cell types, including release from intracellular stores by ryanodine receptor (RyR) and IP3 receptor (IP3R) activation (Clapham, 2007). In cardiac fibroblasts however, no role for RyR has been demonstrated. RyR1-3 have not been able to be detected at the transcriptional level in 18

cultured human or neonatal rat cardiac fibroblasts (Chen et al., 2010; Ding et al., 2017).

In addition, intracellular Ca2+ oscillations in cardiac fibroblasts were not altered by ryanodine or caffeine (Chen et al., 2010). All three IP3Rs are expressed in cardiac fibroblasts and intracellular Ca2+ oscillations can be completely blocked by PLC inhibitors or IP3R inhibitors. Conversely, IP3R agonist, thimerosal, was demonstrated to increase intracellular Ca2+ (Chen et al., 2010).

Many GPCRs are expressed in cardiac fibroblasts and the aforementioned AngII binding to AT1Rs leads to the activation of Gq, leading to IP3-mediated Ca2+ release.

This increase in intracellular Ca2+ is believed to be responsible for AngII-mediated collagen expression (Brilla et al., 1998). In addition to AngII, bradykinin, ATP, and UTP have been demonstrated to increase IP3 production through activation of Gq-coupled

GPCRs (Meszaros et al., 2000). Interestingly, although ETA is coupled to Gq, ET1 treatment failed to increase IP3 production or an increase in Ca2+ concentration

(Meszaros et al., 2000). Upon release of Ca2+ from intracellular stores by IP3, Ca2+ enters through ion channels in the plasma membrane by various mechanisms. One pathway occurs through receptor-mediated Ca2+ entry, which is primarily mediated by transient receptor potential (TRP) channels (Travers et al., 2016).

TRP channels have been implicated in fibroblast activation. This family of channels are composed of several subtypes with the following classifications: TRPC

(canonical), TRPM (melastatin), TRPV (vanilloid), TRPP (polycystin), TRPA (Ankyrin), and TRPML (mucolipin). Most of these channels are activated by various ligands, mechanical stress, or through changes in intracellular Ca2+ (Stempien-Otero et al., 2016).

Three TRP channels have been demonstrated to induce activation of cardiac fibroblasts.

TRPM7, which is specific to human atrial fibroblasts, is the dominant source of Ca2+ during TGFβ-mediated activation (Du et al., 2010). TRPV4 is another TRP channel and was also demonstrated to be involved in TGFβ as well as mechanical stress mediated transdifferentiation (Adapala et al., 2013). While TRPM7 and TRPV4 have been shown to alter fibroblast activation in vitro, they have not been studied extensively in vivo.

TRPC6KO fibroblasts demonstrated impaired activation and fibroblast-specific

TRPC6KO mice reduced cardiac fibroblast activation in vivo; although this led to reduced survival after MI, as the scar formation was impaired.

A classical Ca2+-mediated signaling cascade that appears to be involved in fibroblast activation involves the calcineurin (Cn) – nuclear factor of activated T cells

(NFAT) pathway. Cn is a Ca2+-dependent serine-threonine protein phosphatase that has been shown to be a pro-hypertrophic signaling effector in the heart (Molkentin et al.,

1998). Sustained Ca2+ elevations leads to formation of a heterotrimeric complex composed of Cn A, the catalytic subunit of Cn, Cn B, the regulatory subunit, and calmodulin. Complex formation leads to the activation of Cn, binding to NFAT, leading to its dephosphorylation, and subsequently NFAT translocation to the nucleus and gene transcription (Molkentin, 2013). In cardiac fibroblasts, overexpression of a constitutively active Cn led to transdifferentiation while overexpression of inhibitory molecules for Cn and NFAT, Cain and VIVIT respectively, inhibited TRPC6 mediated α-SMA expression

(Davis et al., 2012). Furthermore, both TGFβ and AngII induces NFAT activity in cardiac and embryonic fibroblasts (Abbasi et al., 2005; Davis et al., 2012; Gooch et al.,

2004; Herum et al., 2013; White et al., 2012). NFAT has been shown to directly regulate the expression of α-SMA along with another transcription factor, serum response factor 20

(SRF), by binding a conserved intronic CArG element in smooth muscle cells (Gonzalez

Bosc et al., 2005).

While cytosolic Ca2+ signaling appears to be required for a variety of profibrotic signals, recently there has been evidence for a critical role in mitochondrial Ca2+.

Elevations in cytosolic Ca2+ is quickly sequestered into the mitochondria through the mitochondrial calcium uniporter channel complex (mtCU) (Kirichok et al., 2004).

Various fibrotic signaling alters the gating of the mtCU to reduce mitochondrial Ca2+ uptake. Deletion of the mitochondrial calcium uniporter (MCU) in fibroblasts led to decreased mitochondrial Ca2+ uptake and increased cytosolic Ca2+ transients, NFAT activation, and myofibroblast differentiation at baseline and after TGFβ or AngII stimulation. In addition, the profibrotic stimuli mediated changes in mtCU gating caused changes in metabolism, which in turn regulated the epigenome to promote fibroblast activation (Lombardi et al., 2019).

CHAPTER 2

G PROTEIN-COUPLED RECEPTOR KINASE 5 IN CARDIAC

PATHOPHYSIOLOGY

G Protein-Coupled Receptor (GPCR) Signaling Overview

Communication at the cellular level is necessary for proper function at the cellular, tissue, and organismal levels. Cells perceive and respond to their environment by being exposed to various stimuli. These extracellular stimuli are then transduced into intracellular signaling pathways leading to a proper response. Cell receptors are the critical protein that allows for the transduction of these stimuli into signaling cascades through activation of second messengers, which amplify the signal. There are a variety of cell surface receptors that have been identified, and of those, GPCRs comprise the largest group with approximately 1000 in its family (Lefkowitz, 2007). In fact, GPCRs comprise the largest protein superfamily in mammalian genomes (Katritch et al., 2013). Adding to their pathophysiological importance, GPCRs are targets for ~35% (475 total) of approved drugs (targeting 108 unique GPCRs) making it the most intensively studied drug target family. In addition, approximately 321 other GPCR targeting drugs are currently in clinical trials, of which 20% are targeting novel GPCRs (Hauser et al., 2017;

Insel et al., 2019). Because of the sheer number of different GPCRs, they are able to transduce a diverse number of signals ranging from hormones, neurotransmitters, ions, photons, odorants, and other stimuli (Hilger et al., 2018).

GPCR Structure

GPCRs, also called seven transmembrane receptors due to their seven membrane spanning domains, can be classified based on sequence similarities of their transmembrane segments: the rhodopsin family (701 members), adhesion family (24 members), frizzled/tase family (24 members), the glutamate family (15 members), and the secretin family (15 members) (Fredriksson et al., 2003). A large number of these receptors have unknown physiological functions and have been coined as orphan GPCRs.

As such, “de-orphanization” of these receptors is a subject of ongoing research to determine if these are suitable pharmacological targets in the context of disease (Howard et al., 2001).

As mentioned, GPCRs have seven transmembrane domains along with an extracellular amino terminus and an intracellular carboxyl terminus (Kobilka, 2007).

Homology among the GPCRs is greatest in the transmembrane domains while there is variation in the carboxyl terminus, intracellular loop spanning TM5 and TM6, and the greatest variation is seen in the amino terminus. The structural similarity of GPCRs are a stark contrast to the structural variety seen in their ligands. GPCR ligands range from subatomic molecules (photons), ions, such as Ca2+, small organic molecules, and proteins

(Ji et al., 1998). Small organic agonists tend to bind GPCRs within the transmembrane segments while hormones and proteins often bind to the amino terminus and extracellular sequences joining the transmembrane domains (Kobilka, 2007). However, ligand size does not appropriately correctly predict the ligand binding site as hormones, glutamate, and Ca2+ all bind their respective receptors in the amino terminus (Ji et al., 1998; Pin et al., 2003). Regardless of ligands, interactions with the effector, the heterotrimeric G

protein, as well as protein that regulate or further transduce the signal, occur intracellularly. GPCRs and G proteins interact at the second and third cytoplasmic loops

(Gether and Kobilka, 1998). The third intracellular loop and carboxyl terminus can interact with GRKs and β-arrestins (Lohse et al., 1990).

GPCR Function and G Protein Activation

Upon agonist binding, the activated receptor undergoes a conformational change allowing for the binding of heterotrimeric G proteins as well as GRKs. While active,

GPCRs act as a guanine nucleotide exchange factor (GEF) on G proteins to promote the exchange of guanosine diphosphate (GDP) with guanosine triphosphate (GTP) that is associated with the Gα subunit. Once bound to GTP, the heterotrimeric G protein undergoes a conformational change, causing dissociation of the α form the β/γ subunits.

These different subunits interact with their respective second messengers, leading to the transduction of the ligand-mediated signal. The α subunit also acts as a GTPase, hydrolyzing GTP back to GDP, and this process allows for the α subunit to re-associate with the β/γ subunits (Oldham and Hamm, 2008).

Despite the large number of different GPCRs, there are only 35 different types of their direct downstream effectors (Milligan and Kostenis, 2006). In humans, there are 21

Gα subunits encoded by 16 genes, 6 Gβ subunits encoded by 5 genes, and 12 Gγ subunits

(Downes and Gautam, 1999). Heterotrimers are typically divided into four main classes based on the type of the Gα subunit: Gαs, Gαi, Gαq, and Gα12/13 (Oldham and Hamm,

2008). These different Gα proteins activate distinct effector proteins, and the molecular

basis for this divergence has not been completely elucidated (Cabrera-Vera et al., 2003).

Briefly, Gαs activates adenylyl cyclase (AC) to increase cyclic AMP (cAMP) while Gαi inhibits AC to decrease cAMP. Gαq activates phospholipase C, which leads to an increase in IP3 and DAG by cleavage of PIP2. Gα12/13 binds to Rho guanine nucleotide exchange factors (Milligan and Kostenis, 2006). Gβγ subunits were initially believed to be passive by binding to Gα and increasing the return of the heterotrimeric G protein complex back to the plasma membrane and increasing specificity of the signal. However,

Gβγ has been shown to interact and activate downstream effectors, such as PLCβ2 and

β3, ion channels, ACs, GRK2, phosphoinositide 3-kinase, and components of the MAPK pathway (Herlitze et al., 1996; Inglese et al., 1995; Katz et al., 1992; Logothetis et al.,

1987; Pitcher et al., 1992; Stephens et al., 1994; Sternweis, 1994; Tang and Gilman,

1991).

GPCR Regulation

While GPCR signaling is critical for proper functioning of cells, continued signaling or overstimulation can be deleterious. Therefore, there are a number of mechanisms to limit GPCR signaling, referred to as desensitization. Desensitization can occur as a short-term process or can continue chronically. When a GPCR is exposed to an agonist, effector pathways are activated quickly within a few milliseconds to a few minutes depending on the GPCR (Lefkowitz, 2013). However, repeated stimulation over minutes leads to a blunted response compared to the initial stimulus (Rajagopal and

Shenoy, 2018). This short-term desensitization involves GPCR phosphorylation, causing

GPCRs that are refractory to stimuli. When GPCRs are exposed to stimuli over a long period of time, the response is significantly blunted due to decreased receptor expression at the plasma membrane. This long-term desensitization caused by GPCR downregulation is associated with receptor internalization and decreased mRNA levels of the receptor (Premont and Gainetdinov, 2007).

An early step in GPCR desensitization involves GPCR phosphorylation at the intracellular carboxyl terminus. GPCRs can be phosphorylated in a homologous fashion, which requires GPCRs to be agonist bound, and is specific for a particular GPCR subtype or in a heterologous manner, in which GPCRs are phosphorylated in the absence of an agonist and is a more generalized effect involving simultaneous loss of agonist responsiveness at multiple GPCR subtypes (Bouvier et al., 1988; Hausdorff et al., 1989).

Heterologous phosphorylation is mediated by protein kinase A (PKA) and PKC which are activated by second messengers cAMP and DAG respectively. In contrast, homologous desensitization is mediated by GRKs. GRK mediated phosphorylation enhances the affinity of the GPCR to interact with β-arrestins, which bind the receptor and prevent further G protein coupling (Rockman et al., 2002). A summary of this homologous desensitization after GPCR activation is depicted in Figure 3.

β-arrestins (βarr1 and βarr2) are ubiquitously expressed proteins that can lead to internalization of GPCRs or assembling signaling cascades. β-arrestin binding to GRK phosphorylated GPCRs are able to recruit endocytic machinery, such as clathrin and the clathrin adaptor AP2, to the GPCR. This allows endocytosis of the receptor where it can be dephosphorylated and desensitized in acidic endosomes and then recycled.

Figure 3. GPCR Activation and Desensitization In the absence of GPCR activation, Gαβγ is bound to GDP and is not associated with the GPCR. Upon ligand binding, Gαβγ associates with the receptor and undergoes nucleotide exchange from GDP to GTP. Gα and Gβγ then dissociate from the GPCR and activates their respective signaling cascades. GRKs then phosphorylate the agonist bound receptor, which causes a conformational change preventing further G protein coupling as well as β-arrestin recruitment. The β-arrestin bound receptors can then be endocytosed through a clathrin mediated process. Adapted from Sato, 2015.

Alternatively, the receptor may be targeted to the endosome for degradation (Drake et al.,

2006). β-arrestins also act as adaptors or scaffolds that bring together elements of GPCR stimulated signaling pathways. Because GPCRs, unlike receptor tyrosine kinases, do not have intrinsic kinase activity, the direct downstream signaling pathways are mediated by catalytic proteins that are assembled by β-arrestins (Rockman et al., 2002). These include members of the Src family, thereby leading to the activation of MAPK pathways in a ligand dependent manner (DeFea et al., 2000a, 2000b; Luttrell et al., 1999). In addition,

β-arrestins can act as a scaffold for members of the MAPK pathways as well, leading to the activation of JNK3 and extracellular signal-regulated kinase (ERK) (DeFea et al.,

2000a; Luttrell et al., 2001; McDonald et al., 2000). Finally, β-arrestin binding to GPCRs is involved in ubiquitination. β-arrestin ubiquitination is required for receptor internalization and ubiquitination of the receptor is required for lysosomal targeting and degradation of the GPCR. β-arrestin is also the adaptor protein that brings the ubiquitination machinery in contact with the receptor (Shenoy et al., 2001). Because β- arrestins have their own signaling pathways, development of biased agonist that are able to activate β-arrestin mediated signaling without activating G protein dependent signaling have therapeutic potential (Rajagopal et al., 2010).

G Protein-Coupled Receptor Kinases (GRKs)

GRK Family Members and Structure

GRKs are a family of seven serine/threonine protein kinases (GRKs 1-7). This is a small family considering that they are responsible for phosphorylating and deactivating

several hundred GPCRs with only few of these that are not regulated by phosphorylation events (Fredriksson et al., 2003). Based on sequence and structural similarity, the seven

GRKs are subdivided into three main subfamilies: the visual or rhodopsin-kinases subfamily (GRK1 and GRK7), β-adrenergic receptor kinases subfamily (GRK2 and

GRK3), and the GRK4 subfamily (GRK4, GRK5, and GRK6) (Sato et al., 2015). All

GRKs have three main regions. First, a short 30-residue proximal amino-terminal region unique to GRKs. This amino terminus is highly conserved, unique to GRKs, and is responsible for recognizing and binding to activated GPCR (Gurevich et al., 2012;

Inglese et al., 1993). Second, the regulator of G protein signaling (RGS) homology domain located in the amino-terminal. Third, the carboxyl terminus has the highest variability. In this domain, GRK1 and GRK7 have a short prenylation sequence while

GRK2 and GRK3 contain a pleckstrin homology domain that has been implicated in phospholipid and Gβγ binding (Carman et al., 2000; DebBurman et al., 1996; Hisatomi et al., 1998; Inglese et al., 1992; Koch et al., 1993; Pitcher et al., 1992). This interaction allows GRK2 and GRK3 to be targeted to activated GPCRs (Haga et al., 1994). GRK4 and GRK 6 have palmitoylation sites as well as lipid binding capabilities while GRK5 has a phospholipid binding domain, composed of an amphipathic helix, which targets it to the membrane (Premont et al., 1996; Pronin et al., 1998; Stoffel et al., 1994). Finally, there is a serine/threonine protein kinase domain that interrupts the RGS homology domain. This kinase domain belongs to the AGC protein kinase subfamily of serine/threonine kinases named after PKA, PKG, and PKC (Gurevich et al., 2012; Inglese et al., 1993).

GRK Tissue and Subcellular Localization

GRK1 and GRK7 are primarily expressed in the retina where GRK1 regulates rhodopsin while GRK7 regulates cone opsin (Hisatomi et al., 1998; Lorenz et al., 1991;

Weiss et al., 1998). These GRKs are also expressed in the pineal gland; albeit at levels roughly 68% compared to the retina as measured by kinase activity (Somers and Klein,

1984). GRK4 is localized primarily to the testes although there are reports that it is expressed in the kidneys, brain, and the uterus (Brenninkmeijer et al., 1999; Felder et al.,

2002; Premont et al., 1996; Sallese et al., 2000). GRKs 2, 3, 5, and 6 are expressed in most tissue types, with GRK2 and GRK5 being the most prominently expressed GRKs in the heart (Dzimiri et al., 2004).

Because the canonical action of GRKs is to counteract chronic GPCR activation by phosphorylating the receptor, GRKs are usually present in the cytosol or close to the plasma membrane. As mentioned above, the carboxyl terminus is responsible for membrane targeting of GRKs to the activated GPCR. Recently, non-canonical activities of GRKs have been elucidated and show that GRKs do not strictly act just as GPCR desensitizers. Members of the GRK4 subfamily (GRK4, GRK5, and GRK6) were all shown to contain a nuclear localization signal (NLS) within their kinase domain, allowing them to translocate into the nucleus (Johnson et al., 2004, 2013; Yi et al., 2002).

In addition, GRK2 was shown to be able to translocate into the mitochondria (Chen et al.,

2013). These non-receptor actions have been implicated in disease processes and are therefore possible therapeutic targets.

GRK Regulation

As with chronic activation of GPCRs, proper regulation of GRK is crucial for proper cellular function. Therefore, various molecular mechanisms are in place in order to regulate GRK activity. GRK1 is capable of auto-phosphorylation, which prevents kinase activity towards rhodopsin (Buczyłko et al., 1991; Palczewski et al., 1993). PKA and PKC is able to directly phosphorylate GRK2, enhancing its activity (Chuang et al.,

1995; Cong et al., 2001). On the other hand, PKC phosphorylation of GRK5 inhibits its activity (Pronin et al., 1998). Calmodulin (CaM) is also able to bind GRK5 causing autophosphorylation leading to an inhibition in its activity at the receptor (Pronin et al.,

1997). Interestingly, this increases its nuclear translocation and increases its ability to phosphorylate non-GPCR targets (Gold et al., 2013).

Phosphorylation of GPCRs by GRKs leads to the recruitment of β-arrestins and their signaling complexes. Src, a mediator of β-arrestin signaling, is able to phosphorylate GRK2, leading to its degradation. In addition, GRK2 is able to be phosphorylated by members of the MAPK pathway, specifically ERK and p38 MAPK, leading to an inhibition of its canonical activity. Interestingly, this phosphorylation event, specifically at Ser670, leads to its mitochondrial translocation (Chen et al., 2013).

G Protein Coupled Receptor Kinase 5 (GRK5)

Pathophysiological Roles of GRK5

The role of GRK5 has been studied in a variety of different tissues due to its universal expression. GRK5 is expressed at high levels in various parts of the brain, such 31

as the septum, septohippocampal nucleus, the anterior thalamic nuclei, the medial habenula, and the locus coeruleus (Erdtmann-Vourliotis et al., 2001; Premont and

Gainetdinov, 2007). The expression of GRK5 increases during neuronal differentiation and appears to be critical as GRK5KO mice have immature spine development and impaired learning and memory (Chen et al., 2011). Furthermore, GRK5KO mice have increased muscarinic responses, such as hypothermia, salivation, and tremor, after treatment with non-selective muscarinic agonist, oxotremorine. This suggests that GRK5 is involved in the regulation of muscarinic receptors in the central nervous system.

Conversely, GRK5 deficiency had no effect on the responsiveness to dopamine, µ- opioids, or serotonin (Gainetdinov et al., 1999). In addition, GRK5 has been shown to phosphorylate α-synuclein causing its oligomerization and accumulation (Pronin et al.,

2000). α-synuclein accumulation leads to the formation of Lewy bodies, which contribute to the development of Parkinson’s disease and Lewy body dementia. Importantly, GRK5 is upregulated and co-localizes with α-synuclein in patients with Parkinson’s disease

(Arawaka et al., 2006; Bychkov et al., 2008). Conversely, GRK5KO mice develop a phenotype resembling Alzheimer’s disease with impaired memory, accelerated development of β-amyloid, and impaired acetylcholine release (Cheng et al., 2010; Liu et al., 2009; Suo et al., 2007).

GRK5 is also involved in the regulation of the inflammatory response. In global

GRK5KO mice, lipopolysaccharide (LPS) induced neutrophil infiltration into the lungs was attenuated. In addition, GRK5KO macrophages demonstrated decreased cytokine production (Patial et al., 2011). Mechanistically, this was due to GRK5’s non-canonical role as an IκBα kinase causing its degradation, release of NFκB, and nuclear shuttling of 32

NFκB (Patial et al., 2009). Interestingly, GRK5’s role in regulating the inflammatory response via NFκB appears to be cell-type dependent. In cardiomyocytes, GRK5 positively regulates NFκB; however in endothelial cells and vascular smooth muscle,

GRK5 inhibits NFκB mediated gene expression (Islam et al., 2013; Sorriento et al., 2008;

Wu et al., 2012). GRK5 is also involved in regulation of the chemokine receptor,

CXCR4, by phosphorylating its adaptor protein heat shock protein (Hsp) 70 interacting protein.

Similar to the central nervous system, GRK5 is involved in the muscarinic response of the airway. GRK5KO mice have impaired relaxation due to excessive muscarinic receptor mediated opposition of smooth muscle relaxation. Interestingly, the cholinergic response appears to be specific to M2 muscarinic receptors as airway contraction was not changed. Furthermore, cardiac muscarinic receptors were not regulated by GRK5, as no change in heart rate was reported, suggesting that regulation of

M2 by GRK5 is also specific to smooth muscle with no effects on peripheral cardiac muscarinic receptors (Walker et al., 2004).

GRK5 in the Cardiovascular System

GRK5 is highly expressed in the cardiovascular system and is the most highly expressed GRK in the heart (Montó et al., 2012). In fact, the expression of GRK5 is highest in the heart out of all tissues (Kunapuli and Benovic, 1993). Importantly, the expression of GRK5 increases by 68% in the heart of patients with dilated cardiomyopathy and 48% in patients with volume overload (Dzimiri et al., 2004). Based on these observations, GRK5 has become a therapeutic target in HF. 33

GRK5 appears to have a role in cardiac development. Zebrafish that are lacking the GRK5 homologue, Grk5l, have developmental defects in the heart. Grk5l KO zebrafish have randomized cardiac cooping and left/right symmetry due to enhanced mammalian target of rapamycin complex 1 (mTORC1) activity. This increase in mTORC1 activity led to an elongation of motile cilia of the heart laterally, causing aberrant organ arrangement. In addition, Grk5l null hearts exhibit generalized atrophy

(Burkhalter et al., 2013; Philipp et al., 2014). GRK5KO mice, unlike GRK2KO mice, are viable and have no basal phenotype (Gainetdinov et al., 1999). However, GRK5 and

GRK6 double KO mice are embryonic lethal, suggesting that GRK6 may be compensating for the lack of GRK5 in GRK5KO mice (Burkhalter et al., 2013).

Like the other GRKs, the canonical function of GRK5 is to phosphorylate agonist occupied GPCRs. As such, cardiomyocyte specific GRK5 overexpressing mice (TK45) have severely attenuated β-adrenergic receptor (β-AR) activation in vivo due to increased

β-AR desensitization (Rockman et al., 1996). However, TK45 mice did not experience a change in contractility while mice overexpressing GRK2 (BK12) had a reduced contractile response after AngII stimulation. Conversely, TK45 mice had diminished response to adenosine while BK12 mice did not (Rockman et al., 1996). These data suggest that GRK5 and GRK2 have varying receptor specificity in regards to their GPCR desensitization roles.

Recently, non-canonical roles for GRK5 have been elucidated and revealed that they are involved in disease progression. As mentioned, GRK5 can bind CaM, leading to its nuclear translocation. GRK5 binds CaM at an affinity 40 times higher compared to

GRK2 (Sallese et al., 2000). This binding process decreases GRK5’s affinity for GPCRs 34

while maintaining its ability to phosphorylate soluble substrates (Chuang et al., 1996;

Freeman et al., 1998). CaM binds both terminal domains of GRK5, blocking its ability to associate with the plasma membrane (Beyett et al., 2019). After being freed from the plasma membrane, the NLS allows nuclear transport. This binding of CaM to GRK5 occurs in an agonist specific manner. Agonists that activate Gq-coupled receptors, specifically α1-adrenergic receptors and AngII-AT1R receptors, lead to the activation of

PLC and subsequently increase intracellular Ca2+. This increase activates CaM, allowing it to bind GRK5 (Gold et al., 2013). Interestingly, stimulation of the ET1 receptor, which is coupled to Gq and is desensitized by GRK5, does not lead to nuclear accumulation of

GRK5. The NLS found within the kinase domain of GRK5 is homologous to homeobox- containing transcription factors (Johnson et al., 2004). These NLS domains are known to bind DNA, and this appears to also occur in GRK5. GRK was shown to bind DNA cellulose and in vivo, GRK5 binds to the Bcl-2 promoter and inhibits its transcription

(Johnson et al., 2013; Liu et al., 2010).

In order to determine the role of GRK5 in cardiovascular disease, various mouse models have been developed and characterized in a variety of disease contexts. TK45 mice are intolerant to left ventricular pressure overload induced by transaortic constriction surgery (TAC). This model of HF led to an exaggerated hypertrophic response and hastened HF progression in GRK5 overexpressing mice compared to WT mice (Martini et al., 2008). This accelerated HF response was attributed to GRK5’s non- canonical activity as mice that overexpressed GRK5 with a mutant NLS at the same levels of the TK45 had a similar phenotype as WT animals after TAC injury. Notably, global and cardiomyocyte-specific GRK5KO mice were protected against pathological

cardiac hypertrophy and HF after TAC and phenylephrine infusion (Gold et al., 2012). In addition, these mice had a diminished fibrotic response as shown by Masson’s Trichrome staining as well as Col1a2 gene expression.

Mechanistically, nuclear GRK5 is able to alter gene transcription by various pathways. First, GRK5 is able to phosphorylate histone deacetylase 5 (HDAC5) leading to its nuclear export. This releases transcription factor, myocyte enhancer factor 2

(MEF2), from HDAC5’s repressive effects, leading to MEF2 mediated pro-hypertrophic gene transcription (Martini et al., 2008; Zhang et al., 2011). Second, GRK5 was shown to potentiate NFAT-mediated gene transcription. GRK5 overexpression increased NFAT activity as shown by utilizing cardiac specific NFAT-luciferase reporter mice crossed with TK45 mice. Conversely, NFAT-luciferase reporter mice crossed with GRK5KO mice demonstrated diminished NFAT activity after TAC. Mechanistically, GRK5’s ability to bind DNA potentiated NFAT activity (Hullmann et al., 2014). Third, as mentioned above, GRK5 is able to regulate NFκB signaling in inflammation. GRK5 upregulation was associated with an increase in NFκB subunits p50 and p65 in cardiomyocytes (Islam et al., 2013). This however is controversial as it is also reported that GRK5 is able to inhibit NFκB signaling by causing nuclear accumulation of IκBα in bovine endothelial cells (Sorriento et al., 2008). Interestingly, NFκB binding elements exist in the GRK5 promoter and NFκB is able to regulate GRK5 expression in cardiomyocytes. This creates a positive feedback loop, at least in cardiomyocytes, leading to pathological signaling.

CHAPTER 3

GRK5 IS A NOVEL REGULATOR OF FIBROBLAST ACTIVATION AND

CARDIAC FIBROSIS

Akito Eguchi B.S. 1; Ryan Coleman B.S.1; Kenneth Gresham PhD 1; Erhe Gao MD, PhD 1; Jessica Ibetti PhD1; J. Kurt Chuprun PhD 1; Walter J. Koch PhD 1,2

1 Center for Translational Medicine, Lewis Katz School of Medicine at Temple University, Philadelphia, PA 2 Department of Pharmacology, Lewis Katz School of Medicine at Temple University, Philadelphia, PA

INTRODUCTION

Heart failure (HF) is a clinical syndrome characterized by the heart’s inability to pump blood sufficiently to meet the metabolic demands of the body. To combat the decrease in cardiac function, various compensatory mechanisms are activated to maintain perfusion. The renin-angiotensin-aldosterone system (RAAS) and sympathetic nervous system (SNS) activate in order to restore lost cardiac output following cardiac injury, such as myocardial infarction (MI) (de Lucia et al., 2018; Pfleger et al., 2019; Sato et al.,

2015). However, sustained activation of these systems exacerbates the condition, causing hemodynamic stress and myocyte death, prompting cardiac remodeling. Cardiac remodeling is a major driving force in the development and progression of HF that includes cardiac hypertrophy, ventricular dilation, and fibrosis (Hashimoto et al., 2018;

Nakamura and Sadoshima, 2018; Tallquist and Molkentin, 2017). The interstitial fibrosis, which decreases compliance as well as promotes arrhythmogenesis, is due to

excessive extracellular matrix proteins deposited by pathologically activated fibroblasts called myofibroblasts. After stress, the quiescent fibroblasts transdifferentiate into myofibroblasts, which have de novo expression of α-smooth muscle actin (αSMA) as well as hyper-secrete extracellular matrix proteins(Davis and Molkentin, 2014; Travers et al., 2016).

During acute tissue injury, profibrotic cytokines, such as angiotensin II (AngII) and transforming growth factor β (TGFβ), are released by inflammatory and mesenchymal cells, which leads to the activation of fibroblasts(Davis and Molkentin,

2014; Stempien-Otero et al., 2016). AngII signals through angiotensin type I receptor

(AT1R), a G protein-coupled receptor (GPCR), coupled to the heterotrimeric G protein,

Gαq (Forrester et al., 2018). AngII-AT1R signaling promotes fibroblast transdifferentiation through activation of calcium (Ca2+) signaling and subsequent gene transcription, primarily through the nuclear factor of activated T-cells (NFAT) as well as release of TGFβ and subsequent autocrine TGFβ signaling (Davis and Molkentin, 2014;

Davis et al., 2012; Forrester et al., 2018). Pharmacological inhibition of AngII in patients with HF has shown to reduce or delay the fibrotic remodeling of the heart (Díez et al.,

2002).

GPCR kinases (GRKs) are a family of serine threonine protein kinases, which canonically recognize and phosphorylate agonist activated GPCR’s in order to terminate signaling. This is the initiation step in the desensitization process, which also involves β- arrestin binding and GPCR internalization (de Lucia et al., 2018; Pfleger et al., 2019;

Sato et al., 2015). GRK5 is a major GRK isoform expressed in the heart and it has been shown to be upregulated in the myocardium of HF patients (Dzimiri et al., 2004). GRK5 38

is a member of the GRK4 subfamily of GRKs, all of which contain a nuclear localization signal (Johnson et al., 2004; Sato et al., 2015). In cardiomyocytes, GRK5 translocation to the nucleus has been shown to be crucial for pathological gene transcription following hypertrophic stress, and this occurs via non-canonical actions (Gold et al., 2012, 2013;

Hullmann et al., 2014; Martini et al., 2008). This occurs in myocytes downstream of Gαq activation that can occur through selective hypertrophic stressors, including α1- adrenergic and AngII mediated signaling pathways (Gold et al., 2013). The gene transcription facilitation induced by nuclear GRK5 in myocytes include NFAT

(Hullmann et al., 2014).

Novel drug classes for HF treatment have been sparse and targeting non-myocytes is a potential alternative approach in the treatment of the disease, especially the cardiac fibroblast. The injured heart contains a variety of signals which promote the transdifferentiation of fibroblasts into myofibroblasts (Humeres and Frangogiannis, 2019;

Stempien-Otero et al., 2016). Our incomplete understanding of these regulatory pathways that promote fibroblast activation has prevented the discovery of novel therapies to target the fibrotic response, which could affect many HF conditions, including HF, with preserved ejection fraction. Interestingly, GRK5 is highly expressed in the cardiac fibroblast and thus, the current study targets this kinase in cardiac fibroblast function in vitro and in vivo and its role in cardiac fibrosis after injury.

METHODS

Experimental Animals

To obtain inducible, fibroblast-specific GRK5 KO mice, collagen1α2- CreER(T) mice (Jackson Laboratories Stock no029235) were crossed with GRK5 fl/fl mice (Jackson

Laboratories Stock no 010960). Pups were backcrossed to generate collagen1α2-

CreER(T)/ GRK5 fl/fl; mice (referred to as GRK5 fibroKO). GRK5 fl/fl mice were used as wild-type (WT) littermate controls. 8-12 week old mice were injected with tamoxifen

(Sigma T5648) intraperitoneally for five days at 100mg/kg/day. All animal studies were conducted with the approval of the Animal Care and Use Committee at Temple

University.

Isolation of Adult and Neonatal Mouse Cardiac Fibroblasts and Myocytes

Hearts were removed from two to three month old mice and adult cardiac fibroblasts (MACFs) and myocytes were isolated as previously described.(Travers et al.,

2017) Briefly, adult mice were anesthetized with 2% isoflurane and injected with 200 U of heparin five minutes before heart excision. The heart was suspended on a Langendorff aparatus and retrogradely exposed to perfusion buffer (120.4 mM NaCl, 14.7 mM KCl,

0.6 mM KH2PO4, 0.6 mM Na2HPO4, 1.2 mM MgSO4, 10 mM Hepes, 4.6 mM NaHCO3,

30 mM taurine, 10 mM BDM (2,3-butanedione monoxime), and 5.5 mM glucose) at

37°C. Hearts were then perfused for 12 min with enzyme solution containing 50 mg of collagenase and 0.005% trypsin in perfusion buffer containing 12.5 µM CaCl2. Solution was then switched to perfusion buffer for another three min. Atria was removed and ventricles were gently minced. Cells were dissociated by pipetting in stop solution

(perfusion buffer containing 10% fetal bovine serum and 12.5 µM CaCl2).

Cardiomyocytes were allowed to settle by centrifugation at 300 rpm for 30 seconds. The

supernatant was collected and centrifuged at 500 xg for five minutes to collect the first fibroblast (non-myocyte) fraction. Cardiomyocytes were resuspended in 10mL DMEM with 10% FBS and subsequently allowed to settle. Supernatant was collected, centrifuged, and combined with the first fibroblast fraction. Fibroblasts were resuspended and plated in growth media (DMEM with 10% FBS) on a 10cm plate coated with 1% gelatin.

Neonatal rat cardiac fibroblasts (NRCFs) were isolated as a byproduct of neonatal rat cardiac myocyte isolation, performed as previously described.(Brinks et al., 2010)

Briefly, hearts were isolated from one- to two-day-old neonatal rats. Hearts were pre- washed in ADS buffer (NaCl 116 mM, HEPES 20 mM, Na2HPO4 0.8 mM, glucose

5.6 mM, KCl 7 mM, and MgSO4-7H2O 0.8 mM, pH 7.35) to remove blood and then divided and placed in dishes with seven ml of ADS. They were minced with sterol razor blades in small pieces and then the whole solutions were transferred in flasks and incubated at 37 °C with seven ml enzyme solution (ADS containing pancreatin 0.6 mg/L, collagenase II 8820 U/L and CaCl2 50 mM) for 10 minutes. The supernatant from this pre-digestion step was discarded and the pieces were incubated with 15 ml of digestion solution for 15-minute intervals at 37 °C. After each interval, the supernatant was collected in 50 ml conical tubes containing 19 ml of F-10 media and 20% FBS pre-heated at 37 °C. The three to six collected fractions were spun down at 1,400g for 10 minutes, the supernatant was discarded and cells were washed with five ml of FBS for each tube.

The cells were then centrifuged at 1,400g for 10 minutes and the supernatant was discarded. The resulting pellet containing the cells was resuspended in HAM's F10 complete media containing 10% horse serum (HS), 5% FBS and 1% penicillin-

streptomycin (P/S), pH 7.4. The cell suspension was filtered through a 70 µm filter and pre-plated on a Nunc Nunclon 100 mm (Thermo Fisher Scientific, Waltham, MA, USA) cell culture dish for two hours to separate the fibroblasts from the myocyte fraction. The fibroblasts attached to the Nunclon dishes were cultured with DMEM with 10% FBS.

Real-time PCR

Total RNA was isolated from MACFs with TRIzol (Thermo Fisher) according to the company’s instructions. After RNA isolation, cDNA was synthesized by reverse transcription of the RNA (iScript cDNA synthesis kit, Bio-Rad). Real-time PCR was performed in triplicated on a CFX96 real-time PCR detection system (Bio-Rad) using

SYBR Green mix (Bio-Rad) and specific primers for mouse α-SMA, collagen I, collagen

III, MMP2, MMP9, and TGFβ. Expression levels were established by comparing to

TPT1 expression, which was similar between groups, for normalization and compared using the ΔΔCt method.

α-SMA: forward 5’-AGATTGTCCGTGACATCAAGG-3’ reverse 5’-TTGTGTGCTAGAGGCAGAGC-3’

Collagen I: forward 5’-ACCTTACCAGACTGAGACTCC-3’ reverse 5’-TTTGGCTCATTCGATCCTGATACCCG-3’

Collagen III: forward 5’-GAAGTCTCTGAAGCTGATGGG-3’ reverse 5’-TTGCCTTGCGTGTTTGATATTC-3’

MMP2: forward 5’-CAGGGAATGAGTACTGGGTCTATT-3’ reverse 5’-ACTCCAGTTAAAGGCAGCATCTAC-3’

MMP9: forward 5’-AATCTCTTCTAGAGACTGGGAAGGAG-3’ 42

reverse 5’-AGCTGATTGACTAAAGTAGCTGGA-3’

TGFβ: forward 5’-CAACAATTCCTGGCGTTACCTTGG-3’ reverse 5’-GAAAGCCCTGTATTCCGTCTCCTT-3’

TPT1: forward 5’-GGAGCTGCAGAGCAGATTAAG -3’ reverse 5’-TAGTCCAGGAGAGCAACCATAC-3’

Surface Sensing of Translation (SUnSET) Assay

WT and GRK5KO MACFs were stimulated with 1µM AngII for varying amounts of time, followed by a pulse of 1µM puromycin for 30 minutes prior to harvesting.

Immunoblotting was performed using an anti-puromycin antibody (Schmidt et al., 2009).

Calmodulin Capture Assay

Calmodulin (CaM)-agarose beads (Sigma) were washed with either 2mM CaCl2 or 2mM EDTA in IP buffer (50mM Tris, 150mM NaCl). 250µg of cell lysates were added to the CaM beads with a final concentration of either 2mM CaCl2 or 2mM EDTA

IP buffer and rotated overnight. Beads were washed with respective buffers, resuspended in 2X Laemmli buffer, and analyzed by immunoblotting (Kaleka et al., 2012).

Immunoblotting

After SDS-PAGE and transfer to nitrocellulose membranes, primary antibody incubations were performed overnight at 4°C. Fluorescent secondary antibodies were obtained from Li-Cor. Membranes were scanned with the Odyssey infrared imaging

system (LI-COR) (Woodall et al., 2016). GRK5 antibody was from Santa Cruz. α-SMA and Puromycin antibody was from Sigma.

In Vivo AngII Infusion and Model of Myocardial Infarction

AngII (1µg/kg/min) dissolved in PBS was continuously infused subcutaneously into mice via an osmotic minipump (ALZET) for four weeks. A control group was infused only with PBS. Mice were anesthetized with isofluorane and pumps were implanted subcutaneously through a sub-scapular incision, which was then closed using

4.0 silk suture (Ethicon). Tissue was collected four weeks post infusion.

MI surgery was performed as described previously and tissue was collected four weeks post MI (Gao et al., 2010).

Transthoracic Echocardiographic Analysis

Transthoracic 2-dimensional echocardiography was performed blinded as previously described using the Vevo 2100 imaging system (Grisanti et al., 2016).

Assessment of Myocardial Fibrosis and Hypertrophy

Collagen levels were measured using the Masson’s Trichrome staining kit (Sigma

HT15) without modifications as previously described (Woodall et al., 2016). Briefly, mice were euthanized four weeks after MI or AngII infusion. Hearts were removed and fixed in 4% paraformaldehyde, embedded in paraffin, and cut into long axis sections

6µM in thickness. Sections were deparaffinized and rehydrated. Before treatment with the Trichrome staining kit, sections were incubated at room temperature in Bouin’s 44

solution (HT101128) overnight. Images of the fibrosis were taken on a Nikon DS-Ri1 in a blinded manner. For each area of the heart, at least 10 random fields were measured.

Images were quantified using CellProfiler, a cell image analysis software, capable of determining fibrotic area in an unbiased manner (McQuin et al., 2018).

Cardiomyocyte hypertrophy was measured using wheat germ agglutinin (WGA) staining, as previously described (Schumacher et al., 2016). Briefly, heart sections were deparaffinized, rehydrated, and washed with phosphate-buffered saline. Sections were then stained with Alexa Fluor 488-conjugated WGA for one hour at room temperature in the dark. Sections were washed three times for five minutes and mounted using

Fluoromount-G mounting media containing DAPI nuclear stain (Southern Biotech).

Immunofluorescence

1x106 MACFs seeded on glass coverslips coated with 1% gelatin were fixed with

4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 5% BSA, and incubated with an anti-αSMA antibody in 5% BSA. Cells were then incubated with respective secondary antibodies.

Drug Treatments

Application of recombinant AngII (1-10uM), TGFβ (10ng/mL), and ET-1

(100nM) for 48 hours was used to induce myofibroblast transdifferentiation. GRK5 nuclear translocation inhibitor malbrancheamide (malb) was used at 1µM 24 hours before

AngII treatments (Beyett et al., 2019).

NFAT Luciferase Reporter and Luciferase Assay

Cardiac fibroblasts were infected with an NFAT reporter adenovirus at an MOI of

10. Medium was changed after 24 hours, and 48 hours after infection, cells were stimulated with AngII for 24 hours. Cells were lysed and luciferase activity measured

(Davis et al., 2012).

Collagen Gel Contraction Assay

Fibroblasts were harvested from a confluent monolayer by Trypsin-EDTA digestion, pelleted, and resuspended in serum free DMEM. Fibroblasts were then seeded into collagen matrices (0.85mg/mL) such that each gel contained 100,000 fibroblasts and cast in 24 well plates. The collagen gels were released from the edges and floating in serum free DMEM with or without AngII. ImageJ software was used to calculate the surface area, which are reported as values normalized to the initial size of the gel (Davis et al., 2012).

Statistical Tests

Data are expressed as mean ± standard deviation. Statistical significance was determined by ANOVA and Tukey’s multiple comparisons test for multivariate experiments and t-test for experiments with two groups.

Results

GRK5 is Required for Fibroblast to Myofibroblast Transdifferentiation

To investigate if GRK5 is required for fibroblast activation, we first isolated primary MACFs from wild type (WT) and global GRK5 knockout (GRK5KO) mice for in vitro analysis (Supplemental Fig 1) (Gainetdinov et al., 1999). WT and GRK5KO

MACFs were then treated with AngII for 48hrs and qRT-PCR was performed.

AngII stimulation caused an upregulation of many myofibroblast associated genes in WT MACFs while AngII did not increase expression of α-SMA, Collagen I, Collagen

III, MMP2, and TGFβ in GRK5KO MACFs (Fig 4A). Further, the AngII induced expression of α-SMA protein in WT MACFs was not seen after AngII treatment of

GRK5KO MACFs (Fig 4B). Immunofluorescence staining for α-SMA stress fiber formation was also inhibited in GRK5KO MACFs compared to WT controls after AngII treatment (Fig 4C). In addition, TGFβ induced an upregulation of α-SMA, MMP9,

Collagen I, and Collagen III. Interestingly, GRK5KO fibroblasts were only resistant to

TGFβ induced α-SMA gene expression (Supplemental Fig 2). α-SMA protein expression was also inhibited by GRK5 deletion as shown by immunofluorescence (Supplemental

Fig 2).

Functionally, the expression of α-SMA fibers in myofibroblasts acts to contract wounds. WT MACFs seeded into a floated collagen matrix demonstrated contraction after AngII stimulation while GRK5KO MACFs did not (Fig 4D). GRK5KO MACFs were also resistant to TGFβ mediated contraction (Supplemental Fig 2). GRK5KO

MACFs also demonstrated decreased wound closure as shown by an in vitro scratch

Figure 4. GRK5 is necessary for fibroblast transactivation into myofibroblasts (A) Myofibroblast gene expression in WT and GRK5KO MACF’s, as measured by qRT-PCR, normalized to transcriptionally controlled tumor protein 1 (TPT1); fold change vs WT Veh. MACFs were stimulated with 1µM AngII for 48 hours. n = 3 per group, 10 images were analyzed per biological replicate. (B) Immunoblot and quantification for α-SMA expression in WT and GRK5KO MACFs stimulated with AngII (normalized to total protein). n = 3 per group (C) Immunofluorescent staining and quantification of α-SMA (green)-positive cells in WT and GRK5KO MACFs stimulated with AngII. Cells were counterstained with DAPI. n = 3 per group. (D) Photographs and quantification of floating collagen gel matrices seeded with WT or GRK5KO MACFs that have contracted after 18 hours of AngII stimulation. n = 5 per group.* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001

assay (Supplemental Fig 3). The contraction of collagen gel matrices and scratch assay closure was not due to altered MACF proliferation in WT versus GRK5KO cells

(Supplemental Fig 4). Finally, myofibroblasts are characterized by their hyper-secretory state. It is currently unknown whether activation of fibroblasts is associated with an increase in protein translation. To determine the rate of protein translation, we performed a SUnSET assay. After four hours of AngII stimulation, there was a significant increase in puromycin incorporation into newly synthesized proteins in WT MACFs while

GRK5KO MACFs had a significantly reduced rate of protein translation, suggesting a more quiescent and fibroblast-like state (Supplemental Fig 5).

Decreased Levels of GRK5 in Fibroblasts Are Beneficial to AngII-Infused Myocardium

The observation that GRK5 was essential for fibroblast activation in vitro suggested that this kinase might participate in the fibrotic response in the heart in vivo. In order to determine the requirement of GRK5 in myofibroblast transdifferentiation and fibrotic response in vivo, we generated a tamoxifen inducible, fibroblast specific, GRK5

KO mouse. These mice were created by crossing mice expressing cre recombinase driven by the Col1a2 enhancer element and GRK5 flox mice (Zheng et al., 2002). Eight-week- old adult male Col1a2-cre/GRK5fl/fl mice and their control littermates were injected intraperitoneally with tamoxifen (100mg/kg per day) for five days to induce a loss of

GRK5 in fibroblasts (GRK5 fibroKO). After a two-week washout period, freshly isolated

MACFs from GRK5 FibroKO mice demonstrated ~80% protein loss compared to

MACFs isolated from WT mice (Fig 5).

Figure 5. GRK5 levels in fibroblasts isolated from GRK5 fibroKO mice. A) Immunoblot analysis and quantification of GRK5 from MACFs isolated from tamoxifen treated GRK5fl/ fl mice or GRK5 FibroKO mice. n = 5 per group ** P ≤ 0.01

To assess the effects of in vivo GRK5 KO on cardiac fibrosis, WT and GRK5

fibroKO mice were subjected to four weeks of AngII (1µg/kg/min) or saline (veh)

treatment. This dose has previously been shown to induce cardiac hypertrophy and

fibrosis without changes in systolic function (Francois Helene et al., 2004; Satoh Kimio

et al., 2011; Takayanagi et al., 2016; Zouein et al., 2013). Mice were followed at two

weeks and four weeks after AngII infusion with echocardiographic assessment of left

ventricular (LV) structure and function. Heart rate was unchanged in both WT and

GRK5fibroKO mice after AngII infusion at four weeks (WT Saline 466±30.6; WT AngII

456±19.7; FibroKO Saline 452±30.8; FibroKO AngII 450±36.3). Systolic function as

measured by LV ejection fraction also remained unchanged as was LV anterior wall

dimension at systole (Fig 6A). WT mice infused with AngII demonstrated significantly

increased LV posterior wall dimension at systole while this increase was absent in GRK5

fibroKO mice (Fig 6A). We also examined the heart and body weights (HW/BW ratio) of

WT and GRK5 fibroKO mice after AngII or veh treatment. Both WT and GRK5 fibroKO

mice had significantly higher heart weights after AngII compared to their respective

Figure 6. GRK5 fibroKO are protected against AngII mediated cardiac hypertrophy and fibrosis (A) Echocardiographic analysis of WT and GRK5 fibroKO mice infused with 1µg/kg/min with AngII for four weeks compared to four weeks of saline infusion. Cardiac function shown by LV ejection fraction and LV wall thickness shown by anterior wall and posterior wall thicknesses at systole. n = 5 per group (B) Quantification of heart weight (HW) normalized to body weight (BW) four weeks after AngII infusion. n = 5 per group (C) Representative images and quantification of WGA and 4′,6-diamidino-2-phenylindole (DAPI)–stained murine heart sections from hearts four weeks after AngII infusion. n = 5 per group. (D) Representative Masson’s Trichrome images and quantification of hearts after four weeks of AngII infusion. Quantification expressed as a percentage of fibrosis from the total area. n = 5 per group.* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001; Scale bar represents 50µm. 51

saline infused control; however GRK5 fibroKO mice had significantly less hypertrophy

(Fig 6B).

We performed histological analysis of both WT and GRK5 fibroKO hearts after four weeks of AngII infusion and compared these to the corresponding mice treated with saline (veh). Wheat germ agglutinin staining of the plasma membrane demonstrated increased cardiomyocyte cross sectional area in both WT and GRK5 fibroKO hearts after

AngII infusion compared to their respective saline controls, however like the HW/BW ratios, GRK5 fibroKO cardiomyocytes had significantly attenuated hypertrophy compared to WT (Fig 6C). AngII induces cardiac fibrosis through activation of fibroblasts. In order to determine the fibrotic response, we utilized Masson’s trichrome staining to stain fibrotic areas within the myocardium. WT mice demonstrated an increase in fibrosis in the LV, atria, and perivascular region after four weeks of AngII while GRK5 fibroKO mice only had a significant increase in LV fibrosis (Fig 6D). Thus, the loss of GRK5 in fibroblasts in vivo protected against AngII mediated cardiac fibrosis.

Fibroblast Specific Loss of GRK5 Protects Against Cardiac Ischemic Injury

In order to determine the effect of fibroblast specific GRK5 knockdown in a more clinically relevant model, we employed a mouse model of MI injury by permanently occluding the coronary artery (Gao et al., 2010). In vivo characterization was done by performing echocardiography on WT and GRK5 fibroKO mice before and after MI.

Control mice presented with decreased LV ejection fraction and LV fractional shortening at two weeks post-MI that worsened at four weeks (Fig 7A). However, GRK5 fibroKO mice displayed partial but significantly improved systolic function compared to WT mice 52

Figure 7. GRK5 fibroKO are protected against MI mediated cardiac dysfunction and fibrosis (A) Echocardiographic analysis of WT and GRK5 fibroKO mice that underwent MI surgery. Cardiac function shown by LV ejection fraction and fractional shortening (top panels). Wall thickness shown by LV anterior wall (LVAW) and posterior wall (LVPW) thicknesses at systole (middle panels). LV dilation shown by internal diameter. n = 5 for sham; 5-8 for MI (B) Quantification of heart weight (HW) normalized to body weight (BW) 4 weeks after MI surgery. n = 5 for sham; 5-8 for MI (C) Representative Masson’s Trichrome images and quantification of hearts 4 weeks after MI surgery. Quantification expressed as a percentage of fibrosis from the total area. n = 5 for sham; 5-8 for MI.* P ≤ 0.05 vs Sham, ** P ≤ 0.01, *** P ≤ 0.001, # P ≤ 0.05 vs WT M; Scale bar represents 50µm.

after MI (Fig 7A). GRK5 fibroKO mice also demonstrated preserved anterior and posterior wall thickness at systole as well as LV internal diameter compared to WT mice

(Fig 7A). There were no survival differences between the MI groups four weeks post-MI.

Four weeks after injury, hearts were harvested and heart weights were measured to assess WT and GRK5 fibroKO remodeling after MI. WT mice exhibited cardiac hypertrophy after four weeks of MI-induced injury compared to sham (Fig 7B). GRK5 fibroKO hearts were protected against cardiac hypertrophy post-MI (Fig 7B). Masson’s trichrome staining for fibrosis showed significant fibrosis in the border zone, remote zone, and perivascular region in WT mice four weeks post-MI whereas GRK5 fibroKO mice only had a significant increase in fibrosis in the border zone (Fig 7C). In all three areas, GRK5 fibroKO hearts displayed a significant decrease in fibrotic area compared to

WT mice four weeks post-MI (Fig 7C). Thus, GRK5 in fibroblasts is critical for the fibrotic response in vivo after ischemic injury.

Non-canonical GRK5 Activity is Responsible for Fibroblast Activation

GRK5 is a member of the GRK4 subfamily of GRKs, which all contain a nuclear localization signal (NLS) (Johnson et al., 2004, 2013; Sato et al., 2015). This allows

GRK5 to translocate to the nucleus where it can modulate gene transcription via non-

GPCR activity. Previous studies from our lab have shown that transgenic cardiomyocyte- specific overexpression of GRK5 in mice leads to nuclear and non-GPCR mediated pathology in the context of pressure-overload induced HF (Martini et al., 2008). Mice overexpressing a mutant GRK5 that cannot enter the nucleus (GRK5ΔNLS) were protected against the pathology seen in the TgGRK5 mice emphasizing the importance of

nuclear GRK5 in pathology (Martini et al., 2008). Our studies have shown that GRK5 translocation to the nucleus in myocytes is downstream of Gq activation and binding of

Ca2+-CaM binding to the N-terminus of GRK5 (Gold et al., 2013; Martini et al., 2008).

One of the non-canonical activities of GRK5 after its accumulation in the nucleus of myocytes is facilitating hypertrophic gene transcription through activation of NFAT

(Hullmann et al., 2014). Interestingly, NFAT is critical in myofibroblast transdifferentiation as well (Davis et al., 2012). In order to see if GRK5 has similar effects in cardiac fibroblasts as in myocytes after injury, neonatal rat cardiac fibroblasts were stimulated with 1µM AngII for 90 min and nuclear fractions were isolated. There was a significant enrichment of GRK5 in the nucleus after AngII stimulation, suggesting that GRK5 is able to translocate to the nucleus in fibroblasts, like in myocytes (Fig 8A).

Interestingly, TGFβ, a non-GPCR ligand, trended towards an increase in nuclear translocation of GRK5 (Supplemental Fig 6). In order to determine if CaM also binds

GRK5 in cardiac fibroblasts, neonatal cardiac fibroblasts were lysed and the lysate was incubated with CaM-conjugated agarose beads in either the presence or absence of Ca2+.

The CaM beads were able to pull down GRK5 only in the presence of Ca2+ (Fig 8B), suggesting that GRK5 binding CaM and subsequent translocation is a Ca2+dependent event, consistent with our previous myocyte studies. Finally, CaM signaling as well as

GRK5 non-canonical activity can activate NFAT mediated gene transcription. In order to see if GRK5 facilitates NFAT activity in the activation of cardiac fibroblasts, we infected

WT and GRK5KO MACFs with an NFAT-luciferase adenovirus. We then stimulated the

Figure 8. Non-canonical GRK5 activity promotes fibroblast activation. (A) Representative immunoblot and quantification of nuclear GRK5 after stimulation of neonatal rat cardiac fibroblasts with AngII (1µM) for 90 min. n = 4 (B) Representative immunoblot and quantification of CaM capture. n = 3 (C) NFAT-luciferase activity from WT and GRK5KO MACFs infected with reporter AdNFAT-luc stimulated with AngII. (D) Immunofluorescent staining and quantification of α-SMA (green)-positive cells in WT and GRK5KO MACFs stimulated with AngII with or without pretreatment with GRK5 nuclear translocation inhibitor Malb. Cells were counterstained with DAPI. n = 3 per group. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001

MACFs with 1 and 10µM AngII for 24 hrs and measured luciferase activity. WT MACFs stimulated with AngII demonstrated a significant increase in NFAT activity at both 1 and

10µM AngII while GRK5KO MACFs were refractory to AngII mediated NFAT activation (Fig 8C). These results suggest that GRK5 is crucial for NFAT-mediated gene transcription during fibroblast activation and this represents a potential contributing mechanism for the amelioration of fibrosis in vivo after chronic AngII treatment and MI above.

We previously reported that malbrancheamide (malb), a fungal indol alkaloid, was able to prevent nuclear translocation of GRK5 in cardiac fibroblasts (Beyett et al.,

2019). Furthermore, malb was able to prevent cardiomyocyte hypertrophy after PE treatment in vitro (Beyett et al., 2019). To determine if the translocation event is necessary for GRK5’s profibrotic function, we stimulated neonatal cardiac fibroblasts with AngII with or without pretreatment with malb. AngII again led to a significant increase in α-SMA positive cells and, interestingly, malb pretreatment significantly impaired fibroblast activation (Fig 8D), implying that nuclear translocation of GRK5 is a critical step required for fibroblast activation. Furthermore, ET-1, a profibrotic agonist that does not induce nuclear localization of GRK5, was able to induce α-SMA expression in both WT and GRK5KO MACFs (Supplemental Fig 6).

Discussion

Fibroblasts make up a significant proportion of the heart with 11% of the heart comprised of fibroblasts compared to 30% for cardiomyocytes (Pinto et al., 2016). In response to cardiac injury, quiescent fibroblasts transdifferentiate into myofibroblast in 57

order to promote healing. However, cardiac myofibroblasts remain active after the healing response and this promotes excessive fibrosis leading to cardiac remodeling, decreased compliance, and arrhythmogenesis. Treatments for HF that have shown efficacy, such as ACE inhibitors, AngII receptor blockers, and mineralocorticoid receptor antagonists, have shown that their therapeutic effects are in part due to their ability to decrease the development of fibrosis; however, no therapeutic intervention directly targets the fibrotic response. Targeting non-myocytes is a potential alternative approach in the treatment of HF given the critical role myofibroblasts play in the initiation and propagation of cardiac fibrosis and remodeling.

GPCRs comprise of roughly 800 human genes and constitute the largest family of proteins targeted by approved drugs (Pfleger et al., 2019). Cardiac fibroblasts in particular express 190 of these GPCRs (Snead and Insel, 2012). The major modality of

GPCR regulation is through GRK activation. GRKs canonically phosphorylate agonist- activated GPCRs to terminate signaling. Because of the sheer number of physiological processes mediated by GPCRs, including angiotensin type 1 receptors, GRKs are crucial in maintaining cardiovascular homeostasis. GRK5 is one of 4 GRKs expressed in the heart and we demonstrate that it is expressed in cardiac fibroblasts and plays a functional role in fibroblast biology.

The role of GRKs, especially GRK2 and GRK5, has been studied at length in the context of HF. These studies have demonstrated that these critical proteins are important inducers of structural and biochemical changes in the heart. Our group as well as others have investigated the functional role that GRK2 has in fibroblasts in the context of HF

(Travers et al., 2017; Woodall et al., 2016). Knockout of GRK2 in fibroblasts conferred a

protective advantage after myocardial ischemia/reperfusion injury. GRK2 fibroKO mice demonstrated preserved systolic function as well as decreased fibrosis (Woodall et al.,

2016). Mechanistically, this was due to a decrease in inflammation. Pharmacological inhibition of GRK2 also was able to prevent fibroblast activation through restoration of

β-adrenergic receptor signaling (Travers et al., 2017). The role of GRK5 in cardiac fibroblasts, however, is not understood. Here we uncovered a previously unknown function of GRK5 in myofibroblast transdifferentiation and cardiac fibrosis in vitro and in vivo. Genetic deletion of GRK5 in cardiac fibroblast prevented AngII mediated fibroblast activation. Using an inducible Cre recombinase driven by a fibroblast-specific regulatory sequence, we showed that knockdown of GRK5 in fibroblasts confers a protective advantage for cardiac function and fibrosis in an AngII infusion model as well as an ischemic injury model.

Recently, our lab as well as others, have elucidated non-canonical functions of

GRKs and have implicated these in cardiac disease processes. We have shown that nuclear GRK5 in cardiomyocytes can bind directly to DNA as well as phosphorylate histone deacytelase 5 to alter gene transcription after pressure overload (Hullmann et al.,

2014). Finally, nuclear GRK5 in cardiomyocytes facilitates NFAT-mediated gene transcription during pathological hypertrophy in a transaortic constriction model of HF.

Herein, we show that the same mechanism appears to be intact in cardiac fibroblasts as

GRK5KO fibroblasts are resistant to activation after AngII and ischemic stress and have diminished NFAT activity compared to WT fibroblasts. However, NFAT activity is not entirely abolished, which demonstrates that other signaling pathways, which feed into

NFAT, are still active. These data are consistent with previous work in cardiac fibroblasts

in which activation of NFAT through overexpression of NFAT itself or its upstream activator calcineurin led to activation of fibroblasts (Davis et al., 2012). Conversely, pharmacological inhibition of NFAT and calcineurin prevented fibroblast conversion

(Davis et al., 2012). In cardiac fibroblasts, NFAT target genes include α-SMA, collagen

I, and collagen III as pharmacological inhibition of NFAT leads to the abolishment of transcription of these genes (Davis et al., 2012; Herum et al., 2013). This is in line with our study, as genetic deletion of GRK5 led to diminished NFAT activity and decreased transcription of α-SMA, collagen I, and collagen III.

Recent studies indicate that sustained elevation of cytosolic Ca2+ can drive the activation of fibroblasts. Neurohormonal signaling molecules which are profibrotic, such as AngII and TGFβ, both trigger an increase in cytosolic Ca2+ (Alevizopoulos et al.,

1997; Ostrom et al., 2003). Furthermore, transient receptor potential (TRP) channels, specifically TRPM7, TRPC6, and TRPV4 which all act to increase cytosolic Ca2+in cardiac fibroblasts, were shown to induce fibroblast transdifferentiation (Adapala et al.,

2013; Davis et al., 2012; Du et al., 2010). In addition, profibrotic stimuli decrease mitochondrial Ca2+uptake thereby increasing cytosolic Ca2+ (Lombardi et al., 2019).

Direct inhibition of mitochondrial Ca2+uptake through genetic deletion of Mcu, the pore- forming unit of the mitochondrial Ca2+uniporter which is responsible for mitochondrial

Ca2+uptake, promoted fibroblast activation both at baseline and after profibrotic stimuli

(Lombardi et al., 2019). Induction of Gαq by AngII leads to an increase in cytosolic Ca2+.

Past studies in cardiac fibroblasts have shown that 1µM AngII can increase intracellular

Ca2+ 24-fold, and that the primary source of this Ca2+was from internal stores (Meszaros et al., 2000). This induces Ca2+-CaM binding to GRK5, causing dissociation from the 60

plasma membrane, and allowing nuclear transport of GRK5. Importantly, we show that this mechanism is active in fibroblasts and contributes to pathology mediated by GRK5.

Our studies show for the first time that prevention of Ca2+ -CaM binding to GRK5 with a small molecule, malb, can prevent fibroblast activation and is consistent with the nuclear localization of GRK5 mediating AngII-dependent fibroblast activation and transdifferentiation. Importantly, we did not see an increase in GRK5 protein levels after

AngII stimulation, implying that translocation is the primary driving event for fibroblast activation rather than its upregulation.

In both of our AngII and MI models of fibrosis, we saw a reduction in fibrosis that was accompanied by a reduction in the extent of cardiac hypertrophy in GRK5 fibroKO mice. This amelioration of the cardiomyocyte hypertrophic response has also been previously reported in Tgfbr1/2 and Hsp47 deletion in fibroblasts (Khalil et al.,

2017, 2019). This suggests that activation of fibroblasts and extracellular matrix deposition/myofibroblast mediated paracrine factors may be required to support the hypertrophic response in cardiomyocytes. Indirect evidence exists suggesting that a stiffer ECM matrix promotes cardiomyocyte hypertrophy; however, further studies are needed to determine the exact mechanism by which myofibroblasts are able to regulate myocyte biology (Terracio et al., 1991).

Inhibition of AngII signaling in patients with HF by angiotensin converting enzyme

(ACE) inhibitors or angiotensin receptor blockers (ARBs) have shown to reduce or delay the fibrotic remodeling of the heart (Díez et al., 2002). AngII is believed to be involved with the fibrotic response both directly and indirectly. Cardiac fibroblasts stimulated with

AngII induces TGFβ expression as well as downstream fibrotic genes, such as Col1a

(Campbell and Katwa, 1997; Gray et al., 1998; Lee et al., 1995). AngII is also able to activate canonical TGFβ signaling through activation of SMADs in cardiac fibroblasts

(Gao et al., 2009). This is in line with our data as AngII stimulation in our WT MACFs caused a significant upregulation of TGFβ transcript levels while GRK5KO MACFs had significantly reduced TGFβ expression. AngII is also able to directly activate cardiac fibroblasts. AngII stimulation can cause differentiation with or without the presence of

TGFβ receptor, suggesting that AngII can independently activate fibroblasts.6 However, inhibition of calcineurin and NFAT downstream of AngII abolished its effects. In our studies, we cannot rule out that GRK5 directly mediated AngII-dependent fibroblast activation or whether it participates indirectly through TGFβ release. An interesting result of our study is that loss of fibroblast GRK5 did not alter survival after MI, suggesting no changes in the incidence of cardiac rupture, which can occur when fibrosis and fibroblast activation is severely impaired (Davis et al., 2012, 2015; Khalil et al., 2019; Molkentin et al., 2017; Oka et al., 2007). Our results thus indicate that GRK5 is not involved in scar formation. Most importantly, because these animals were protected against interstitial fibrosis and cardiac hypertrophy, GRK5 and its nuclear accumulation and activity are an attractive target for the treatment of HF.

Supplemental Figure 1. Immunoblot probing for GRK5 in MACFs isolated from WT and GRK5KO mice.

Supplemental Figure 2. GRK5KO MACFs are resistant to TGFβ mediated α-SMA expression (A) Myofibroblast gene expression in WT and GRK5KO MACF’s, as measured by qRT- PCR, normalized to transcriptionally controlled tumor protein 1 (TPT1); fold change vs WT Veh. MACFs were stimulated with 10ng/mL TGFβ for 48 hours. n = 3 per group (B) Immunofluorescent staining and quantification of α-SMA (green)-positive cells in WT and GRK5KO MACFs stimulated with TGFβ. Cells were counterstained with DAPI. n = 3 per group. 10 images per n. (C) Photographs and quantification of floating collagen gel matrices seeded with WT or GRK5KO MACFs that have contracted after 18 hours of TGFβ stimulation. n = 5 per group.* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001

Supplemental Figure 3. GRK5KO MACFs demonstrate decreased wound closure by in vitro scratch assay. Representative image after scratch was initially made and 24 hours after scratch (A) and quantification (B). n = 5 per group. ** P ≤ 0.01

Supplemental Figure 4. GRK5KO MACFs do not demonstrate changes in proliferation. WT and GRK5KO MACF proliferation measured by CyQUANT cell proliferation assay over 4 days in the presence of 10% FBS. Fibroblasts were initially seeded at either high density (2000 cells/well) or low density (200 cells/well). n = 3 per group.

Supplemental Figure 5. GRK5KO MACFs have decreased protein translation rates after AngII stimulation compared to WT MACFs. Immunoblot and quantification of puromycinylated proteins from MACFs stimulated with 1µM AngII. n = 3 per group. * P ≤ 0.05

Supplemental Figure 6. GRK5 nuclear effects mediate activation. (A) Representative immunoblot and quantification of nuclear GRK5 after stimulation of neonatal rat cardiac fibroblasts with TGFβ (10ng/mL) for 30 min. n = 2. (B) Immunoblot and quantification for α-SMA expression in WT and GRK5KO MACFs stimulated with ET-1 (normalized to total protein). n = 3 per group. *** P ≤ 0.001

CHAPTER 4

CONCLUSIONS AND SIGNIFICANCE

Extended Discussion and Future Directions

HF is the clinical end point of a variety of cardiovascular diseases and is characterized by the hearts inability to pump blood sufficiently to meet the metabolic demands of the body. This condition can be caused by abnormalities in its ability to pump blood out due to systolic dysfunction or in its ability to relax properly due to diastolic dysfunction. In either case, fibrosis is a major contributing factor in HF pathogenesis.

Currently, pharmaceutical therapies are palliative, as the main therapies used in HF (β- blockers, ACE inhibitors, ARBs, etc.) do not cure the disease. In fact, patients with severe HF undergo surgical options with left ventricular assist devices acting as a short- term option, and cardiac transplant as the only long-term therapeutic option. Novel drug classes for the treatment of HF have been sparse. No new class of clinically efficacious

HF drugs was introduced between 1999 (mineralocorticoid receptor blocker) and 2014

(Angiotensin Receptor Blocker plus Neprolysin Inhibitors) (Sacks et al., 2014). In addition, no curative strategies for HF have been developed. Targeting non-myocytes is a potential alternative approach in the treatment of HF, given the critical role myofibroblasts play in the initiation and propagation of cardiac fibrosis and cardiac remodeling.

Anti-fibrotic therapies thus far have been underwhelming in the treatment of HF.

While RAAS inhibitors have been shown to reduce cardiac fibrosis in humans, the fibrosis persists in these patients with heart failure, suggesting novel therapies are needed.

TGFβ signaling is critical in fibroblast activation and subsequent fibrosis and TGFβ 69

inhibitors are efficacious attenuating cardiac fibrosis in animal models (Engebretsen et al., 2014). Unfortunately, these drugs are associated with adverse cardiac events, suggesting that they may not be applicable clinically (Frantz et al., 2008). Furthermore, pirfenidone and tranilast, drugs which inhibit TGFβ and other growth factors, are used clinically in other diseases involving fibrosis, such as idiopathic pulmonary fibrosis

(Edgley et al., 2012). While efficacious in animal models of cardiac fibrosis, they have not been shown to reduce fibrosis in human patients (Holmes et al., 2002). Endothelin receptor inhibitors have also been shown to attenuate fibrosis in animal models (Clozel and Salloukh, 2005). In addition, there are three drugs, bosentan, macitentan, and ambrisentan, that are FDA-approved drugs in the treatment of pulmonary hypertension.

While initially promising in small human cohorts, follow-up studies showed that these drugs were either neutral or harmful in patients with HF (Anand et al., 2004; Prasad et al.,

2006; Sütsch et al., 1998). There are also a plethora of anti-fibrotic drug candidates ranging from inflammatory modulators to MMP inhibitors that have failed to translate to human therapy (Fang et al., 2017). Because of the many failures in identifying drug targets in fibrosis, deciphering the molecular networks involved in fibroblast activation is necessary to determine a better candidate in the prevention and/or reversal of cardiac fibrosis.

We demonstrate that GRK5 deletion is able to prevent fibroblast activation after

AngII stimulation in vitro and fibrosis after MI or AngII infusion in vivo. Because GRK5 ablation in fibroblasts prevented perivascular fibrosis, this suggests that GRK5 inhibition may be beneficial in a wide variety of fibrotic disease including renal and pulmonary fibrosis and cirrhosis. While prevention of fibrosis is beneficial, it is not therapeutically

applicable in patients. It would be interesting to determine if GRK5 ablation after fibroblast activation has been initiated would be able to reverse the phenotype. In addition, genetic deletion of fibroblast GRK5 in vivo after fibrotic stimuli (MI or AngII infusion) would establish if GRK5 is a drugable target in the context of fibrosis.

From a translational perspective, targeting fibroblast specifically is a challenge.

Drug delivery to specific cell types has proven to be a challenge, although there are studies attempting to specifically target fibroblast using nano-particles in the context of ischemic HF (targeting fibroblasts for cellular reprograming) and in cancer (targeting tumor associated fibroblasts) (Ferreira et al., 2018; Liu et al., 2019). This technology is in its infancy and has not been implemented in human disease. Fortunately, GRK5 has been shown to be pathological in a variety of disease contexts (Premont and Gainetdinov,

2007). Specifically in HF, GRK5 inhibition in both fibroblasts and cardiomyocytes of mouse models after cardiac stress has been shown to be beneficial (Gold et al., 2012;

Hullmann et al., 2014). Therefore, specific cellular targeting in this circumstance is not as crucial. Various GRK5 inhibitors have been proposed and shown to have therapeutic potential. The overexpression of the amino terminal region of GRK5 (GRK5-NT) was shown to inhibit NFκB transcription in a cardio-myoblast cell line (Sorriento et al.,

2010). In vivo, adenoviral delivery of GRK5-NT ameliorated cardiac hypertrophy and cardiac dysfunction in hypertensive rats by inhibiting the CaM-Cn-NFAT axis (Sorriento et al., 2018). In addition to peptide inhibitors, some small molecule inhibitors of GRK5 have been identified. The FDA approved anti-inflammatory, anti-allergic immunomodulatory drug amlexenox used to treat canker sores was shown to bind GRK5 and inhibit its kinase activity (Homan et al., 2014; Pfleger et al., 2019). Additionally,

malbrachamide was shown to prevent GRK5 from complexing with Ca2+-CaM, preventing its nuclear transport (Beyett et al., 2019). Numerous studies are currently being undertaken to determine how these small molecules bind GRK5 in order to design more efficacious inhibitors (Cho et al., 2013; Homan et al., 2015a, 2015b).

While the canonical role of GRKs is to desensitize GPCRs, there is a growing list of non-canonical activities of GRKs involving a non-GPCR interactome. In cardiomyocytes, GRK2 is able to translocate to the mitochondria via the chaperone protein Hsp90 after being phosphorylated by ERK (Chen et al., 2013). GRK2 is then able to induce pro-death signaling and metabolic stress (Sato et al., 2018). Conversely, GRK5 is shuttled to the nucleus where it can exert changes in gene transcription. Here, we demonstrate that the Gq-Ca2+-CaM axis in inducing GRK5 nuclear translocation is intact in cardiac fibroblasts. In addition, we have preliminary data suggesting that this process may not be entirely specific to Gq activation. Cardiac fibroblasts stimulated with TGFβ have increased GRK5 enrichment in the nucleus (Supplemental Fig 6). TGFβ has previously been shown to increase cytosolic Ca2+, which was prevented by a TGFβ receptor kinase inhibitor (Mukherjee et al., 2012). Mechanistically, this influx of Ca2+ has been demonstrated to be from various sources. Pharmacological inhibitors as well as antibodies against the IP3R prevented TGFβ induced Ca2+ influx suggesting that this was

IP3 mediated at least in mesangial cells (McGowan et al., 2002). Furthermore, TGFβ has been shown to cause release of Ca2+ from mitochondria in prostate carcinoma cells

(Gizatullina et al., 2003). In fibroblasts, it appears that TGFβ causes an upregulation of the mitochondrial Ca2+ uptake 1 (MICU1) to increase the threshold needed to allow mitochondrial Ca2+ uptake through the mitochondrial Ca2+ uniporter. This reduces 72

mitochondrial Ca2+ uptake, thereby causing an increase in cytosolic Ca2+ (Lombardi et al.,

2019). GRK5 may be a nodal point in Ca2+ signaling and further studies are needed to determine the various physiological activators of GRK5 nuclear translocation.

In cardiomyocytes, GRK5 is able to potentiate NFAT binding to DNA as well as phosphorylate HDAC5, thereby de-repressing its effect on MEF2 (Hullmann et al., 2014;

Martini et al., 2008). As shown, GRK5 regulates NFAT activity in cardiac fibroblasts and therefore contributes to fibroblast activation. This, however, appears to be tissue specific.

Lung fibroblasts that had genetic ablation of Cn, the direct activator of NFAT, demonstrated increased proliferation, migration, and contractility compared to WT fibroblasts (Lieberman et al., 2019). Intriguingly, this study did not directly examine myofibroblast specific markers in Cn null fibroblasts (Lieberman et al., 2019). HDACs are also involved in myofibroblast transdifferentiation. In lung fibroblasts, pracinostat, an

HDAC inhibitor, was shown to attenuate fibroblast activation after TGFβ stimulation

(Jones et al., 2019). Furthermore, trichostatin A, a pan-HDAC inhibitor, and HDAC4 knockdown inhibited TGFβ mediated expression of α-SMA in healthy human lung fibroblasts (Guo et al., 2009). GRK5 may alter chromosome structure because it is an

HDAC kinase and its role in altering the opening and closing of chromatin should be investigated. Stimulating fibroblasts with or without AngII and performing histone chromatin immunoprecipitation (ChIP) – sequencing (seq) as well as assay for transposase-accessible chromatin using sequencing (ATAC-seq) would provide insight into if or how GRK5 is altering the chromatin landscape and genome accessibility.

Additionally, there are undoubtedly additional cytosolic and nuclear interacting partners of GRK5. These can be uncovered by utilizing a proteomic approach. 73

GRK5 has been demonstrated to bind directly to DNA in a neuronal cell line, inhibiting bcl-2 transcription and expression (Liu et al., 2010). Because of the presence of a conserved DNA binding domain within the NLS of GRK5, there are sure to be DNA binding partners for GRK5. Performing ChIP-seq for GRK5 and aligning those results with RNA polymerase ChIP-seq in fibroblasts would shed light on the direct transcriptional role that GRK5 has in regulating fibroblast activation. In addition, GRK6, another GRK within the GRK4 subfamily, also contains an NLS, is expressed in mouse embryonic fibroblasts, and shares 70.1% amino acid homology with GRK5 (Yang Jian et al.; Zheng et al., 2012). The data suggesting that GRK6 is able to compensate for the loss of GRK5 indicates that there may be an overlap in function. It would be interesting to determine if GRK6 has a functional role in fibroblast biology as this has not yet been investigated.

Conclusion

Overall, our work investigated the role of GRK5 in fibroblast activation in vitro and in vivo using two differing models of cardiac fibrosis. We demonstrate that GRK5 is a key regulator of myofibroblast differentiation and its genetic deletion inhibited the fibrotic response. Further, our work provides evidence that the non-canonical roles of

GRK5 are pathological and contribute to fibrosis. In conclusion, our work identified a novel regulator of fibroblast activation and showed that GRK5 is a therapeutic target in the treatment of HF.

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