Regulation of Cardiac Fibroblast Funtion Via Cyclic Amp, Collagen I, Iii, and Vi: Implications for Post-Myocardial Infarction Remodeling

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REGULATION OF CARDIAC FIBROBLAST FUNTION VIA CYCLIC AMP, COLLAGEN I, III, AND VI: IMPLICATIONS FOR POST-MYOCARDIAL INFARCTION REMODELING

A dissertation submitted to Kent State University in cooperation with the Northeastern Ohio Universities College of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Jennifer Elaine Naugle May, 2006

TABLE OF CONTENTS

List of Figures……………………………………………………………… iii Acknowledgements………………………………………………………… v Abstract…………………………………………………………...…………vi List of abbreviations………………………………………………...... viii

Chapters I. Introduction Role of cardiac fibroblasts in the heart………………………… 1 G-protein coupled receptor signaling………………………….. 2 Role of myofibroblasts in the heart……………………………. 12 Collagen composition of the myocardial ECM…………………16 Collagen receptors and cardiac fibroblasts…………………….. 19 Receptors associated with type VI collagen and myofibroblast differentiation………………………………. 22 Myocardial infarction and pathological remodeling…………… 23 Specific aims and hypotheses………………………………….. 29

II. Mechanism of angiontensin II-induced cAMP production and functional consequences Introduction…………………………………………………….. 31 Methods…………………………………………………...... 34 Results………………………………………………………….. 39 Discussion………………………………………………...... 65

III. Type VI collagen induces cardiac myofibroblast differentiation: implications for post-infarction remodeling Introduction…………………………………………………….. 74 Methods…………………………………………………………76 Results………………………………………………………….. 80 Discussion……………………………………………………… 92

IV. Temporal changes in type VI collagen, myofibroblast content, and integrin receptors in rats post-myocardial infarction Introduction…………………………………………….. ………103 Methods…………………………………………………………105 Results………………………………………………….. ………108 Discussion……………………………………………….………114

V. Overall Conclusions……………………………………………… 126 VI. Bibliography…………………………………………...………… 136

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LIST OF FIGURES

1. Angiotensin II signaling in cardiac fibroblasts…………………………………….. 5

2. β-adrenergic signaling in cardiac fibroblasts……………………………………...... 8

3. Angiotensin II potentiates β-adrenergic signaling in cardiac fibroblasts…………... 11

4. Differentiation of fibroblasts into mature myofibroblasts………………………….. 14

5. Structural differences between fibrillar collagen and type VI collagen……………. 18

6. Integrin receptor structure and interaction with ECM proteins……………………. 21

7. The role of cardiac fibroblasts in the progression of cardiac fibrosis……………… 27

8. Gq-Gs cross-talk is dependent upon Gq and phospholipase C activation………….. 41

9. Ionomyocin-induced intracellular transients enhance cAMP production………….. 45

10. Angiotensin II and ionomycin enhance forskolin-stimulated cAMP production…. 48

11. Intracellular Ca2+ transients enhance cAMP production and are blocked by Ca2+

chelation…………………………………………………………………………….51

12. Cardiac fibroblasts express multiple AC isoforms with different subcellular

distributions……………………………………………………………………... 54

13. Calmidazolium and overexpression of AC6 inhibit cross-talk………………….. 57

14. Elevation of cAMP inhibits differentiation to cardiac myofibroblasts………….. 60

15. Gq-Gs cross-talk impacts cardiac fibroblast collagen synthesis………………… 64

16. Collagen I production is inhibited by elevations in cAMP via forskolin……….. 67

17. Proposed Gq-Gs cross-talk mechanism…………………………………………. 73

18. Type VI collagen induces cardiac myofibroblast differentiation……………….. 82

19. Collagen substrates and ANG II treatment differentially affect cardiac fibroblast

proliferation……………………………………………………………………… 85

20. Treatment with ANG II induces type VI collagen expression…………………... 88

21. Coronary ligation in rats induces a collagen-rich infarcted myocardium……….. 91

22. Type VI collagen is elevated following post-myocardial infarction remodeling…. 94

23. Enhanced myofibroblast content in the infarcted rat myocardium……………… 97

24. Effects of the ECM on CF activation and the progression of cardiac fibrosis….. 102

25. Type VI collagen is elevated 7 and 14 days post-myocardial infarction.……….. 110

26. Myofibroblast content is significantly increased by 7 days post-MI……………. 113

27. αv integrin receptor subunit levels do not change within two weeks post-MI….. 116

28. Collagen VI interacts with the α3 integrin receptor in focal adhesions………… 119

29. α3 integrin is elevated 3 days and 16 weeks post-MI………………………….... 122

30. Temporal changes in type VI collagen, myofibroblast content, and α3 integrin

following MI……………………………………………..……………………... 125

31. Factors effecting CF activation during the progression of cardiac fibrosis……... 131

ACKNOWLEDGEMENTS

I would like to thank everyone who has helped me and supported me throughout this process, especially my family and friends, who have always been there for me no matter what. My parents, Robert and Nancy Naugle, have been a wonderful, guiding force in my life and have always believed in me. My graduate mentor, Gary Meszaros, has challenged me to become a well-rounded scientist, and the rest of my doctoral committee has aided me greatly in many areas. My colleague and friend Erik Olson has also been there for me as a fellow scientist and as my pseudo-big brother. My best friend and love of my life,

Andrew Bryant, has made this so much easier by reminding me not to take for granted the important things in life. I also would like to thank God for all of the blessings he has bestowed upon me, and for surrounding me with such amazing people in my life.

ABSTRACT

Cardiac fibroblasts (CFs) are the major non-contractile cells present in the

myocardium, and are primary regulators of synthesis and secretion of extracellular matrix

(ECM) proteins. Both proliferation and differentiation of CFs can potentially result in

excess ECM protein production and cardiac fibrosis, a condition characterized by a

stiffening of the myocardium. This condition is common after myocardial infarction and

develops during heart failure, resulting in compromised cardiac function. Hormonal

input, as well as input from the surrounding ECM can affect CF proliferation and/or

differentiation, and an increase in either one of these parameters will result in elevated

ECM production. Consequently, limiting prolonged fibroblast activation and the

subsequent detrimental ECM production after myocardial infarction or heart failure might

help to preserve left ventricular function.

The specific ECM composition in the myocardium likely imparts significant effects

on CF function. However, to date little is known about the effect of the ECM on CF function or the signaling pathways utilized by ECM molecules. In the adult, the myocardium is primarily composed of types I and III collagen, in addition to lower levels of types IV, V, and VI collagen. Extensive remodeling of the ECM occurs following myocardial infarction, and the resulting ECM composition can influence cardiac fibroblast activation in addition to affecting cardiac performance.

My goals are to determine the mechanism of Gq/Gs cross-talk and the functional

consequences in CFs, to determine the functional effects of specific types of collagen on

vii

CF differentiation and proliferation, and to identify the collagen composition and myofibroblast content post-myocardial infarction.

viii

LIST OF ABBREVIATIONS

AC; adenylyl cyclase

ANG II; angiotensin II

α-SMA; α-smooth muscle actin

β-AR; β-adrenergic receptor

BAPTA/AM; 1,2-bis(o-Aminophenoxy)ethane-N,N,N,N’-tetra acetic acid

Tetra(acetoxymethyl) Ester

Ca2+; calcium

cAMP; cyclic adenosine monophosphate

CAV; caveolae

CF; cardiac fibroblast

ECM; extracellular matrix

FSK; forskolin

IN; ionomycin

IP3; inositol 1,4,5--trisphosphate

ISO; isoproterenol

MI; myocardial infarction

PCR; polymerase chain reaction

PKA; protein kinase A

PKC; protein kinase C

TG; thapsigargin

TGF-β; transforming growth factor-β

CHAPTER ONE

INTRODUCTION

Role of cardiac fibroblasts in the heart:

Cardiac fibroblasts (CFs) comprise approximately 20% of the myocardial mass and

account for over half of the cell number in the heart. In the adult myocardium, CFs are interspersed with cardiac myocytes, and this network of cells is embedded in the extracellular matrix (ECM). CFs are responsible for synthesis and secretion of ECM proteins, as well as secretion of the enzymes responsible for ECM degradation, the matrix

metalloproteinases (MMPs) (Camelliti et al., 2005). CFs can secrete cytokines and

growth factors which can act in an autocrine or paracrine manner. These cells can be

stimulated by a variety of growth factors, hormones, and cytokines. Activation of CFs

involves proliferation and/or differentiation to myofibroblasts, both of which can lead to

excess ECM production and eventually cardiac fibrosis. Cardiac fibrosis is characterized

by overproduction of ECM components and stiffening of the myocardium, and is often a

secondary condition that develops in response to myocardial infarction, hypertension, or

heart failure. The excessive ECM present in cardiac fibrosis is detrimental to cardiac

function by decreasing compliance and serving as an obstacle for the cardiac conduction

system. Decreased compliance, or stiffening of the myocardium, causes the heart to work

harder with each beat in order to pump the same amount of blood, and interruption of the

cardiac conduction system makes the heart prone to arrhythmias, both of which are acutely and chronically detrimental to the heart.

G-protein coupled receptor signaling:

G-protein coupled receptors (GPCRs) are plasma membrane receptors that span

the lipid bilayer seven times and couple to various heterotrimeric G proteins to initiate

intracellular signaling events (Kim and Iwao, 2000; Hur and Kim, 2002). Heterotrimeric

G proteins are composed of three subunits (α, β, and γ), and when stimulated, the α and

βγ subunits can dissociate from one other to activate downstream signaling molecules.

Upon binding of an agonist, GPCRs promote release of GDP from the Gα subunit and

binding of GTP, which converts the G protein to its active state. G proteins remain active

until the intrinsic GTPase activity of the α subunit promotes hydrolysis of GTP to GDP

and re-association of the subunits. The GPCR is deactivated via phosphorylation by

GPCR kinases (GRKs) which then allows for recruitment of β-arrestins, and uncoupling

of the receptor from the G protein (Ferguson, 2001).

G proteins are classified into four main classes each with a general signaling

mechanism: Gs, which activates adenylyl cyclase, Gi/o, which inhibits adenylyl cyclase,

Gq, which stimulates phospholipase C, and G12, which regulates small GTP binding

proteins (Hur and Kim, 2002). More recently, it has been observed that the βγ subunits

can also regulate downstream targets such as adenylyl cyclase and tyrosine kinases,

including phosphatidylinositol-3 kinase and the mitogen activated protein kinase

(MAPK) pathway. Typically, specific GPCRs preferentially couple to a particular G

protein under physiological conditions. Several GPCR pathways exist in CFs; two major pathways include the β-adrenergic receptor (β-AR) pathway and the angiotensin II (ANG

II) receptor pathway.

Angiotensin II signaling: ANG II is an octapeptide hormone that plays a significant role in the regulation of systemic blood pressure via the renin-angiotensin system. This hormone mainly affects vascular smooth muscle cells by increasing contraction in order to elevate blood pressure. ANG II has specific non-vascular targets including effects on CFs mediated through the cardiac renin-angiotensin system (Touyz and Berry, 2002). These effects include induction of CF proliferation (Booz et al., 1994;

Olson et al., 2005), differentiation to myofibroblasts (Swaney et al., 2005; Naugle et al.,

2006), as well as ECM production (Brilla et al., 1994).

ANG II binds to two physiologically relevant cell membrane receptors, the AT1 and AT2 receptors. The AT1 receptor is highly expressed throughout the body and helps regulate effects on blood pressure, vasoconstriction, cardiac contractility, and aldosterone release. This receptor is a significant participant in both cardiovascular and renal diseases, whereas the AT2 receptor is expressed at higher levels during fetal development and appears to have physiologically antagonistic effects when compared to the AT1 receptor (Kim and Iwao, 2000). In the myocardium, ANG II-dependent cardiac fibroblast proliferation (Booz et al., 1994; Olson et al., 2005) and collagen production (Brilla et al.,

1994) are mediated through the AT1 receptor. This receptor subtype couples to Gq to activate phospholipase C which results in production of inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 liberates calcium from the endoplasmic reticulum, and calcium

Figure 1. Angiotensin II signaling in cardiac fibroblasts. Angiotensin II (ANG II) binds to the AT1 receptor, which couples to Gq. The Gq α subunit dissociates from the βγ subunits and stimulates phospholipase Cβ (PLCβ), which cleaves phosphatidylinositol- 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol trisphosphate (IP3). IP3 2+ binds to the IP3 receptor on the endoplasmic reticulum, and stimulates Ca release from intracellular stores. Together, DAG and Ca2+ activate protein kinase C (PKC), which phosphorylates a number of downstream targets.

in combination with DAG activates protein kinase C (PKC; see Figure 1). ANG II- induced release of intracellular calcium results in contraction of vascular smooth muscle cells (Touyz and Berry, 2002), which plays a significant role in chronic hypertension. In

CFs, activation of the Gq pathway leads to activation of many intracellular signaling proteins that mediate various functional responses.

β-AR signaling and cyclic AMP output: β-ARs are endogenously stimulated by

the circulating catecholamines norepinephine and epinephrine, which are released by the

adrenal medulla. Three types of β-AR exist: β1, β2, and β3; however, in the heart, the

β1- and β2-ARs predominate. Cardiac myocytes possess both of these subtypes although

β1-ARs are the primary subtype (greater than 80%) present (Buxton and Brunton, 1985;

Kuznetsov et al., 1995; Steinberg, 1999). This receptor is responsible for mediating the

positive inotropic and chronotropic effects of catecholamines on the myocardium.

Cardiac fibroblasts on the other hand, express primarily β2-ARs (Meszaros et al., 2000;

Turner et al., 2003), and activation of this receptor leads to CF proliferation via

transactivation of the epidermal growth factor receptor and activation of extracellular

signal-regulated kinases 1/2 (ERK1/2) (Kim et al., 2002).

The β-ARs couple to Gs to activate adenylyl cylase and produce the second

messenger cyclic AMP (cAMP). cAMP then induces activation of protein kinase A

(PKA), which phosphorylates a number of other proteins as well as initiating negative feedback. PKA stimulates activity of phosphodiesterases, which converts cAMP to 5’

AMP and phosphorylates β-ARs which eventually leads to uncoupling from the G protein

Figure 2. β-adrenergic receptor (β-AR) signaling in cardiac fibroblasts. Isoproterenol (ISO) is a non-specific β-AR agonist (a synthetic catecholamine) that binds to the β-AR to activate Gs. The Gs α subunit dissociates from the βγ subunits and activates adenylyl cyclase (AC), a membrane bound enzyme that utilizes ATP to form the second messenger cyclic AMP (cAMP). cAMP then activates protein kinase A, which phosphorylates a number of downstream targets.

and subsequent receptor internalization, both of which effectively turn off the signaling cascade (see Figure 2).

The cellular localization of the β-AR has a significant effect on the regulation and function of this receptor. β-ARs are membrane receptors that can localize to specific membrane compartments depending on the conditions. In cardiac myocytes, both the β1 and β2 receptors are present in caveolae (CAV), which are cholesterol-rich invaginations of the plasma membrane containing the protein caveolin. β2 receptors are confined to the

CAV in cardiac myocytes whereas the β1 receptors and the Gsα subunit are distributed between CAV and non-CAV plasma membrane. Upon agonist stimulation, the β2 receptor moves out of the CAV domain, but the distribution of the β1 receptor does not change in cardiac myocytes. In CFs, the β2 receptors are localized chiefly to the caveolae and are not contained in any non-CAV plasma membrane (Rybin et al., 2000).

CAV domains are characterized by the expression of the caveolin protein, of which there are three types, caveolin-1, -2, and -3. In vivo, much has been learned from caveolin knock out (KO) mice and the role of caveolin in disease. Caveolin-1 KO mice exhibit hyperactivation of the ERK1/2 pathway in CFs as well as increasing interstitial fibrosis. Although caveolin-1 is not expressed in cardiac myocytes, the caveolin-1 KO mice present with myocyte hypertrophy and degeneration, which indicates that the CFs most likely have paracrine effects on myocyte function (Cohen et al., 2003). Caveolin-3 is muscle specific, and these KO mice demonstrate hypertrophic cardiomyopathy and hyperactivation of the ERK1/2 pathway in myocytes (Cohen et al., 2004). Disrupting this

Figure 3. ANG II potentiates β-AR signaling in cardiac fibroblasts. Cardiac fibroblasts were treated with 100 nM ANG II, 100 nM isoproterenol (ISO), or the combination of ANG II and ISO. ISO alone stimulated cAMP production 10 fold over control, whereas ANG II alone had no effect (as expected). However, ANG II augmented the ISO response by 10-fold over ISO alone, which suggests a mechanism of cross-talk between these two signaling pathways in cardiac fibroblasts (Meszaros et al., 2000).

25 * 20

5 Cyclic AMP production (fold change over control) 0

particular intracellular signaling compartment in vivo has dramatic effects on the development of cardiac hypertrophy and fibrosis.

GPCR cross-talk: Meszaros et al. have described a novel signaling “cross-talk” phenomenon between two key GPCR signal transduction pathways in cardiac fibroblasts

(Meszaros et al., 2000). Isoproterenol (ISO), a β-AR agonist, activates Gs and stimulates cAMP production 10-fold over basal levels (Figure 3). ANG II by itself does not alter cAMP production in cardiac fibroblasts, but in combination with ISO it potentiates the β-

AR response to 20-fold over basal levels (Meszaros et al., 2000). Elucidating the mechanism by which ANG II augments cAMP signaling is invaluable in determining the functional consequences of this signaling phenomenon. Several investigators have shown that stimulation of the β-AR with ISO induces proliferation in rat CFs via multiple mechanisms (Colombo et al., 2001; Kim et al., 2002). As both ANG II and catecholamine levels are increased in the circulation during heart disease, potentiation of cyclic AMP signaling by ANG II may augment CF proliferation as well as playing a potential role in other functional outputs such as differentiation to myofibroblasts and

ECM production.

Role of myofibroblasts in the heart:

Myofibroblasts are a specialized subtype of fibroblast critically involved in wound

healing in many tissues. Differentiation of fibroblasts to myofibroblasts involves de novo

expression of both α-smooth muscle actin (α-SMA) and a splice variant of fibronectin

Figure 4. Differentiation of fibroblasts into mature myofibroblasts. Fibroblasts contain cytoplasmic actin but do not form adhesion complexes with the ECM. When fibroblasts are exposed to mechanical force, they differentiate into proto-myofibroblasts which contain stress fibers composed of cytoplasmic actin, as well as adhesion complexes with the ECM. Proto-myofibroblasts also begin to express the ED-A fibronectin splice variant, and the combination of ED-A fibronectin, mechanical force, and stimulation with transforming growth factor-β (TGF-β) then converts the proto-myofibroblast into a mature myofibroblast. Myofibroblasts express α-smooth muscle actin de novo and incorporate it into stress fibers. These fully differentiated cells also contain larger adhesion complexes that interact with the ECM, and aid in the cell’s ability to generate contractile force (Figure taken from Tomasek et al., 2002).

referred to as ED-A fibronectin. In order to be classified as a mature myofibroblast, the

α-SMA expressed must be organized into stress fibers (see Figure 4, taken from

(Tomasek et al., 2002)). This reorganization of the cytoskeleton allows for a slow, sustained contraction, which is responsible for wound closure, while the hypersecretory nature of the myofibroblasts allows for large amounts of ECM protein deposition in the wound site, which aids in wound stabilization (Tomasek et al., 2002). Following wound healing and scar formation, myofibroblasts typically die via apoptosis (usually about 20 days after wound induction), and the resident fibroblasts then maintain the ECM. If this apoptotic event does not occur, the hypersecretion of ECM proteins by the myofibroblasts can lead to aberrant scarring and pathological fibrosis (Desmouliere et al.,

1997). Consequently, we and others hypothesize that myofibroblasts are the cell type responsible for production of excess ECM during pathological fibrosis.

Cardiac fibroblasts can be induced to differentiate into myofibroblasts in vitro by the cytokine transforming growth factor-β (TGF-β) (Petrov et al., 2002) as well as via mechanical force (Wang et al., 2003). An increase in the amount of myofibroblasts induced by TGF-β correlates with an elevation in collagen production (Petrov et al.,

2002), and production of several actin-associated proteins involved in microfilament arrangement in the contractile apparatus. Myofibroblasts are often referred to as a terminal cell type, however, it has been demonstrated that “reversal” of the myofibroblast phenotype is possible. Treatment of cultured corneal myofibroblasts with a combination of fibroblast growth factor and heparin caused these cells to revert back to a fibroblast phenotype lacking α-SMA (Maltseva et al., 2001). It is critical to understand the process

of myofibroblast differentiation in order to fully comprehend the progression of fibrosis and ultimately devise therapies that target this hypersecretory fibroblast phenotype.

Collagen composition of the myocardial ECM:

Collagen types I and III comprise approximately 90% of the myocardial collagen content, while types IV, V, and VI make up the remainder (Borg et al., 1982; Eghbali et al., 1989; Weber et al., 1994; Agocha et al., 1997; Heeneman et al., 2003). Types I and III collagen are fibrillar collagens, which play an important role in withstanding the tensile and shear stresses continuously exerted on the myocardium (see Figure 5). CFs have the ability to synthesize and secrete both collagens type I and III in vivo (Weber and Brilla,

1991). The cardiac fibrosis that occurs following myocardial infarction, hypertension, or heart failure, has been attributed to increases in both collagen I and III (Weber and Brilla,

1991; Gonzalez et al., 2002; Koshy et al., 2003), however, levels of the minor collagen types during these pathological conditions have not been extensively studied.

Collagen type VI, a non-fibrillar collagen present in the adult heart, has been less well

studied. Type VI collagen is more accurately a glycoprotein that associates with itself to

form tetramers, and when assembled has the appearance of a beaded filament (see Figure

5). Only a small portion of collagen VI molecules are triple helical, the carboxy- and

amino-terminal ends are comprised of large globular domains (Bashey et al., 1992; Pfaff

et al., 1993). Although few studies have been performed investigating the levels of type

VI collagen in the heart, some evidence implicates collagen VI in cardiac pathologies.

Collagen type VI has been demonstrated to be increased in the hearts of both diabetic and

Figure 5. Structural differences between fibrillar collagen and type VI collagen. A) Fibrillar collagen (such as collagens I, II, III, or V) is synthesized from three α chains wound together into a triple helix, and this is referred to as pro-collagen. Once collagen is secreted from the cell, proteases cleave both the carboxy- and amino-terminal ends of the pro-collagen, rendering it a mature collagen molecule. Collagen molecules can then assemble together to form collagen fibrils, and these fibrils then compile together to form cable-like bundles called collagen fibers. B) Type VI collagen also is composed of three α chains that wind together into a triple helix, however, collagen VI contains large globular proteins on both the carboxy- and amino-terminal ends that do not get cleaved extracellularly. The triple helical domain of native type VI collagen contains RGD domains, which do not have access to integrin receptors. Type VI collagen associates into dimers, and when it is secreted from a cell, it is secreted as tetramers. These collagen VI tetramers then associate end to end, and have the appearance of a beaded filament.

A. FIBRILLAR COLLAGEN B. TYPE VI COLLAGEN

triple helical pro-collagen Globular proteins

Collagen molecule cleaved extracellularly triple helical region (contains RGD domains)

Collagen molecules assemble

Dimer Tetramer

Collagen fibers

Beaded filaments

hypertensive rats (Spiro and Crowley, 1993), and in human hypertrophic cardiomyopathy

(Kitamura et al., 2001). In addition, due to the fact that two of the three genes for collagen VI are located on chromosome 21, there are implications for trisomy 21 patients who typically possess cardiac defects (Klewer et al., 1998). Another study demonstrated that type VI collagen and myofibroblasts are concurrently elevated in areas of renal fibrotic injury in human tissue (Groma, 1998). The role of type VI collagen in these pathologies has not been conclusively determined, yet it does not appear to contribute to the structural ECM integrity.

Collagen receptors and cardiac fibroblasts:

In addition to serving as structural support, the ECM and more specifically collagen

can bind to cell surface receptors and initiate intracellular signaling cascades. Integrins

are ECM receptors that are heterodimers composed of both α and β subunits (see Figure

6). Of the 25 combinations of α and β subunits that have been identified in vivo, only 4 integrin receptors have been classically identified as collagen-binding integrins: α1β1,

α2β1, α10β1, and α11β1 (Vogel, 2001). Ligands for both the α2β1 and α11β1 integrins tend to be fibrillar collagens, while α1β1 preferentially binds basement membrane

collagen (type IV) and type XIII, and α10β1 is binds type II and type VI collagen (Vogel

2001, Tulla, 2001 #148). Many integrin receptors bind ECM proteins via three key amino

acid residues—arginine, glycine, and aspartate, which is referred to as an RGD domain

(Schnee and Hsueh 2000). Cardiac fibroblasts possess several RGD-binding integrins:

α3β1, α5β1, αvβ1, αvβ3, and α8β1 (Thibault et al., 2001). Other cell surface receptors

Figure 6. Integrin receptor structure and interaction with ECM proteins. Integrin receptors, which are composed of α (represented in blue) and β (represented in green) subunits, interact with both ECM proteins such as collagen, as well as intracellular signaling molecules. These receptors provide a way for the ECM to transmit signals across the cell membrane and initiate intracellular events. Collagens (represented in red) are abundant ECM proteins that can be degraded by matrix metalloproteinases (MMPs; represented by scissors). The balance between synthesis and degradation of the ECM provides a dynamic matrix that can produce numerous cellular effects via signaling through integrin receptors.

Extracellular space

Cell membrane Cytoplasm

have been established as functional collagen receptors such as discoidin domain receptors

(DDRs) and glycoprotein VI (found only in platelets). DDRs have two different isoforms,

DDR1 and DDR2, of which DDR1 has been shown to bind collagens I through VI and

VIII, whereas DDR2 binds fibrillar collagens, specifically types I and III (Vogel, 2001).

DDRs are tyrosine kinase receptors that form homodimers upon ligand binding and have intrinsic catalytic activity. DDR2 receptors, but not DDR1 have been identified on cardiac fibroblasts, and have been proposed as an accurate marker for fibroblasts in vivo

(Goldsmith et al., 2004).

Receptors associated with type VI collagen and myofibroblast differentiation

Type VI collagen has been identified as a ligand for α3β1 and the integral membrane

proteoglycan NG2 in corneal fibroblasts (Doane et al., 1998). The other integrin receptors

that bind native type VI collagen include both α1β1 and α2β1 (Jongewaard et al., 2002).

However, it has been demonstrated that type VI collagen does not bind to integrin

receptors via RGD domains. Due to the structure of collagen VI, the RGD domains

present in the triple helical domain are obstructed by the large globular proteins present

on each end of a native type VI collagen molecule (see Figure 5), therefore these RGD

domains do not have access to integrin receptors (Pfaff et al., 1993). It has been

demonstrated in corneal fibroblasts that attachment to native type VI collagen is not

RGD-dependent (Doane et al., 1992). In contrast, pepsin digested collagen VI (with

globular ends cleaved off) can bind to integrins via RGD domains, and therefore exhibit

different binding capabilities than native type VI (Pfaff et al., 1993). Consequently, studying native type VI collagen represents a more physiological setting.

The αv integrin subunit (more specifically αvβ3 and αvβ5) has been shown to be critically involved in myofibroblast differentiation of human oral, dermal, and kidney fibroblasts. Differentiation to myofibroblasts was inhibited in these cell lines via blockade of αvβ3, αvβ5 or β1 integrin (Lygoe et al., 2004). Another study demonstrated that treatment of CFs with ANG II or TGF-β caused an increase in the level of α8β1 integrin, concurrent with differentiation to myofibroblasts (as measured by α-SMA elevation) (Thibault et al., 2001). All of the receptors mentioned above are potential mediators of type VI collagen-induced myofibroblast differentiation via type VI collagen.

Myocardial infarction and pathological remodeling:

Several pathologies can lead to alterations in myocardial remodeling, one of which is

myocardial infarction (MI). MI occurs when a coronary vessel is occluded and the

surrounding tissue becomes ischemic. This ischemia initially induces an inflammatory

response, which then triggers a wound healing and remodeling phase. Remodeling of the injured myocardium involves breakdown of the ECM and clearing of debris from

necrotic tissue; new ECM is then synthesized to replace the dying cells in order to

stabilize cardiac performance (replacement fibrosis). Repairing the injured myocardium is highly dependent upon the presence of myofibroblasts. These cells act to both contract

the wound and deposit large amounts of collagen and other ECM proteins to stabilize the

injured area as well as aid in scar formation (reparative fibrosis). Interstitial fibrosis is

also evident at sites remote from MI, which can have a detrimental effect on myocardial compliance (Weber and Brilla, 1991; Weber et al., 1994; Manabe et al., 2002). Despite the necessary healing process, formation of a collagen-rich scar as well as interstitial fibrosis can lead to alterations in cardiac conduction and contractility, which can contribute to arrhythmias and heart failure, respectively (Sun et al., 2000). MI can be induced in animal models by ligation of the left anterior descending coronary artery

(LAD), causing widespread cardiac myocyte necrosis, and extensive remodeling of the ventricular wall. The rat coronary ligation is a well established model to study remodeling events at the cellular, systemic, and whole organ level.

There is considerable debate as to when myofibroblasts appear and subsequently undergo apoptosis during remodeling, with different models yielding distinct results.

Myofibroblasts appear in the infarcted heart around day 3, and are abundant by day 7 post-MI in rats (Sun et al., 2000). In mice, proliferation of myofibroblasts peaked at day 4 post-MI and diminished by day 14 (Virag and Murry, 2003). Myofibroblasts have been shown to persist in infarcted hearts for at least 28 days post-MI in rats (Sun et al., 2000), and up to 17 years post-MI in human tissue (Willems et al., 1994). Collagen accumulates in the rat heart beginning at day 7 post-MI (Sun et al., 2000) and can continue for months

(Cleutjens et al., 1995). Elevations in collagens I and III mRNA are evident as soon as 3 days following myocardial infarction, which contributes significantly to the ensuing fibrosis.

MI is an injury to the heart that stimulates an inflammatory response. This inflammatory response is evident soon after MI in rats; macrophages have been shown to

infiltrate the infarcted region beginning at day 2 and remained elevated through day 7, but were absent by day 28 post-MI. Macrophages are an inflammatory cell type that can secrete cytokines, the major one being TGF-β. Expression patterns of TGF-β mRNA post-MI closely mirrored the appearance of macrophages. TGF-β mRNA was elevated by day 2, peaked on day 7 post-MI, and was attenuated at the later time-points of 14 and 28 days, although this message remained higher than control hearts (Sun et al., 2000).

Macrophage infiltration is an indicator of the inflammatory phase, which is typically an acute reaction, although long-term effects are possible.

Cells interact with the ECM in a variety of ways, and one of the best described interactions is via integrin receptors. Integrin receptors bind specific ECM components, and the interactions present are dependent upon both the ECM composition as well as the integrin receptor subtypes expressed on the cell types (see Figure 6). The α1 and α3 integrin receptors appeared (but were not abundant) in cardiac fibroblasts in the infarcted region at days 4, 7, and 14 post-MI. However, α5 integrin was not detectable at any of these time points examined (Nawata et al., 1999). Recently, a study by Bouzeghrane et al. demonstrated upregulation of the α8β1 integrin receptor in myofibroblasts present in the fibrotic myocardium (Bouzeghrane et al., 2004). Several integrin receptors exist that could be potentially important mediators of ECM-cell interactions during the post-MI remodeling process. Cardiac remodeling includes not only the synthesis but also the degradation of the ECM, with the catabolic events being mainly mediated by matrix metalloproteinases (MMPs). MMPs are zinc-dependent proteases that digest ECM components, and are essential in pathological remodeling (see

Figure 6). Several types of MMPs are expressed in the myocardium and play roles in both the early and late remodeling phases following an MI. MMPs-2 and 9, which are gelatinases that are responsible for breaking down degraded collagen, are elevated within days of an acute MI, and MMP-2 remains elevated for up to 8 weeks post-MI, whereas

MMP-9 begins to decrease by this time-point. MMP-1 and 13, which are collagenases that are responsible for degrading collagen, are elevated following MI, however MMP-1 levels decline after 7 days post-MI, whereas MMP-13 levels only begin to decline after

21 days (Vanhoutte et al., 2006).

Cardiac remodeling following MI is dependent upon many factors such as the cell types present, the net accumulation of specific ECM proteins, as well as how the cells interact with the ECM. The altered cardiac remodeling post-MI is initially beneficial to preserve cardiac function; however, eventually this process becomes detrimental and leads to cardiac fibrosis (see Figure 7). Much remains to be learned, particularly concerning the events that govern the role of myofibroblast differentiation in cardiac remodeling, and clarifying this process could lead to potential therapeutic targets acting to ultimately limit fibrosis.

Figure 7. The role of cardiac fibroblasts in the progression of cardiac fibrosis. Hypertension and myocardial infarction can promote activation of cardiac fibroblasts (CFs). CFs are activated by proliferating or differentiating to myofibroblasts, both of which lead to increases in collagen production. Excessive collagen deposition is referred to as cardiac fibrosis, which is characterized by stiffening of the myocardium and compromised cardiac function.

SPECIFIC AIMS AND HYPOTHESES

1A To define mechanisms that mediate ANG II-induced augmentation of cyclic AMP production by ISO in CFs. 1B To measure the effects of cAMP signaling on cardiac fibroblast differentiation and collagen production.

Hypotheses: I hypothesize that potentiation of cyclic AMP by activators of Gq signaling is mechanistically dependent upon elevation of intracellular calcium. I propose that elevation of cyclic AMP will inhibit both myofibroblast differentiation and collagen production in CFs.

2A To determine how specific collagen substrates (types I, III, VI) affect in vitro cardiac fibroblast proliferation and differentiation to the myofibroblast phenotype. 2B To determine whether type VI collagen and myofibroblast content are elevated in 20 weeks post-myocardial infarction hearts.

Hypotheses: I hypothesize that type VI collagen will promote differentiation, whereas types I and III collagen will stimulate proliferation of CFs. I predict that both myofibroblast content and type VI collagen will be elevated in the infarcted areas 20 weeks post-MI.

3A To determine the temporal changes in type VI collagen and myofibroblast content using an in vivo model of myocardial remodeling. 3B To determine whether expression of specific integrin receptors change in the myocardium post-MI.

Hypotheses: I hypothesize that myofibroblasts will appear in the infarcted myocardium within one week of an MI, concurrent with an elevation in type VI

collagen. In addition, I propose that one or more α integrin receptor subunits will be elevated within days following an MI.

CHAPTER TWO

MECHANISM OF ANGIOTENSIN II-INDUCED cAMP PRODUCTION AND FUNCTIONAL CONSEQUENCES

INTRODUCTION

Cardiac hypertrophy is associated with increased cardiac mass, a gradual decline in contractile function and eventual heart failure. The remodeling associated with these changes involves an altered balance of synthesis and degradation of extracellular matrix

(ECM) by cardiac fibroblasts and can lead to abnormal accumulation of ECM in the interstitial space (i.e. fibrosis) (Eghbali and Weber, 1990; Weber et al., 1994). Several G protein-coupled receptors (GPCRs) that signal through Gq have been implicated in the pathogenesis of cardiac hypertrophy and failure (Ramirez et al., 1997; Adams et al.,

1998). Conversely, GPCRs that signal via Gs may inhibit collagen deposition by cardiac fibroblasts (Dubey et al., 1998).

Major pro-fibrotic signals in the heart are cytokines, such as TGF-β, and the peptide hormone ANG II, both of which increase collagen synthesis by cardiac fibroblasts (Lee et al., 1995; Weber, 1997) as well as differentiation to myofibroblasts (a hypersecretory subtype of fibroblast characterized by the expression of α-smooth muscle actin). ANG II also inhibits matrix metalloproteinase expression by cardiac fibroblasts, thereby attenuating degradation of ECM proteins (Brilla et al., 1995). An anti-fibrotic role for cAMP is supported by evidence that adenosine and prostacyclin inhibit cardiac

fibroblast proliferation and collagen synthesis through activation of A2b and prostanoid receptors, respectively, and each couple via Gs to enhance cAMP production (Yu et al.,

1997; Dubey et al., 2001). GPCR agonists that stimulate production of cAMP were recently shown to inhibit serum or TGF-β-stimulated α-smooth muscle actin (α-SMA) expression and collagen formation by human lung fibroblasts (Kolodsick et al., 2003; Liu et al., 2004). β-adrenergic receptors have been shown to stimulate fibroblast proliferation via epidermal growth factor receptor transactivation (Kim et al., 2002) but their role in regulating differentiation to myofibroblasts and collagen synthesis in cardiac fibroblasts is poorly documented.

Adenylyl cyclase (AC) catalyzes the synthesis of cAMP and its expression limits the ability of a cardiac cell to maximally produce this second messenger (Post et al.,

1995; Gao et al., 1998). Nine different transmembrane AC isoforms exist, each with a different amino acid sequence, tissue and chromosomal distribution, and regulation

(Hanoune and Defer, 2001). Differences in regulation include stimulation or inhibition by

Gβγ, Ca2+, and various protein kinases. AC5 and AC6, which are the predominant

isoforms expressed in cardiac myocytes (Ishikawa et al., 1994), represent a subfamily of

ACs that are related in structure and regulation. These isoforms are inhibited by PKA,

Ca2+, Gi, Gβγ, and nitric oxide (McVey et al., 1999; Hill et al., 2000; Hanoune and Defer

2001). By contrast, AC1, AC3, and AC8 are stimulated by Ca2+/calmodulin (yet AC3 can

also be inhibited by calmodulin kinase-II) (Choi et al., 1992; Wei et al., 1996; Hanoune

and Defer, 2001). AC2 and AC4 are activated by Gβγ and AC2 and AC7 can be activated

by phosphorylation by PKA and/or PKC (Hanoune and Defer, 2001). Thus, the AC

isoform expression in a cell determines the interaction between cAMP production and other signal transduction cascades.

Meszaros et al. have recently described a signaling “cross-talk” between two key

GPCR signal transduction pathways in cardiac fibroblasts (Meszaros et al., 2000).

Isoproterenol, a β-adrenergic receptor (β-AR) agonist, activates Gs and stimulates cAMP production 10-fold over basal levels. ANG II activates Gq-coupled angiotensin receptors that, by themselves, do not alter cAMP production in cardiac fibroblasts, but in combination with ISO potentiates the β-AR response, resulting in a 2-fold potentiation of

ISO-stimulated cAMP production. The goal of the present study was to identify the molecular mechanism and physiological consequence of the cross-talk between these two signaling pathways in cardiac fibroblasts. The current data indicate that Gq-mediated elevation of intracellular Ca2+ induced by ANG II potentiates Gs-stimulated cAMP

formation, probably via stimulation of AC3. Moreover, we show that the potentiation is

functionally relevant: combined treatment of cardiac fibroblasts with ANG II and ISO

inhibits ANG II-promoted collagen synthesis and differentiation to myofibroblasts more

so than ISO alone. Thus, we identify an endogenous signaling pathway by which

intracellular Ca2+ enhances cAMP production, inhibits myofibroblast differentiation, and

limits collagen synthesis, and perhaps fibrosis, in the heart.

METHODS

Materials: Cell culture reagents were obtained from Invitrogen. Primary antibodies to

AC isoforms and all secondary antibodies were obtained from Santa Cruz Biotechnology

(Santa Cruz, CA). Antibodies to caveolin were from BD Pharmingen. ANG II, UTP,

Fura-2/AM, forskolin, and BAPTA/AM were obtained from Calbiochem. Antibodies for

α-SMA was obtained from Sigma, and antibodies for collagen type I were obtained from

Rockland (Gilbertsville, PA). All other drugs and reagents were of reagent grade and obtained from Sigma.

Preparation and culture of adult rat cardiac fibroblasts: Cardiac fibroblasts were prepared from adult male 250-300g Sprague Dawley rat hearts. The hearts were rapidly excised, the ventricles were isolated, minced, pooled and placed in a collagenase

(100U/ml)/ trypsin (0.6 mg/ml) digestion solution. After eight 10-minute collagenase digestions, fibroblasts were spun for 10 minutes at 1000 rpm and resuspended in

Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 1% penicillin/ streptomycin, 1% fungizone and 10% fetal bovine serum (FBS, Gemini BioProducts).

After a 45 minute period of attachment to uncoated culture plates at 37 °C and

10% CO2, cells weakly attached or unattached were rinsed free and discarded. After 2-3

days, confluent cultures were amplified by trypsinization and seeding onto new dishes.

Cell cultures were maintained in 10% CO2 in DMEM and 10% FBS at 37 °C. For

signaling assays, only early passage (< 3) cells grown to 80-90% confluency were used.

The purity of these cultures is greater than 95% cardiac fibroblasts as determined by

positive staining for vimentin and negative staining for α-smooth muscle actin and von

Willebrand factor, as previously described (Gustafsson and Brunton, 2000).

Adenoviral Gene Transfer to Cardiac Fibroblasts: Wild-type Gq was cloned into the

PACCMVpLpA shuttle vector for production of adenovirus. We found that a titer of 200

virus particles per cell was optimal for Gq expression without cytotoxicity, as determined by trypan blue exclusion and morphological examination with phase-contrast light microscopy. We used 1000 viral particles per cell of an adenovirus construct to maximally overexpress the murine adenylyl cyclase type 6 (AC6) gene. Cardiac fibroblasts were infected with the appropriate virus and incubated at 37 °C 18–24 h in serum-free DMEM prior to the assays. Preliminary experiments using LacZ expression and β-galactosidase staining indicated that the efficiency of the adenoviral construct to increase gene expression was 40% (data not shown).

Quantitation of cAMP production: Cardiac fibroblasts cultured on 24 well plates were washed three times with serum-free and NaHCO3-free DMEM supplemented with 20

mM HEPES, pH 7.4 (DMEH). Cells were equilibrated for 30 min then assayed for cAMP

accumulation by incubation with drugs of interest in the presence of 0.2 mM

isobutylmethylxanthine (IBMX) for 10 min. When antagonists or inhibitors were used,

these agents were equilibrated with cells for 15 min before stimulation with agonists for

15 minutes. Assay medium was aspirated and 250 µl of 0.1 M hydrochloric acid (HCl)

was immediately added to each well to terminate reactions. HCl extracts were assayed for

cAMP content by Direct ELISA kit (Assay Designs). AC activity was measured in

cardiac fibroblast membranes as previously described (Ostrom et al., 2001). Briefly, membranes were prepared by rinsing cells twice in ice cold PBS then scraping cells into hypotonic homogenizing buffer (30 mM NaHEPES, 5 mM MgCl2, 1 mM EGTA, 2 mM

DTT, pH 7.5) and homogenizing with 20 strokes in a Dounce homogenizer.

Homogenates were spun at 300g for 5 min at 4 °C. Supernatants were transferred to a

clean centrifuge tube and spun at 5,000g for 10 min. Pellet was suspended in membrane buffer (30 mM NaHEPES, 5 mM MgCl2, 2 mM DTT, pH 7.5) to attain approximately 1 mg/ml total protein concentration. The assay was conducted by adding 30 µl membranes into tubes containing assay buffer (30 mM NaHEPES, 100 mM NaCl, 1 mM EGTA, 10 mM MgCl2, 1 mM IBMX, 1 mM ATP, 10 mM phosphocreatine, 5µM GTP, 60 U/ml creatine phosphokinase and 0.1% bovine serum albumin, pH 7.5.) and drugs of interest.

The mixture was incubated for 15 min at 30 °C and reactions were stopped by boiling for

5 min. cAMP content of each tube was assayed by ELISA as described above.

Intracellular Ca2+ measurements: Fibroblasts (0.25 x 105 cells) were plated on 22 mm

glass coverslips. The cells were washed once in phosphate buffered saline (PBS) and

incubated in 1 ml DMEM containing 1 µM Fura2/AM (Molecular Probes, Eugene, OR)

at 37 °C for 30 min. Cells were then washed once with DMEM and placed in a 37 °C

chamber containing 1.5 ml HEPES buffered saline (HBS: 130 mM NaCl, 5 mM KCl, 10

mM glucose, 1 mM MgCl2, 1.0 mM CaCl2, 25 mM HEPES, pH 7.4), such that groups of

58 cells could be viewed using an inverted Olympus IX70 microscope.

Spectrofluorometric measurements were collected using Delta Scan System

spectofluorometer (Photon Technology), where the field was excited at 380nm and

340nm and the emission ratio collected at 511nm. Agonists were administered from

1000X stocks to maintain a constant volume of 1.5 ml.

Reverse Transcriptase-PCR to Identify AC Isoforms: Total RNA was extracted from

cardiac fibroblasts grown to 80–90% confluency on 15-cm plates using Trizol reagent

(Invitrogen, Carlsbad, CA). A DNase reaction was performed to eliminate DNA

contaminants and the RNA was reverse transcribed using Superscript II (Invitrogen) and poly-T priming. Individual isoform-selective primer pairs were used to amplify each isoform of AC. Primers were based on rat or murine sequences as previously described

(Ostrom et al., 2002). PCR reactions with each primer pair were performed on cDNA, genomic DNA (positive control), and minus reverse transcriptase (negative control) template using 35 cycles and an annealing temperature of 56 °C. Sequence analysis was used to confirm the identity of all PCR products.

Purification of Caveolin-enriched Membrane Fractions: Cardiac fibroblasts were fractionated using a detergent-free method adapted from Song et al. (Song et al., 1996), and described previously (Ostrom et al., 2000). The faint lightscattering band resulting from sucrose density centrifugation was collected from the 5–35% sucrose interface. The bottom 4 ml of the gradient (45% sucrose) was collected as non-caveolar membranes.

Immunoblot Analysis for adenylyl cyclase: Individual fractions or whole cell lysates were separated by SDS-polyacrylamide gel electrophoresis (Nu-PAGE, Invitrogen).

Equal volumes of fractions were loaded so that each lane represented similar proportions from the cells, resulting in ∼10-fold lower amounts of protein loaded in the caveolin- enriched membrane fraction lanes. Proteins were transferred to a polyvinylidene difluoride membrane (Millipore) by electroblotting and probed with the appropriate primary antibody. Bound primary antibodies were visualized by chemiluminescence. In some experiments, the optical density of bands was calculated using a digital imaging system and LabWorks software (UVP Bioimaging Systems) and reported as arbitrary optical density units.

Immunoblot analysis for α-SMA and collagen type I: Whole cell lysates were collected

and Western blot analysis performed. Cells were scraped and maintained in lysis buffer

containing: 62.5 mM HCl-Tris, 2 mM EDTA, 2.3% SDS, 10% glycerol (pH 6.8), and

protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 1 μg/ml pepstatin A, and 5μg/ml aprotinin). Protein content was quantified by the BCA assay

(Pierce). Equal amounts of protein samples (10 μg) were boiled for 5 minutes in 2X sample buffer (100 mM Tris base, 20% glycerol, 2% SDS, 0.01% bromophenol blue), separated by standard SDS-polyacrylamide gel electrophoresis (SDS-PAGE; 10% acrylamide gel was used for α-SMA blots, and 7.5% for collagen I blots) and transferred by electroblotting to nitrocellulose membranes. Western blot analysis was performed utilizing standard techniques, using 5% milk in 0.1% Tween-20/TBS as a blocking reagent. Membranes were washed in 0.1% Tween-20/TBS 4-5 times for 10 minutes following incubation with both primary and secondary antibodies and bands were visualized with enhanced chemiluminescence. The band intensity of the indicated proteins was quantified by densitometric scanning using a Kodak 1D Digital Science

Imaging System.

Assay of Collagenase-sensitive [3H]Proline Incorporation: [3H]Proline incorporation by

cardiac fibroblasts was measured according to modified methods of Guarda et al.

(Guarda et al., 1993). Briefly, cells were transferred to 12-well plates and then serum

starved in 0% FBS was added for 24 h. [3H]proline (1 μCi/well, Perkin Elmer Life

Sciences), along with, where indicated, drugs of interest and 2.5% FBS for 24 h. Cells

were lifted by trypsinization and protein precipitated overnight with 20% trichloroacetic

acid. Protein was pelleted by centrifugation and washed 3 times with 1.0 ml of 5% trichloroacetic acid + 0.01% proline. Pellets were dissolved with 0.2 M NaOH and the solutions titrated to neutral pH with 0.2 M HCl. Collagenase type 2 (2 mg/ml:

Worthington Biochemical Corp.) in Tris/CaCl2/N-ethymaleimide buffer was added to

each tube and samples were incubated for 1 h at 37 °C. Samples were then placed on ice

and proteins precipitated with 10% trichloroacetic acid for 1 h. Samples were centrifuged

at 14,000 rpm for 10 min and the collagenase-sensitive [3H]proline in the supernatant was

determined by liquid scintillation counting.

RESULTS

Cross-talk between ANG II and β-AR Signaling Pathways Is Mediated by

Phospholipase C and Gq: Meszaros et al. (Meszaros et al., 2000) have described that

ANG II, perhaps acting via Gq, potentiates β-AR and Gs-mediated cAMP production in

cardiac fibroblasts. In the first series of experiments we assessed whether crosstalk

between Gq and Gs was dependent upon Gq-promoted phospholipase C (PLC) activity

(promoted by Gq activation). We incubated cardiac fibroblasts with the specific PLC

inhibitor, U73122 (5 μM), for 30 minutes prior to 15 minute hormonal stimulation with

ANG II (100 nM) or uridine triphosphate (UTP, 30 μM) in combination with ISO (1 μM,

Fig. 8). UTP provides a second class of agonist to assess cross-talk because it is an efficacious activator of the Gq/PLC/inositol trisphosphate pathway in these cells

(Meszaros et al,

Figure 8. Gq-Gs cross-talk is dependent upon Gq and PLC activation. A) Cardiac fibroblasts were treated with PLC inhibitor U73122 (5 μM) for 5 min. Cells were co- stimulated with 100 nM ANG II + 1 μM ISO as well as with 30 μM UTP + ISO. Cell lysates were extracted with 0.1 M HCl and cAMP was determined by an ELISA. Data are mean ± S.E. expressed as fold-change over ISO. *Statistically significant differences (p ≤ 0.01) by one-way analysis of variance. B) Cardiac fibroblasts were infected with adenoviruses containing the lacZ gene (control) and the wild-type α subunit of Gq (10 virus particles/cell) for 18–24 h prior to 15-min reactions and cAMP determinations. Data are mean ± S.E. expressed as fold-change over control. *Statistically significant differences versus control virus ANG II + ISO (p ≤ 0.01) by Student’s t test.

A. 50 B. * 2.5 * * 40 2.0

1.5 30

1.0 20

0.5 10

Control Adenovirus WT Gq Adenovirus

2000). Neither ANG II nor UTP alone caused altered cAMP accumulation. At the concentration used in these experiments, ISO routinely produces a 10–12-fold increase in cAMP levels in cardiac fibroblasts (Meszaros et al., 2000). ANG II and UTP (activator of

Gq signaling) potentiated the response to ISO by 1.8 ± 0.1 and 2.0 ± 0.1-fold (relative to

ISO alone), respectively. Whereas U73122 alone did not affect basal cAMP accumulation or the ISO response, this inhibitor (at a concentration that reduced stimulated phosphoinositide hydrolysis 75–80%) completely eliminated the potentiation of cAMP accumulation by ANG II and UTP. These data suggest that Gq-linked activation of PLC is required for Gq potentiation of Gs-AC activity.

To assess whether activation of Gq might mediate the effects of ANG II on β-AR signaling, we used adenoviral-mediated gene transfer of Gq to increase its expression in cardiac fibroblasts (Fig. 8B). Increased expression of Gq enhanced inositol phosphate accumulation by both ANG II (1.6-fold increase over control) and UTP (2.6-fold increase over control) and significantly enhanced the ANG II potentiation of ISO-stimulated cAMP (3.9 ± 0.5-fold over 1 μM ISO alone) compared with control cells incubated with the null (PACCMVpLpA) virus (2.5 ± 0.3-fold over ISO alone). Thus, results in Fig. 8 are consistent with the conclusion that activation of GPCRs that activate Gq and PLC enhance β-AR/Gs-mediated cAMP formation.

To determine whether signaling by Gβγ subunits generated by activation of Gq mediates the observed cross-talk, we used an adenovirus to express the C-terminal peptide of G-protein coupled receptor kinase 2 (GRK2; β-AR kinase 1, or βARK1). This peptide (βARKct) binds to free Gβγ subunits, and inhibits both Gβγ signaling and GRK2

activation (Drazner et al., 1997). We exposed cardiac fibroblasts to either βARKct (10 virus parts per cell) or null virus for 18 hours and measured cAMP accumulation stimulated for 15 minutes by ISO with and without ANG II. Expression of βARKct did not attenuate ANG II-induced potentiation of ISO-stimulated cAMP accumulation in cardiac fibroblasts (null virus: ISO 10.8 ± 0.2-fold over basal, ANG II + ISO 19.4 ± 2.2;

βARKct virus: ISO 9.2 ± 0.5, ANG II + ISO 20.8 ± 0.6) but did induce a 4-fold increase in ISO potency, consistent with its action blocking GRK2 activation (Drazner et al.,

1997).

Another possible pathway by which ANG II could affect cAMP production is via receptor tyrosine kinase signaling. However, cross-talk was not inhibited in cells treated with AG1478 (10 μM), a specific tyrosine kinase inhibitor (ANG II + ISO, 2.0 ± 0.1-fold over ISO alone; ANG II + ISO + AG1478, 2.0 ± 0.1) or with PP2 (1 μM), a selective Src inhibitor (data not shown). These findings and those of Fig. 8 indicate that ANG II enhances ISO-mediated cAMP production via the action of Gq and PLC rather than Gβγ or tyrosine kinase-associated signaling, an important distinction given that ANG II activates both G protein and tyrosine kinase pathways in cardiac fibroblasts (Dostal et al.,

1996; Hou et al., 2000).

Requirement for Elevation of Intracellular Ca2+ but Not PKC: Because Gq activation

by ANG II elevates intracellular Ca2+ levels and activates PKC in cardiac fibroblasts

(Hou et al., 2000), we examined the role of both responses as potential mechanisms by

which ANG II influences cross-talk with Gs. We utilized the Ca2+ ionophore, ionomycin

Figure 9. Ionomycin-induced intracellular Ca2+ transients enhance cAMP production. A) Cardiac fibroblasts were loaded with Fura-2/AM (1 μM) and treated with the calcium ionophore ionomycin (IN) at varying concentrations. As anticipated, IN elevated cytosolic Ca2+ in a dose-dependent manner. The fluorescence ratios are representative data of 3 similar experiments. B, cells were co-treated with 100 nM ISO + IN for 15 min in the presence of 0.1 mM isobutylmethylxanthine at the indicated doses prior to cAMP measurements. Data are mean ± S.E. expressed as fold-change over ISO. Statistically significant differences were determined versus ISO alone by Student’s t test (*, p ≤ 0.05; **, p ≤ 0.01).

(10 and 100nM), to elevate intracellular Ca2+ levels in a concentration-dependent manner

(Fig. 9A) that synergistically enhanced ISO-induced cAMP production (Fig. 9B) by 2.7 ±

0.4 to 4.0 ± 0.9-fold compared with 100 nM ISO alone (indicated by the dashed line) using 10 and 100 nM ionomycin, respectively. Ionomycin alone did not alter basal cAMP production. Conversely, pharmacological inhibition of PKC by GF109203X (10 μM) failed to inhibit the ANG II-induced potentiation of the ISO response (ANG II + ISO 2.0

± 0.1-fold over ISO alone, plus GF109203X 1.9 ± 0.1) but did reduce UTP-stimulated phosphor-ERK immunoreactivity (basal, 36 ± 6.1 OD; 100 μM UTP, 1037 ± 41; UTP +

GF109203X, 180 ± 14.4). Similar negative results were seen with the PKC inhibitors calphostin C and staurosporine (data not shown). Thus, Gq-Gs cross-talk appears to be mediated by the effect of ANG II to elevate intracellular Ca2+ and not activation of PKC.

ANG II and Ionomycin Potentiate Forskolin-induced cAMP Production: To test

whether the potentiation effect of ANG II on ISO-stimulated cAMP formation might

occur via an effect distal to β-AR, we utilized forskolin, which directly activates AC

(albeit with some dependence upon Gs). Forskolin (1 μM) alone stimulated cAMP production by 8–10-fold over basal levels, a level of response similar to that seen with

0.1–1 μM ISO. ANG II potentiated forskolin-induced cAMP levels (over forskolin alone) in a concentration-dependent manner in the range tested (1–100 nM), reaching statistical significance at 10 and 100 nM (1.6 and 2-fold over forskolin alone, respectively, Fig.

10A). Likewise, ionomycin (100 nM) significantly potentiated forskolin-induced cAMP levels (from 8.3 ± 1.3-fold alone to 25.5 ± 7.4-fold in combination, relative to controls,

Figure 10. ANG II and ionomycin enhance forskolin-stimulated cAMP production. A) The cells were given varying concentrations of ANG II for 15 minutes in combination with 1 μM forskolin (FSK) for 15 min prior to cAMP determinations. Data are mean ± S.E. expressed as fold-change over control. Statistically significant differences were determined versus FSK alone by one-way analysis of variance (*, p ≤ 0.05; **, p ≤ 0.001). B) Cardiac fibroblasts were co-treated with ionomycin (IN) + 1 μM FSK for 15 min and cAMP was determined. Data are mean ± S.E. expressed as fold-change over control. *Statistically significant differences versus FSK alone (p ≤ 0.05) by Student’s t test.

Fig. 10B). These results indicate that the effect of ANG II is distal to the receptor and that ionomycin can mimic this effect by elevating intracellular Ca2+.

Effects of Ca2+ Store Release and Ca2+ Buffering on Gq/Gs Cross-talk: We next sought

to determine whether Gq/Gs cross-talk might occur via Gq-promoted Ca2+ release from

intracellular stores and if we could inhibit the cross-talk by buffering intracellular Ca2+.

Thapsigargin (0.1-100 nM), which inhibits the sarcoendoplasmic reticular Ca2+-ATPase pump, elevated intracellular Ca2+ levels in a concentration-dependent manner (Fig. 11A).

Thapsigargin at a concentration of 100 nM was maximally efficacious in stimulating

rapid Ca2+ release; this concentration also enhanced forskolin-induced cAMP production

(Fig. 11B) with slight but statistically insignificant effects on basal cAMP production. We

obtained similar results when these experiments were performed in the absence of

extracellular Ca2+ (data not shown), indicating that Ca2+ release from internal stores was

sufficient to observe the enhancement in cAMP formation. These results, together with those from the studies with ionomycin, indicate that potentiation of the cAMP signal can occur from elevation of intracellular Ca2+, either by storage release or influx promoted by

a Ca2+ ionophore. Conversely, preincubation with 1 μM BAPTA/AM, a Ca2+ chelating

agent, for 30 min prior to agonist stimulation prevented ANG II-induced Ca2+ transients

(Fig. 11C) and blocked the potentiative effects of ANG II on forskolin-mediated cAMP

production (Fig. 11D). Thus, elevation of intracellular Ca2+ is both necessary and sufficient for potentiation of cAMP production by ANG II. The results seen with forskolin indicate that Ca2+ works at the level of Gs/AC rather than β-AR.

Figure 11. Intracellular Ca2+ transients enhance cAMP production and are blocked by Ca2+ chelation. A) Cardiac fibroblasts were loaded with 1 μM Fura-2/AM and the 340/380 fluorescence ratios were monitored. The cells were given varying concentrations of thapsigargin (TG), which produced dose-dependent increases in intracellular Ca2+. Data are representative of 3 similar experiments. B) Cells were co-treated with similar doses of TG (as in A) along with 1 μM FSK. A dose-dependent increase in cAMP levels was observed with increasing concentrations of TG. Data are mean ± S.E. expressed as fold-change over control. *Statistically significant differences versus FSK alone (p ≤ 0.01) by Student’s t test. C) Cells were incubated in the absence and presence 1 μM BAPTA/AM for 30 min prior to monitoring intracellular Ca2+ levels in the presence of 100 nM ANG II. BAPTA/AM effectively buffers intracellular Ca2+ levels when stimulated with 100 nM ANG II for 15 minutes. Data are representative of three similar experiments. D) Cardiac fibroblasts were treated with 1 μM BAPTA/AM for 30 min, and then with the indicated hormone treatments for 15 min. BAPTA/AM effectively blocked the FSK/ANG II potentiation of cAMP. Data are mean ± S.E. expressed as fold-change over control. *Statistically significant differences versus FSK alone (p ≤ 0.01) by Student’s t test. **Statistically significant differences versus FSK + ANG II by Student’s t test (p ≤ 0.001).

2.9 A. B. * 50 2.7 100nM TG 2.5 40

2.3 30 10nM TG 2.1 20 340/380 Ratio 1.9 10 1.7 1 nM TG Thapsigargin 0 0 50 100 150 200 250 Time (sec) control μ M TG μ M FSK 1 nM TG 1 1 0.1 nM TG nM10 TG 100 nM TG

+ 1 μM FSK C. 18 2.9 D. * 15 2.8

12 2.7 ** 2.6 No Bapta 9 340/380 Ratio 2.5 6 2.4 3 2.3 ANG II 1μM Bapta 0 0 50 100 150 200 250 300 350

Time (sec) FSK control FSK + ANG ANG + FSK BAPTA; F + A F A + BAPTA;

AC Isoform Expression in Cardiac Fibroblasts: Because the identity of AC isoforms expressed in cardiac fibroblasts could result in specific signaling characteristics, we sought to define the AC isoforms expressed in these cells and in particular to assess isoforms that are regulated by Ca2+. RT-PCR analysis using isoform-specific primers revealed that cardiac fibroblasts express mRNA for AC2, AC3, AC4, AC5, AC6, AC7, and AC8 (Fig. 12A). Each of the primer pairs amplified appropriate genomic DNA sequence but did not yield PCR products when RNA (no reverse transcriptase) was used as template (data not shown). PCR reactions using primers for AC1 and AC9 yielded products that were not of the expected size or sequence. We also conducted immunoblot analysis to detect expression of AC proteins. Because AC immunoreactivity in cardiac myocytes is enriched in buoyant, caveolin-rich fractions in a manner that improves immunological detection (Schwencke et al., 1999; Ostrom et al., 2001), we fractionated cardiac fibroblasts to isolate caveolin-rich fractions and performed immunoblot analyses.

As shown in Fig. 12B, caveolin-1 immunoreactivity was detected in buoyant fractions

(caveolin-rich fraction) and was excluded from non-buoyant fractions. Immunoreactivity for AC3 and AC5/6 (the antibody used does not distinguish between AC5 and AC6) was detected primarily in caveolin-enriched membrane fractions, whereas immunoreactivity of AC2, AC4, and AC7 was detected only in non-caveolin-enriched membrane fractions

(Fig. 12B). No immunoreactivity was detected for AC8 or AC9 (Fig. 12B). Thus, cardiac fibroblasts express numerous AC isoforms but the isoforms appear to be differentially localized in caveolin-rich membrane microdomains.

Figure 12. Cardiac fibroblasts express multiple AC isoforms with different subcellular distributions. A) RT-PCR analysis was performed using AC isoform-specific primer pairs and cDNA, mRNA (no reverse transcriptase negative control, not shown), or genomic DNA (positive control, not shown). Arrows indicate the size of the expected PCR product from each primer pair. Image is representative of three experiments. B) expression of AC isoforms was assessed by immunoblot analysis of caveolin-rich (cav) and non-caveolin rich (non-cav) membrane fractions from cardiac fibroblasts (see “Methods”). Each fraction was separated by SDS-PAGE, transferred to membrane, and probed with antibodies specific for AC1, AC2, AC3, AC4, AC5/6, AC7, AC8, AC9, and caveolin 1. Nonspecific immunoreactive bands are partially evident at the top of images for AC5/6 and AC8. Each image is representative of at least three experiments.

AC1 AC2 AC3 AC4 AC5 AC6 AC7 AC8 AC9 A.

AC Isoform Specificity of Gq-Gs Cross-talk: We hypothesized that expression of a Ca2+- calmodulin-stimulable isoform of AC was essential for the cross-talk we observe. Thus, we pretreated cardiac fibroblasts with a specific calmodulin inhibitor (10 μM calmidazolium) and found that it significantly attenuated cross-talk induced by ANG II plus ISO or by ionomycin plus forskolin (Fig. 13, A and B, respectively). We also measured Ca2+ and forskolin-stimulated AC activity in membrane preparations from cardiac fibroblasts with and without exogenously added calmodulin; 3 μM free Ca2+ in the presence of forskolin (10 μM) did not stimulate AC activity unless calmodulin (0.1

μM) was added to the reaction (Fig. 13C). This Ca2+-stimulable AC activity was inhibited by the inclusion of a calmodulin inhibitor (1 μM fluphenazine, which did not alter basal or forskolin-stimulated AC activity). These results indicate that Ca2+-calmodulin activation of AC is a plausible mechanism for the effect of ANG II and ionomycin to enhance cAMP production by β-AR.

We next probed whether it was possible to attenuate cross-talk between ANG II and cAMP production by altering the balance of Ca2+-stimulated versus Ca2+-insensitive

AC isoforms by using an adenovirus to overexpress an isoform of AC that is not Ca2+- stimulable, AC6 (Hanoune and Defer, 2001). Incubation of cardiac fibroblasts with the

AC6 adenovirus for 18 h resulted in a 2-fold increase in maximal forskolin-stimulated cAMP production (Fig. 13D). Based on evidence that the extent of AC overexpression is proportional to the increase in maximal forskolin response (Gao et al., 1998), the conditions used here approximately doubled the total cellular content of AC and generated cardiac fibroblasts expressing predominantly AC6. Immunoblot analyses

Figure 13. Calmidazolium and overexpression of AC6 inhibit cross-talk. A) Cardiac fibroblasts were pretreated with the calmodulin inhibitor calmidazolium (CDZ; 10 μM) for 30 min prior to reactions. The potentiation effect produced by the combination of ANG II and ISO (A+I) was significantly blocked in the presence of CDZ, but did not have a significant effect on ISO alone. Data are means ± S.E. expressed as fold-change over control. *Statistically significant differences versus ANG II + ISO (*, p ≤ 0.05) by Student’s t test. B) Cells were pretreated with CDZ for 30 min prior to reactions. CDZ significantly inhibited cAMP potentiation induced by 100 nM ionomycin (IN) and 1 μM forskolin (FSK), but did not have a significant effect on FSK alone. Data are means ± S.E. expressed as fold-change over control. *Statistically significant differences versus IN + FSK (I+ F; *, p ≤ 0.05) by Student’s t test. C) AC activity was measured in cardiac fibroblast membranes with or without calmodulin (0.1 μM) and with or without fluphenazine (1 μM). Data are expressed as fold change over basal AC activity. #Statistically significant difference (p ≤ 0.05) versus Fsk alone; **Statistically significant difference (p ≤ 0.01) versus control by Student’s t test. D) Production of cAMP in response to forskolin or forskolin plus ANG II was measured in control and AC6- overexpressing cardiac fibroblasts. ANG II potentiation of FSK was significant (p ≤ 0.01) by Student’s t test) in control cells but not significant (p = 0.14) in AC6 overexpressing cells. Immunoblot analysis indicated an appreciable increase in AC5/6 protein in caveolin-enriched membrane (Cav) fractions of cardiac fibroblasts incubated with AC6 adenovirus (inset).

indicated an appreciable increase in detectable AC5/6 protein in caveolin-enriched membrane fractions (Fig. 13D, inset). ANG II potentiation of forskolin-stimulated cAMP was unchanged by AC6 overexpression in terms of absolute amount of cAMP stimulated over forskolin alone (14.2 ± 1.4 pmol of cAMP/mg of protein in control cells versus 17.7

± 8.8 in AC6 cells), but was significantly inhibited in terms of the fractional effect (p =

0.14, Fig. 13D). Thus, the potentiative effect of ANG II appeared not to extend to the overexpressed AC6. The inability of ANG II to potentiate forskolin-stimulated cAMP production to the same degree in AC6-overexpressing cells was not related to a limited capacity of cardiac fibroblasts to synthesize cAMP in this latter condition, as combined treatment of the AC6-overexpressed cells with ISO and forskolin-stimulated cAMP levels to 27.6 ± 3.48 pmol/mg protein, a level nearly 3-fold higher than the combination of forskolin and ANG II. Thus, cross-talk is decreased in cells manipulated to express increased levels of AC6, an AC isoform not activated by Ca2+. Taken together, the data in

Figs. 8–13 implicate the central role of both Ca2+ influx and a Ca2+-calmodulin- stimulable AC isoform in mediating the observed Gq-Gs cross-talk.

Elevation of cAMP inhibits differentiation into cardiac myofibroblasts: To examine the functional consequences of elevated cAMP signaling, we studied one aspect of CF activation—differentiation to myofibroblasts. Differentiation to myofibroblasts was induced by treatment with either 100 nM ANG II or 200 pM TGF-β as measured by α-

SMA expression (Figure 14). Co-treatment with 1 μM forskolin inhibited both ANG II

(Figure 14A and B) and TGF-β-induced myofibroblast differentiation (Figure 14A).

Contrary to the positive effect of elevated of cAMP on CF proliferation, increasing

Figure 14. Elevation of cAMP inhibits differentiation to cardiac myofibroblasts. Cardiac fibroblasts were treated with either 100 nM ANG II or 200 pM TGF-β with or without 1 μM forskolin (FSK) for 48 hours. α-SMA protein expression was measured as a marker of differentiation to cardiac myofibroblasts. A) Immunofluorescence demonstrates that ANG II induces α-SMA (myofibroblasts), and co-treatment with FSK inhibited the differentiation to myofibroblasts. B) Western blot analysis confirm results from panel A: FSK, a direct activator of adenylyl cyclase inhibited both ANG II and TGF-β-induced α- SMA expression. A representative Western blot is shown above, and the graph below represents a summary graph of densitometric measurements from 3 different experiments in cells taken from 3 different animals.

A. B. Con ANG TGF FSK A+F T+F CONTROL ANG II α-SMA

1.6

1.4

1.2

1.0

0.8 Fold change control over 0.0 β

FSK ANG II + FSK F T + A + F M FSK Control μ 1 100nM ANG 100nM 200pM TGF-200pM

cAMP has an inhibitory effect on differentiation to the hypersecretory myofibroblast phenotype.

Role of Cross-talk on Collagen Synthesis: To further investigate the functional relevance of the cross-talk between ANG II signaling and the cAMP pathway, we assessed the synthesis of collagen by cardiac fibroblasts. Increased cellular cAMP levels can inhibit cardiac fibroblast-mediated production of collagen (Dubey et al., 1997). Thus, we tested whether simultaneous incubation with cAMP elevating agents and ANG II would inhibit collagen synthesis to a greater extent than incubation with cAMP-elevating agents alone.

FBS-stimulated collagenase-sensitive [3H]proline incorporation was measured in cardiac fibroblasts as an index of collagen formation in the absence and presence of ANG II and in the absence and presence of ISO. 2.5% FBS stimulated collagen synthesis 2.6 ± 0.2- fold over basal levels. ANG II alone increased collagen synthesis (data not shown) but in the presence of 2.5% FBS induced no significant increase over FBS alone (Fig. 15A).

ISO (100 μM) inhibited FBS-stimulated collagen synthesis (31 ± 9% inhibition, Fig. 15,

A and B). However, in the presence of ANG II, ISO inhibited proline incorporation 48 ±

8%, an effect that was significantly different (p < 0.01) from serum-stimulated levels

(Fig. 15, A and B). Moreover, the ANG II enhancement of the ISO effect was reduced to nonsignificant levels by chelation of Ca2+ (with 0.1 μM BAPTA/AM) or inhibition of calmodulin (with 0.1 μM calmidazolium). Thus, the data support the conclusion that the enhanced β-AR-mediated cAMP production that ANG II promotes via elevation of Ca2+ and signaling via calmodulin can blunt collagen synthesis.

Figure 15. Gq-Gs cross-talk impacts cardiac fibroblast collagen synthesis. A) Collagen synthesis, measured by collagenase-sensitive [3H]proline incorporation as described under “Methods,” was stimulated by adding 2.5% FBS to serum-starved cells in the absence and presence of ISO (100 nM), ANG II (10 nM), or ISO plus ANG II for 24 h. The Ca2+ chelator BAPTA/AM (0.1 μM) and the calmodulin inhibitor calmidazolium (0.1 μM) were also added in the presence of ISO and ANG II. B) Data for ISO, ISO plus ANGII, and BAPTA/AM and calmidizolium are presented expressed as the percent inhibition of the FBS-stimulated collagen synthesis. *Statistically different p ≤ 0.05; **, p ≤ 0.01 by Student’s t test as compared with 2.5% FBS or FBS + ANG II stimulated, respectively.

63 % Inhibition% % Inhibition% fold over basal fold over basal Collagen production Collagen production ISO ISO ANG II Calmid Calmid 2.5% FBS 2.5% BAPTA-AM BAPTA-AM ANG II + ISO ANG ANG II + ISO ANG +ISO + ANG II +ISO + ANG II

+2.5% FBS

Effect of cAMP on collagen I production: Since cardiac fibroblasts are responsible for production of type I collagen, the main collagen present in the heart, we wanted to determine the effects of elevated cAMP specifically on the production of type I collagen.

Isolated CFs were treated with 100 nM ANG II or 200 pM TGF-β in combination with 1

μM forskolin. Both ANG II and TGF-β increased the production of collagen I, and forskolin decreased control levels as well as ANG II and TGF-β induced collagen I protein expression (Figure 16). Taken together, the data suggest that coincidental Gs and

Gq signals can interact to modulate cardiac fibroblast signaling and function, and thereby promote a Gs-mediated inhibition of both myofibroblast differentiation and collagen synthesis.

DISCUSSION

The focus of the present work was to define the mechanism of Gq-Gs cross-talk, which we previously described in adult rat cardiac fibroblasts (Meszaros et al., 2000).

The striking potentiation of β-AR signaling in the presence of ANG II might occur via several different mechanisms. ANG II activates AT1 receptors in cardiac fibroblasts, thereby activating both Gq-coupled and tyrosine kinase signaling pathways (Dostal et al.,

1996; Hou et al., 2000). Several pieces of evidence indicate that this cross-talk is mediated by activation of Gq: 1) at least 2 other Gq-coupled receptors (P2Y and bradykinin) are capable of inducing similar potentiation (Fig. 8A and (Meszaros et al.,

2000)); 2) blockade of PLC eliminates potentiation of the ISO response by either ANG II or UTP (Fig. 8A); 3) overexpression of Gq enhances cross-talk (Fig. 8B); 4) chelation of

Figure 16. Collagen I production is inhibited by elevations in cAMP via forskolin. Cardiac fibroblasts were cultured and serum-starved for 48 hours and subsequently treated with 100 mM ANG II or 200 pM TGF-β in combination with 1 μM forskolin (FSK) for 48 h before protein isolation (see Methods for lysis buffer composition). Western blot analysis was carried out, and densitometry and quantitation was performed on the pro-α1 (I) band (top two bands). Values represent mean ± SEM of three experiments and compared by using Students t test. #Statistically significant (p ≤ 0.05) in response to forskolin.

a key second messenger of the Gq pathway, intracellular Ca2+, results in attenuation of cross-talk; and 5) pharmacological agents that directly increase cytosolic Ca2+ concentrations (ionomycin, thapsigargin) also potentiate cAMP accumulation (Figs. 9 and 10). These data, along with evidence that exclusion of extracellular Ca2+ does not affect cross-talk (data not shown), indicate that ANG II-induced potentiation of β-AR signaling is triggered by Gq activation and Ca2+ release from internal stores and that Ca2+ influx is not required for enhanced cAMP production. Consistent with this conclusion, pharmacological inhibition of the other major pathway activated by Gq, PKC, had no effect on cross-talk. Our studies with forskolin allowed us to test the target at which Ca2+ acts to increase β-AR signaling. As a direct activator of AC, albeit requiring Gs activation for full effect (Green and Clark, 1982), forskolin-stimulated cAMP formation was potentiated by signals that elevate intracellular Ca2+ (e.g. ANG II, ionomycin, or thapsigargin, Figs. 10 and 11). These results led us to focus on the role of AC in Gq-Gs cross-talk. We were surprised to discover that cardiac fibroblasts express mRNA for seven of the nine transmembrane AC isoforms (Fig. 12). This expression of multiple isoforms contrasts with results from cardiac myocytes, which appear to express predominantly 2 isoforms, AC5 and AC6 (Ishikawa et al., 1994). Of the isoforms expressed in cardiac fibroblasts, the protein for AC3 appears to be predominantly expressed, particularly in caveolin-rich fractions (Fig. 12B).

Given its level of expression and action as a Ca2+-stimulable isoform, AC3 is likely critical for the cross-talk that we observe in cardiac fibroblasts. The other AC isoforms capable of being activated by Ca2+, AC1 and AC8, were not detected by

immunoblot analysis (although AC8 mRNA was detected in PCR studies). Previous data using heterologously expressed protein indicate that AC3 stimulation by Ca2+ is dependent upon coincidental activation of AC by either Gs or forskolin (Choi et al.,

1992), consistent with what we observe in cardiac fibroblasts. An important role for a

Ca2+-stimulable AC isoform in the observed cross-talk is inferred by data from vascular smooth muscle (Webb et al., 2001) and directly supported by our studies overexpressing

AC6. By increasing AC6 expression, we altered the balance of AC isoform expression in favor of an isoform not stimulated by Ca2+ and found that the Gq-Gs cross-talk was fractionally reduced. Taken together, these data provide a clear-cut example of AC isoform expression imparting distinct signaling characteristics and regulation of cell function.

The membrane microdomains in which expression of AC isoforms occurs can influence cell responses (Ostrom et al., 2000; Xiang et al., 2002). Caveolae are a subset of lipid rafts, plasma membrane microdomains enriched in sphingolipid and cholesterol, and are the site of enrichment of many membrane-associated signaling molecules

(Anderson, 1998). We found that among numerous AC isoforms expressed in cardiac fibroblasts, only AC3 and AC5/6 localize in buoyant, caveolin-rich domains. These data are the first of which we are aware that show that native AC isoforms localize differently with respect to caveolae/lipid rafts. Smith et al. (Smith et al., 2002) recently reported that

AC7 and AC8 heterologously expressed in HEK-293 cells localized differently with respect to caveolin-rich domains (AC8 in caveolar fractions, AC7 in non-caveolar fractions). The reason for the difference between the data from Smith et al. (Smith et al.,

2002) and the present studies (we observe AC8 in non-caveolar fractions, Fig. 12) is unclear, but localization of GPCR and AC isoforms in caveolar fractions can be highly cell-specific and can differ between exogenously and endogenously expressed protein

(Ostrom et al., 2002). More work is needed to understand the functional importance, if any, of the observed pattern of AC isoform expression in different plasma membrane microdomains of these cells.

Our studies of collagen synthesis and myofibroblast differentiation (Figs. 14-16) suggest the physiological importance of Gq-Gs cross-talk. Agents that elevate cellular cAMP attenuate collagen synthesis by cardiac fibroblasts (Dubey et al., 1998). In our studies, elevation of cAMP via forskolin significantly inhibited ANG II and TGF-β- induced myofibroblast differentiation, and by doing so is indirectly limiting the potential

ECM production by CFs. In addition, ISO alone (in the absence of a phosphodiesterase inhibitor) could significantly inhibit collagen synthesis, and the addition of ANG II, which produced a 2-fold increase in ISO-stimulated cAMP accumulation, led to a greater reduction in collagen synthesis. These results appear to conflict with observations that β-

AR antagonists can reduce fibrosis in heart failure (Asai et al., 1999; Brilla 2000).

However, the beneficial effects of β-AR blockade are likely dominated by improvements in cardiac contractility (which would lead to decreased levels of renin-angiotensin and possibly other hormones) and myocyte survival, which in turn influence ECM formation and turnover as well as fibroblast proliferation. Our findings are consistent with data on certain other Gs-linked agonist action on cardiac fibroblasts (Dubey et al., 1997; Dubey et al., 1999) and with a previous report that the β-AR antagonist, propranolol, elevates

collagen deposition in pulmonary fibroblasts, cells not subjected to the mechanical forces of a contractile organ (Lindenschmidt and Witschi, 1985). In addition, our studies examine fibroblasts over a short period (24-48 h) and in isolation, removed from circulating catecholamines and the mechanical forces of cardiac contraction.

In conclusion, the current results demonstrate that ANG II enhances β-AR signaling via activation of Gq and elevated intracellular Ca2+. We propose that a Ca2+- stimulable isoform of AC, likely AC3, is the regulatory site of this Ca2+/calmodulin- dependent potentiation and acts as an integrator of these signaling pathways (see Figure

17 for proposed mechanism). Moreover, cross-talk between the Gq and Gs pathways (i.e. enhanced cAMP production) appears to have functional consequences for regulation of both myofibroblasts and the ECM in the myocardium, acting to limit collagen synthesis.

Figure 17. Proposed Gq-Gs cross-talk mechanism. In cardiac fibroblasts, ANG II acts at its receptor which couples to Gq. Activation of Gq causes stimulation of phospholipase Cβ (PLCβ), and production of inositol trisphosphates (IP3) and diacylglycerol (DAG). IP3 2+ binds to the IP3 receptor (IP3R) and causes release of Ca from the endoplasmic reticulum (ER). Both DAG and Ca2+ activate protein kinase C (PKC). A β-adrenergic receptor agonist such as isoproterenol binds to its receptor, which couples to Gs. Activation of Gs stimulates the membrane-bound enzyme adenylyl cyclase (AC) to produce cAMP. Simultaneous activation of both the Gq and the Gs pathways results in a potentiation of cAMP production. We propose that the Ca2+ being released into the cytoplasm is acting on a Ca2+-sensitive isoform of AC (likely AC3) present in the caveolar domain of the plasma membrane.

CHAPTER THREE

TYPE VI COLLAGEN INDUCES CARDIAC MYOFIBROBLAST DIFFERENTIATION: IMPLICATIONS FOR POST-INFARCTION REMODELING

INTRODUCTION

Cardiac fibroblasts (CFs) are the major non-contractile cells present in the myocardium and the primary regulators of extracellular matrix (ECM) secretion (Brilla et al., 1995; Dostal et al., 1996; Villarreal, 1998). Proliferation and differentiation of CFs can cause excess ECM protein production and cardiac fibrosis, a condition characterized by a loss of myocardial compliance (Weber and Brilla, 1991; Brilla, 2000; Burlew and

Weber, 2002). This condition is common in patients who have experienced myocardial infarction and during heart failure, and often leads to further loss of cardiac function

(Capasso et al., 1990; Cleutjens et al., 1995; Funck et al., 1997; Sun et al., 2000).

Although activation of CFs and cardiac ECM remodeling is necessary following myocardial injury, limiting prolonged fibroblast activation and subsequent detrimental

ECM production is a potential approach to preserving left ventricular function.

A key aspect of CF activation is differentiation into myofibroblasts, which are specialized cells that play critical roles in wound healing and repair in several tissues

(Cleutjens et al., 1995; Lorena et al., 2002). It has been suggested that these cells are responsible for the majority of ECM deposition (Faouzi et al., 1999; Chai et al., 2003) and that these differentiated fibroblasts are the primary mediators of fibrosis in the heart

(Weber et al., 1997). In normal wound healing, differentiation to myofibroblasts is necessary for repair and stabilization, but these cells eventually undergo apoptosis. If myofibroblasts remain in the injured area for an extended period of time, excessive ECM production occurs resulting in fibrosis.

The specific collagen types produced by fibroblasts and myofibroblasts may, in turn, affect the activation and subsequent function of these cells. In adults, the myocardial

ECM is primarily composed of collagens type I and III (Borg et al., 1982; Eghbali et al.,

1989; Weber et al., 1994; Agocha et al., 1997) with collagens IV, V, and VI comprising the remainder of the collagen network (Heeneman et al., 2003). During cardiac pathologies, investigators have detected an increase in both types I and III collagen, which contribute significantly to the observed cardiac fibrosis (Weber and Brilla, 1991;

Gonzalez et al., 2002; Koshy et al., 2003). Although type VI collagen is considered to be a minor type in the adult heart, type VI levels have been demonstrated to significantly increase in the hearts of both hypertensive and diabetic rats (Spiro and Crowle,y 1993). In addition, interstitial fibrosis and cardiac dysfunction related to hypertrophic cardiomyopathy have been positively correlated with elevation in levels of type VI and type III collagen (Kitamura et al., 2001).

The fact that collagen VI is significantly elevated in these specific cardiac pathologies prompted us to hypothesize that this collagen type plays a critical role in pathological remodeling via induction of myofibroblast differentiation. The goals of our study were to determine the effects of types I, III, and VI collagen on CF differentiation and proliferation, the effect of angiotensin II (ANG II) on type VI collagen production by

CFs, and whether type VI collagen and cardiac myofibroblast levels change in vivo during post-MI cardiac remodeling.

METHODS

Preparation of substrates - isolation of type VI collagen, solubilization of types I and

III collagen and coating of plates: Type VI collagen was isolated according to the methods previously outlined (Doane et al., 1992). Acid-soluble collagens were removed by treatment of bovine corneas with acetic acid. The acid-insoluble fraction was homogenized in 6M urea and ammonium sulfate precipitated, then resuspended in 5%

SDS in borate buffer. The sample was chromatographed using a Sepharose 4B column, and fractions were analyzed using SDS-PAGE to identify the presence of purified type

VI collagen. Type VI collagen was solubilized in 20 mM carbonate buffer (pH 9.6).

Types I and III collagen were both purchased commercially, type I collagen from

Vitrogen (solubilized in 0.012 N HCl) and type III collagen from Chemicon International

(solubilized in 0.5 M acetic acid). All collagens were used at a final concentration of 20

μg/ml and added to non-coated plastic wells.

Immunocytochemical staining and fluorescent imaging: Cardiac fibroblasts were seeded onto collagen-coated 18-well glass “dot” slides at approximately 10,000 cells per dot (each with an area of 28.3 mm2) and incubated for 24 hours in serum-free DMEM.

Subsequently, the cells were washed and incubated with 100 nM ANG II for an additional 24 hours. The cells were washed, fixed in 3.5% paraformaldehyde, permeabilized in 0.1% Triton X-100, blocked in 2% goat serum, and incubated with a

mouse monoclonal anti-α-smooth muscle actin primary antibody at 1:400 (Sigma-

Aldrich) for 1 hour at room temperature. The cells were next washed and incubated with a goat anti-mouse secondary antibody conjugated to Alexa Fluor 488 (Molecular Probes,

Eugene, OR) at 1:400 for 1 hour, washed, mounted with Vectashield anti-fade reagent containing DAPI (Vector Laboratories, Burlingame, CA), and visualized by an Olympus

IX-70 microscope. Digital images were taken using 10x-40x objectives. Additional control conditions were routinely carried out, including secondary antibody alone as well as cell-free backgrounds with primary and secondary antibodies (alone and in combination).

Assessment of cell proliferation: Cardiac fibroblasts were grown on 96-well plates pre- coated with collagens I, III, and VI in the presence and absence of 100 nM ANG II for 24 hours. To assess cellular proliferation, a BrdU assay was performed according to manufacturer’s protocol (Roche Applied Science, Indianapolis, IN). Briefly, 10 μM BrdU was included in the growth medium following the indicated treatments, and an ELISA was performed using an anti-BrdU antibody. Results were analyzed using spectrophotometry at a wavelength of 450 nm.

Western blot analysis in isolated CF cultures: Whole cell lysates were collected and

Western blot analysis performed. Cells were scraped and maintained in lysis buffer containing: 62.5 mM HCl-Tris, 2 mM EDTA, 2.3% SDS, 10% glycerol (pH 6.8), and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 1 μg/ml pepstatin A, and 5μg/ml aprotinin). Protein content was quantified by the BCA assay

(Pierce). Equal amounts of protein samples (15 μg) were boiled for 5 minutes in 2X

sample buffer (100 mM Tris base, 20% glycerol, 2% SDS, 0.01% bromophenol blue), separated by standard SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto to nitrocellulose membranes. Western blot analysis was performed utilizing standard techniques, using 5% milk in 0.1% Tween-20/TBS as a blocking reagent. Membranes were washed in 0.1% Tween-20/TBS 4-5 times for 10 minutes each following incubation with each antibody and bands were visualized with enhanced chemiluminescence. (Primary antibodies used include: anti-phospho-ERK1/2 at 1:1000 from Santa Cruz Biotechnology, Santa Cruz, CA; anti-collagen VI at 1:5000 from

Research Diagnostics, Inc.; α-SMA: mouse anti-α-smooth muscle actin at 1:2000, from

Sigma-Aldrich. The band intensity of the indicated proteins was quantified by densitometric scanning using a Kodak 1D Digital Science Imaging System.

Surgical procedures for infarct induction: Rats were administered butorphanol (10 mg/kg) and atropine (0.04 mg/kg, i.m.) 10 min before being placed in a flow-through chamber and exposed to 100% O2 - 4% isofluorane. The anesthetized rat was then intubated with a 14 g Venacath and ventilated with a Harvard Rodent Ventilator at a tidal volume and frequency of 2.6 ml and 74 breaths/min, respectively, anesthesia maintained with 0.5-1.5% isofluorane (oxygen flow of 700 ml/min). The heart was exposed via a left thoracotomy between the 4th and 5th ribs and the left coronary artery ligated 1-2 mm ventral to the left atrial margin with a 6-0 Prolene suture. After coronary ligation, lungs were hyperinflated with 10 mL of oxygen, and the chest closed with a 2-0 cat gut suture.

Xylocaine (1%, 0.2 mL) was injected into the surgical site to control arrhythmias and to relieve pain. Muscle and skin were closed in layers with a 4-0 vicryl suture. Immediately

after surgery, rats were given buprenorphine (BP, 0.03 mg/kg) for pain and normal saline

(10 ml) s.c. for volume replacement. BP was administered twice more for pain at 12 hr intervals. This animal protocol was approved by the NEOUCOM Institutional Animal

Care and Use Committee.

Tissue procurement, histology, immunohistochemistry and immunoblotting: Animals were sacrificed at 20 weeks following infarction. The atria were excised and the ventricular heart tissue was rinsed twice in ice-cold PBS, fixed in 4% paraformaldehyde for 30 minutes on ice, rinsed again, incubated in 7% sucrose at four degrees for a minimum of 4 hours, and frozen in Tissue-Tek tissue freezing medium (Miles, Inc,

Elkhart, IN). Transverse sections were cut from both the infarcted and non-infarcted

(taken from the anterior wall adjacent to the infarct) zones of the heart at a thickness of

10 micrometers on a Leica cryostat and placed on albumin-coated slides.

Histology: Trichrome staining was carried out according to Masson’s Trichrome method (Manual of Histologic Staining Methods of the Armed Forces Institute of

Pathology) and utilized to examine tissue morphology and composition by staining cell cytoplasm red, cell nuclei purple and collagen (non-specific) blue.

Immunohistochemistry: Slides for immunostaining were blocked in 2% goat serum for 1 hour at room temperature (RT), incubated in the primary antibody (type VI collagen: rabbit anti-collagen VI at a 1:100 dilution, obtained from Research Diagnostics,

Inc.; α-SMA: mouse anti-α-smooth muscle actin at 1:400, from Sigma-Aldrich; rabbit anti-desmin at 1:100, a gift from Dr. Carole Moncman (Moncman et al., 1996)) for 1-2 hours at (RT) and then washed with 0.1% Tween/PBS extensively for 1 hour. Slides

were then incubated with secondary antibodies, either Alexa Fluor 488 (Green and Clark,

1982) or Alexa Fluor 568 (Koshy et al., 2003), at a dilution of 1:200 for 1 hour at RT prior to mounting in Vectashield Mounting Medium (Vector Laboratories) containing

DAPI for nuclear visualization.

Immunoblotting: Heart tissue was rinsed twice in ice-cold PBS and the infarct region separated from the non-infarcted region. Tissue samples were minced, placed in lysis buffer and incubated on ice for 15 minutes prior to homogenization with a polytron.

This process of homogenization was repeated two more times. Samples were centrifuged at 4500 g for 10 minutes and the supernatant removed and frozen at -20°C. Western blot analysis was carried out as described above.

RESULTS

The type VI collagen substrate markedly induces myofibroblast differentiation in vitro:

Cardiac fibroblasts were plated onto glass slides or tissue culture plates coated with collagen I, III, or VI, and myofibroblast differentiation (as assessed by α-SMA protein expression and stress fiber formation) was examined via immunofluorescence. CFs plated on the type I collagen substrate (Figure 18, panel A) displayed low levels of α-SMA immunostaining which was similar to the level seen in CFs plated on glass (not shown).

The type III collagen substrate caused a slight induction of α-SMA expression (Figure

18, panel B). However, the cells plated on the type VI collagen substrate exhibited a marked elevation of α-SMA and highly organized stress fibers (Figure 18C). The key characteristic of mature myofibroblasts is the organization of α-SMA into stress fibers

Figure 18. Type VI collagen induces cardiac myofibroblast differentiation. A-F) Glass slides were coated with 20 μg/ml of type I, III, or VI collagen. Cardiac fibroblasts were plated at a density of 10,000 cells per dot (28.3 mm2) in serum-free media either on glass or the indicated collagen substrates for 24 hours. CFs were treated with 100 nM ANG II 24 hours after plating and remained in culture for a total of 48 hours. α-SMA expression was detected via immunofluorescence; nuclei were DAPI stained (blue fluorescence) and α-SMA was identified by Alexa Fluor 488 (green fluorescence). Photographs are representative of three similar experiments using cells from separate rats. Secondary antibody alone controls were also performed and negligible staining was observed.

(Villarreal, 1998), which was most evident on the type VI collagen substrate. ANG II induced a slight increase in α-SMA expression in CFs plated on the type I and III collagen substrates compared to the substrates alone (Figure 18D, E). However, ANG II treatment did not further induce α-SMA expression in CFs over that seen with the type

VI collagen substrate alone (Figure 18F), indicating that type VI by itself maximally induced the in vitro differentiation of CFs to myofibroblasts. The above results were confirmed via Western blotting for α-SMA. Treatment with ANG II increased expression of α-SMA on type I collagen by 7% (over collagen type I alone) and 82% on type III collagen (over collagen III alone) as assessed by Western blotting.

Type I and III collagen stimulate cardiac fibroblast proliferation via distinct mechanisms: Assessment of cell proliferation was carried out via BrdU incorporation in fibroblasts plated on collagen types I, III or VI substrates. Administration of ANG II increased proliferation by 157.3 ± 8.6% of basal levels (Figure 19, panel A) after 24 hours. The types I and III collagen substrates alone markedly increased proliferation of

CFs by 240.7 ± 10.3% and 271.7 ± 21.8% of basal levels, respectively. The type VI collagen substrate induced proliferation to a level similar to that of ANG II (143.3 ±

13.6% of basal), but not to the extent stimulated by the types I and III substrates.

Stimulation by ANG II enhanced CF proliferation on the types I and III substrates in an additive manner, but failed to enhance mitogenesis on the type VI substrate. Since

ERK1/2 is a major mitogenic signal in CFs, we measured ERK1/2 activation in response to the collagen substrates. Collagen I alone significantly increased ERK1/2 activity 5.7 ±

2.4 fold over control (p<0.05, Figures 19B and D), whereas collagen III and VI caused no

Figure 19. Collagen substrates and ANG II treatment differentially affect cardiac fibroblast proliferation. A) Cardiac fibroblasts were grown on 96-well plates pre-coated with 20 mg/ml of collagen I, III, or VI in the presence or absence of 100 nM ANG II for 24 hours. To assess DNA synthesis, 10 mmol/L 5-bromo-2’-deoxyuridine (BrdU) was included during the growth period, and incorporation was assessed by a BrdU incorporation assay and spectrophotometry. Data are expressed as a percent of basal ± SEM and are representative of three similar observations in cells taken from separate rats. *Statistically significant vs basal, p<0.001. †Statistically significant vs collagen I substrate alone, p<0.01. B and C) CFs were plated on either collagen I, III, or VI substrates or tissue culture plates. Cells were seeded in 10% serum for 24 hours and then incubated without serum for another 24 hours. Where indicated, CFs were stimulated with 100 nM ANG II for 10 minutes and whole cell protein lysates were collected and subjected to SDS-PAGE and Western blot analysis. Blots were probed for phospho- ERK1/2, stripped and reprobed for total ERK1/2 as a protein loading control. Blots are representative of either 3 (panel B) or 2 (panel C) similar experiments. D) Densitometric analysis was performed on the blot from panel B and normalized to total ERK. Data are expressed as the mean fold change ± SEM over controls. *Statistically significant vs. control by one-way ANOVA (p < 0.05). E ) Densitometric analysis was carried out on the blot from panel C and normalized to total ERK, with the data expressed as the mean fold change ± SEM relative to controls.

ANG

or little change in ERK phosphorylation. We next assessed ANG II-induced ERK activation on each collagen substrate (Figures 19C and E). ANG II on plastic had no effect on ERK activation. Following a 10 minute treatment with ANG II, ERK activation was enhanced in CFs plated on collagen I and III substrates by 1.3 ± 0.2 and 1.3 ± 0.3 fold, respectively, over ANG II alone. In contrast, ANG II did not induce ERK phosphorylation over control when CFs were plated on collagen VI, indicating that type

VI collagen in combination with ANG II does not induce proliferation via ERK activation.

Isolated cardiac fibroblasts produce type VI collagen in response to ANG II: We next aimed to confirm that isolated CFs are an inducible source of collagen VI in vitro.

Unstimulated CFs produced type VI collagen (Figure 20, panel A), and ANG II treatment induced an elevation of type VI protein expression (Figure 20B), as indicated by Alexa

Fluor 488 fluorescence. Analysis by Western blotting confirmed that collagen VI protein was expressed by unstimulated CFs (Figure 20C, lanes 1, 2), and production was enhanced by stimulation with ANG II by 3.0 ± 0.4 fold (Figure 20C, lanes 3, 4). Western blot assessment for total ERK served to demonstrate equal loading of protein samples

(Figure 20D). Thus, CFs produce significant amounts of hormonally inducible type VI collagen.

Type VI collagen is elevated in vivo following myocardial infarction: To determine the morphology and collagen content of the myocardium following a 20-week ligation of the left anterior descending coronary artery, the infarcted heart was separated into non- infarcted and infarcted regions, and trichrome staining was carried out on 10 mm thick

Figure 20. Treatment with ANG II induces type VI collagen expression. A-B) Cardiac fibroblasts were plated on glass slides in serum-free media for 24 hours. Cells were either stimulated with 100 nM ANG II (B) or remained untreated for another 24 hours (A) prior to fixation. CFs were stained for type VI collagen, mounted, and visualized under a 40X fluorescence objective. C) CFs were plated on tissue culture plates and treated with 100nM ANG II for 24 hours. Protein lysates were collected and samples were electrophoresed in duplicate and immunoblotted using an anti-collagen type VI antibody. D) Blots were additionally stripped and re-probed for total ERK as a loading control. The bands were visualized with ECL and the image is representative of three similar experiments.

transverse cryosections. The non-infarcted region of the ligated hearts exhibited an organized staining pattern of muscle tissue (red: muscle and cytoplasm; purple: nuclei) with little evidence of collagen (blue) deposition (Figure 21A); conversely, trichrome staining in the infarcted region indicated a disrupted network of cells, along with increases in total collagen (Figure 21B). The infarcted region appeared to have some dead or dying cells, an expected result due to the extent of the damage that was incurred in this area. These data confirmed successful induction of fibrosis via coronary ligation and prompted us to investigate the specific collagen subtypes involved in pathological remodeling.

We next quantitated the region-specific type VI collagen expression in the infarction model. The age-matched control hearts (Figure 22, panels A and D, lane 1) contained low, but observable, levels of type VI collagen. Immunohistochemisty revealed a modest increase in type VI collagen in the non-infarcted region (22B) and a marked elevation in the infarcted region (Figure 22C). Quantitative Western analysis determined these increases in type VI collagen to be 4.5 fold and 17 fold over control for the non- infarcted (Figure 22D lane 2) and infarcted regions (Figure 22D lane 3) of this heart, respectively. Thus, the remodeling that occurs post-MI leads to interstitial fibrosis and elevation of type VI collagen throughout the myocardium, with the highest levels being evident in the infarcted region.

Myofibroblast differentiation is enhanced in the infarcted myocardium: Our next goal was to determine whether myofibroblast content increased concurrently with type VI collagen in the infarcted rat myocardium. α-SMA staining was observed in the

Figure 21. Coronary ligation in rats induces a collagen-rich infarcted myocardium. Myocardial infarction was induced in adult male rats via permanent occlusion of the left anterior decending coronary artery. Ten micrometer thick cryosections were obtained and mounted on albumin-coated glass slides. Masson’s trichrome staining was performed on the infarcted hearts (each section included an infarcted region and the adjacent non- infarcted region). A) The non-infarcted region contains some collagen (blue staining), yet still contains a viable network of muscle cells. B) The infarcted region includes a large amount of collagen, yet lacks an organized muscle cell structure.

A. Non-Infarct Region B. Infarct Region

endomysium of the non-infarcted tissue region (Figure 23, panel A). Both the staining intensity and pattern was altered in the infarcted region (Figure 23B); a more punctate cellular staining pattern, which was of the approximate size of fibroblasts and myofibroblasts, was evident throughout the infarcted tissue. To rule out the possibility that the increase α-SMA expression in the infarcted myocardium was due to the presence of vascular smooth muscle cells (VSMCs), we measured levels of the intermediate filament desmin (Figure 23C), which is expressed in VSMCs but not myofibroblasts

(Wang et al., 2003). Figure 23B and C contain the same field of tissue, and demonstrate an elevation of α-SMA in the infarcted region (Figure 23B), whereas only a minimal amount of desmin staining was evident (Figure 23C). Co-expression of α-SMA and desmin in VSMCs in a blood vessel is presented in Figure 23D and E (see white arrows for location), confirming reliability of the antibodies. Western blot analysis validated the immunostaining results: the age-matched control heart (Figure 23H, lanes 1, 2) exhibited low levels of α-SMA expression, whereas both the non-infarcted (Figure 23H, lanes 3, 4) and infarcted regions (Figure 23H, lanes 5, 6) contained elevated α-SMA levels (1.4 ±

0.2 and 2.7 ± 0.2 fold over control, respectively). In addition, demonstrates that expression of desmin did not vary across the indicated conditions (Figure 23I). Overall, myofibroblast content was highest in the infarcted myocardium, and our data demonstrate that these hypersecretory cells remain in the injured myocardium for at least 20 weeks post-infarction.

Figure 22. Type VI collagen is elevated following post-myocardial infarction remodeling. A-C) Rat hearts were extracted and ventricles removed. The ventricular tissue was fixed, frozen, and 10 μm cryosections were cut (each section included an infarcted region and the adjacent non-infarcted region) and mounted on albumin-coated slides. Tissue sections were stained for type VI collagen, and age-matched control hearts (A) were compared to the non-infarcted (B) and infarcted region (C). Photographs were taken with identical exposure times and aperature settings, and are representative of three similar experiments taken from 3 separate rats. Appropriate secondary antibody alone controls were also performed using Alexa Fluor 568 and confirmed specificity of the antibody (data not shown). D) Infarcted hearts were obtained as described above, and the non-infarcted region was dissected from the infarcted region. Whole tissue protein lysates were isolated and homogenized by a 10 second burst with a polytron, from both the posterior wall (non-infarcted region) and anterior wall (infarcted region). These samples (15 μg each)were subjected to SDS-PAGE and Western blotting for type VI collagen. Each lane represents a different tissue sample. Lane 1: age-matched control, lane 2: non-infarcted region, lane 3: infarcted region.

DISCUSSION

Our studies demonstrated that the type VI collagen substrate was a potent inducer of myofibroblast differentiation in vitro, whereas the types I and III collagen substrates alone caused significant CF mitogenesis. Treatment of cells plated on each collagen substrate in combination with ANG II caused a modest additive effect (over substrate alone) in the case of collagen I and III, an effect that was not apparent in the type VI collagen substrate group. The differential effects that these collagen types have on CF proliferation and differentiation is supported by the idea that at any given time most cells are capable of either proliferation or differentiation (but not both), a fate that depends upon the regulatory factors present (Grotendorst et al., 2004). Thus, it is important to note that in our study types I and III collagen, the most abundant types in the heart, appear to drive proliferation of undifferentiated cardiac fibroblasts and are poor inducers of myofibroblast differentiation. On the other hand, type VI collagen augments differentiation to a greater extent than cell division. To gain a better understanding of the signaling mechanism responsible for proliferation, we examined ERK1/2 activation, which has been shown to be a critical signal underlying hormone-stimulated CF proliferation (Schorb et al., 1995; Kim et al., 2002; Stockand and Meszaros, 2002). The type VI collagen substrate both alone and in combination with ANG II was a poor activator of ERK1/2, which is consistent with our observations, indicating that type VI collagen preferentially induces myofibroblast differentiation over proliferation.

Figure 23. Enhanced myofibroblast content in the infarcted rat myocardium. Ventricular tissue was obtained as described in Figure 21, and the non-infarcted (A) and infarcted regions (B) were stained for α-SMA. The infarcted rat tissue was also stained with desmin (C). Positive staining was detected for both α-SMA (D) and desmin (E) in a blood vessel wall (white arrows denote co-expression). Alexa Fluor 568 (F) and Alexa Fluor 488 (G) alone displayed minimal background fluorescence. All photographs were taken with identical exposure times. H-I) Protein lysates were extracted from both age- matched control and infarcted hearts, and separated by SDS-PAGE. Western blotting was performed using specific antibodies against α-SMA (H) or desmin (I). The image is representative of three separate experiments from three different animals.

The various factors that control transformation of cardiac fibroblasts to myofibroblasts are not fully understood. However, specific hormones have been shown to induce fibroblast differentiation, and inhibiting the actions of these hormones has proven to be effective in limiting fibrosis. In pressure overloaded rats, inhibition of transforming growth factor-β with neutralizing antibodies resulted in decreased types I and III collagen mRNA transcription, myofibroblast number and myocardial fibrosis (Kuwahara et al.,

2002). Their study demonstrated that anti-TGF-β antibodies inhibited both proliferation and differentiation of cardiac fibroblasts and abolished the increase in left ventricular end-diastolic pressure and the ratio of early to late filling velocity, both of which are direct measures of diastolic cardiac function. In addition to hormonal factors, our data demonstrate that specific extracellular matrix proteins may play critical roles in promoting myofibroblast differentiation. Therefore, targeting ECM-induced signal transduction may be an alternative approach to effectively reduce cardiac fibrosis by preventing excessive myofibroblast activity and/or differentiation.

Several studies have been performed in non-cardiac cells that provide insight into how myofibroblast differentiation occurs. Differentiation of corneal fibroblasts to myofibroblasts is induced by the type VI collagen substrate, which appears to be dependent upon the β1 integrin receptor (Doane, unpublished observations). In addition, the type VI collagen substrate also reduces apoptosis in these cells (Howell and Doane,

1998). Faouzi et al. (1999) presented evidence of significant elevations of type VI and type IV collagen in both hepatic carcinoma tissue and the cells isolated from these tumors. In their study, the rise in both collagen isotypes was accompanied by an increase

in myofibroblasts, rather than endothelial cells, although there was no discussion or speculation as to a potential causative link between type VI collagen and myofibroblast differentiation (Faouzi et al., 1999). A coexistence of type VI collagen and myofibroblasts was also reported following renal injury. Expression of type VI collagen was demonstrated to be significantly elevated in diabetic glomeruli, as well as in areas of renal fibrotic injury (Groma, 1998). Increased amounts of α-SMA expressing cells were evident in renal fibrotic interstitium, along with elevated type VI collagen. Given the above studies, the coexistence of type VI collagen and myofibroblast differentiation suggests a potential link between this specific collagen type and the process of myofibroblast differentiation.

The mechanism by which type VI collagen induces myofibroblast differentiation has not currently been established, however, there are several possibilities that require further investigation. Integrin receptors, which are heterodimers composed of one α and one β subunit, are responsible for mediating intracellular signaling in response to many types of collagen, as well as other ECM proteins. Specifically, α3β1 and the integral membrane proteoglycan NG2 on corneal fibroblasts have been identified as receptors that associate with type VI collagen during development (Doane et al., 1998). More recently, a study by Bouzeghrane et al. demonstrated upregulation of α8β1 in myofibroblasts present in the fibrotic myocardium (Bouzeghrane et al., 2004). In addition, evidence exists showing that the αv subunit (more specifically αvβ3 and αvβ5) is critically involved in myofibroblast differentiation in fibroblast cell lines (Lygoe et al., 2004).

Thus, several avenues exist to explore the mechanism by which type VI collagen induces cardiac myofibroblast differentiation and will be the focus of our future studies.

Evidence from two clinical studies indicates that type VI collagen is elevated during hypertension, heart failure and diabetes, conditions in which cardiac fibrosis is prevalent and accelerated (Spiro and Crowley, 1993; Kitamura et al., 2001). However, the possibility that type VI collagen might actually drive the fibrotic process was not considered. We postulated that diseases which increase type VI collagen deposition creates conditions favorable for myofibroblast differentiation, and that these differentiated cells are the major players in both normal and pathological remodeling. In the current study, we demonstrate that type VI collagen expression is elevated during post-MI remodeling, which is accompanied by an increase in myofibroblast content in the damaged myocardium. Our data support a novel role for type VI collagen in post-MI remodeling by augmentation of myofibroblast differentiation (see Figure 24).

Understanding the mechanisms by which type VI collagen enhances myofibroblast differentiation may provide a basis to limit excessive myofibroblast activity and pathological fibrosis following myocardial infarction.

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Figure 24. Effects of the ECM on CF activation and the progression of cardiac fibrosis. During hypertension, MI, or heart failure, CFs become activated by either proliferating or differentiating into myofibroblasts. When activated, CF collagen production increases, and if these cells become overactive, excess matrix deposition can lead to cardiac fibrosis. Types I and III collagen promote CF proliferation, whereas type VI collagen is a potent inducer of myofibroblast differentiation, both of which can lead to production of collagen.

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CHAPTER FOUR

TEMPORAL CHANGES IN TYPE VI COLLAGEN, MYOFIBROBLAST CONTENT, AND INTEGRIN RECEPTORS POST-MYOCARDIAL INFARCTION IN RATS

INTRODUCTION

Myocardial infarction (MI) is an acute injury to the myocardium, however, the effects of this event can persist for years following the initial insult. Cardiac fibrosis is the result of excessive extracellular matrix (ECM) production, and typically occurs subsequent to

MI or hypertension (Manabe et al., 2002). Cardiac myofibroblasts are the primary cell type responsible for wound healing, and they play a major role in the overproduction of

ECM proteins and cardiac fibrosis (Weber et al., 1994; Faouzi et al., 1999; Chai et al.,

2003). We have recently demonstrated that type VI collagen is significantly elevated 20 weeks post-MI concurrent with an increase in myofibroblast content. In addition, type VI collagen has the ability to induce cardiac fibroblast differentiation to myofibroblasts in vitro (Naugle et al., 2006). Thus, the goal of this study was to investigate the temporal changes in type VI collagen, myofibroblast content, and integrin receptor expression at 3,

7, and 14 days post-MI to gain insight into whether type VI collagen could affect myofibroblast differentiation in the in vivo setting.

A number of studies have demonstrated that both types I and III collagen are elevated post-MI and contribute significantly to the observed cardiac fibrosis (Cleutjens et al.,

1995; Cleutjens et al., 1995; Sun et al., 2000; Manabe et al., 2002). However, relatively

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few investigations have focused on the other collagen types in the heart. Type VI collagen is present in the adult myocardium, but it is a non-fibrillar collagen that is structurally distinct from the fibrillar collagens I and III. Fibrillar collagen is composed of three α chains wound into a triple helix, and these collagens assemble into highly cross- linked fibrils (Gonzalez et al., 2002; Bosman and Stamenkovic, 2003). Type VI collagen contains a very small triple helical domain, with large globular proteins on both the carboxy- and amino-terminal ends, and does not assemble into fibrils (Bashey et al.,

1992; Pfaff et al., 1993). The structural differences among these collagens imply that they possess different functions. We and others have demonstrated that type VI collagen is elevated in specific cardiac pathologies such as MI, hypertension, and hypertrophic cardiomyopathy (Spiro and Crowley, 1993; Kitamura et al., 2001; Naugle et al., 2006).

Our previously published data suggest that type VI collagen participates in remodeling by promoting the differentiation of CFs to myofibroblasts.

Myofibroblasts are a key cell type involved in cardiac remodeling; their main function is healing the wound and secreting large amounts of ECM to stabilize the wound by creating an infarct scar. Typically, once their wound healing responsibilities are complete, myofibroblasts undergo apoptosis, and the resident fibroblasts then take over maintenance of the ECM (Tomasek et al., 2002). However, when myofibroblasts persist, this chronic hypersecretion of ECM proteins leads to fibrosis. In human myocardial scars, it has been demonstrated that myofibroblasts persist for up to 17 years post-infarction

(Willems et al., 1994). In rats, myofibroblasts appear by day 3, and are present through day 28 post-MI (Sun et al., 2000); however, in mice, proliferation of myofibroblasts

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peaks at day 4, and returns to control levels by day 14 post-MI (Virag and Murry, 2003).

The temporal appearance of myofibroblasts post-MI differs depending on the animal model used, so it is important to thoroughly investigate this event under highly consistent conditions. Much remains to be learned regarding the cellular and structural mediators of in vivo myofibroblast differentiation.

Collagens and other ECM proteins can initiate intracellular signaling events via binding to integrin receptors. Integrin receptors, located in focal adhesions (areas of the membrane with clusters of integrin receptors bound to the ECM), contain both α and β subunits that dimerize and bind to specific ECM proteins (Hynes 2002; Sepulveda et al.,

2005). Type VI collagen has been shown to interact with the α3β1 integrin receptor in corneal fibroblasts (Doane et al., 1998), and the αv integrin subunit has been shown to be critical in the process of differentiation to myofibroblasts in three different fibroblast cell lines (Lygoe et al., 2004). A key initial step to elucidate mechanisms by which the ECM affects CF function is to measure the temporal expression of type VI collagen and specific integrins to determine a possible correlation with myofibroblast differentiation during post-MI remodeling.

METHODS

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volume and frequency of 2.6 ml and 74 breaths/min, respectively, anesthesia maintained with 0.5-1.5% isofluorane (oxygen flow of 700 ml/min). The heart was exposed via a left thoracotomy between the 4th and 5th ribs and the left coronary artery ligated 1-2 mm ventral to the left atrial margin with a 6-0 Prolene suture. After coronary ligation, lungs were hyperinflated with 10 mL of oxygen, and the chest closed with a 2-0 cat gut suture.

Xylocaine (1%, 0.2 mL) was injected into the surgical site to control arrhythmias and to relieve pain. Muscle and skin were closed in layers with a 4-0 vicryl suture. Immediately after surgery, rats were given buprenorphine (BP, 0.03 mg/kg) for pain and normal saline

(10 ml) s.c. for volume replacement. BP was administered twice more for pain at 12 hr intervals. This animal protocol was approved by the NEOUCOM Institutional Animal

Care and Use Committee.

Tissue procurement, immunohistochemistry, and immunoblotting: Animals were sacrificed at 3 days, 7 days, 14 days, and 16 weeks following infarction. The atria were excised and the ventricular heart tissue was rinsed twice in ice-cold PBS, fixed in 4% paraformaldehyde for 30 minutes on ice, rinsed again, incubated in 7% sucrose at 4 °C for a minimum of 4 hours, and frozen in Tissue-Tek tissue freezing medium (Miles, Inc,

Elkhart, IN). Transverse sections were cut including both the infarcted and non-infarcted

(taken from the posterior wall) zones of the heart at a thickness of 10 μm on a Leica cryostat and placed on albumin-coated slides.

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Inc.; α-SMA: mouse anti-α-smooth muscle actin at 1:400, from Sigma-Aldrich; α3 integrin: mouse anti-α3 integrin at 1:200 dilution, from Chemicon) for 1-2 hours at RT and then washed extensively in 0.1% Tween/PBS for 1 hour. Slides were then incubated with secondary antibodies, either Alexa Fluor 488 (Green and Clark, 1982) or Alexa

Fluor 568 (Koshy et al., 2003), were incubated at a dilution of 1:200 for 1 hour at RT prior to mounting in Vectashield Mounting Medium (Vector Laboratories) containing

DAPI for nuclear visualization.

Cross-linking assay: Cardiac fibroblasts were isolated as described in Chapter 2, and plated on dishes coated with 20 μg/ml of purified collagen I, III, VI, or vehicle alone.

Cells were washed three times with PBS and then incubated in 0.4 mM 3,3’-Dithio bis

(sulfo-succinimidylpropionate) (DTSSP; a cross-linker) for 10 minutes. The purpose of the cross-linker is to link extracellular matrix to the cell surface receptors to which they are bound (which isolates integrins in focal adhesions bound to the ECM). Cardiac fibroblasts were rinsed once in 10 mM Tris-PBS for 2 minutes, rinsed two times with

PBS and then incubated in 0.5 ml of lysis buffer (as described in Chapter 3) for 10 minutes. After dissolving away the cells, lysis buffer was aspirated and cells rinsed with

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PBS two times. 30μL of lysis buffer was added to each 60 mm dish, and remaining protein (in focal adhesions) was scraped and collected. Protein lysates were collected and subjected to SDS-PAGE and Western blot analysis. Blots were probed for α3 integrin to demonstrate interaction with the indicated collagens.

RESULTS

Type VI collagen is elevated 7 and 14 days post-MI: Whole tissue lysates were obtained from the infarcted (anterior wall) and non-infarcted (posterior wall) regions of the heart, as well as from the same regions of the sham operated control hearts. At 3 days (3D) post-MI (Figure 25A), there were no significant changes in the levels of type VI collagen, however at 7 days (Figure 25D) post-MI type VI collagen levels were significantly elevated in both the non-infarcted and infarcted regions when compared to the sham operated control (Figure 25B). Collagen VI remained significantly elevated at 14 days

(14D) post-MI in both the non-infarcted and infarcted regions (Figure 25C).

Myofibroblast content is elevated 7 days post-MI: Due to our previous evidence that type VI collagen and myofibroblast content are concurrently elevated in the 20 week post-MI heart (Naugle et al., 2006), we next wanted to determine if myofibroblasts

(measured by α-SMA expression) were elevated 3, 7, or 14 days post-MI. Three days following MI, there were no significant changes in α-SMA levels (Figure 26A). At 7 days post-MI, α-SMA levels dramatically increased in both the non-infarcted and infarcted regions (Figure 26B), although by day 14 the α-SMA levels returned to that of the sham controls (Figure 26C). Thus, the α-SMA expression exhibits a biphasic pattern

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Figure 25. Type VI collagen is elevated 7 and 14 days post-myocardial infarction. Myocardial infarction (MI) in rats was induced by ligating the left anterior descending coronary artery. This model produced an infarcted left ventricle and significant fibrosis. At 3, 7, and 14 days post-MI, rat hearts were extracted and ventricles removed. The non- infarcted region was dissected from the infarcted region. Whole tissue protein lysates were isolated and homogenized by a 10 second burst with a polytron, from both the posterior wall (non-infarcted region) and anterior wall (infarcted region) of the left ventricle. These samples were subjected to SDS-PAGE and Western blotting for type VI collagen. Above are representative Western blots, and below are summary graphs of results from at least 4 rats for each condition. Type VI collagen is represented by two bands on the Western blots (arrows), one at 200 kD and one at 160 kD. Densitometry was performed on the 160 kD band. *Indicates statistical significance versus sham, p<0.05; #p<0.001 versus sham; †p<0.001 versus non-infarcted region.

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Shams Shams Non-Inf Inf Inf Non-Inf Non-Inf Inf Inf A. Non-Inf B. 3D 7D

3.0 3.0 #†

2.0 2.0 *

1.0 1.0 Fold Change Fold Change 0.0 0.0 3D Sham 3D Non-Inf 3D Inf 7D Sham 7D Non-Inf 7D Inf

Shams Non-Inf Inf Inf C. Non-Inf 14D

* 3.0 * 2.0

1.0

Fold Change 0.0 14D Sham 14D Non-Inf 14D Inf

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of expression—it is elevated at 7 days post-MI, decreased at day 14, and then significantly increased again at a longer time-point of 20 weeks post-MI.

αv integrin levels do not change within two weeks post-MI: MI was induced as described above, and the levels of αv integrin were analyzed via Western blotting at 3, 7, and 14 days post-MI. There were no significant differences between the infarcted hearts and the sham controls at any of the time-points examined (Figure 27A, B, C).

Types VI and III collagen interact with the α3 integrin receptor in cardiac fibroblasts:

Cardiac fibroblasts were plated overnight on purified collagens I, III, VI, or vehicle treated control. Focal adhesions were isolated using a cross-linking agent, and the cells were dissolved away prior to lysis and scraping of the focal adhesion proteins (see methods section for further detail). The α3 integrin receptor in cardiac fibroblasts bound to both types III and VI collagen, but not to collagen I (Figure 28). Since type VI collagen is elevated post-MI and interacts with the α3 integrin receptor in vitro, our next goal was to determine the post-MI expression levels of α3 integrin.

α3 integrin receptors are elevated 3 days post-MI: The α3 integrin receptors were elevated in both the non-infarcted and infarcted regions at 3 and 7 days post-MI, with the

3 days expression reaching statistical significance (Figure 29A and B). By 14 days post-

MI, the levels of α3 integrin had returned to the level of the sham controls (Figure 29C).

Since we know that both type VI collagen and α-SMA levels are elevated at a longer time-point of 16-20 weeks, we next measured the levels of α3 integrin at 16 weeks

(16wk) post-MI. α3 integrin was slightly elevated in the non-infarcted region, and

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Figure 26. Myofibroblast content is significantly increased by 7 days post-MI. MI in rats was induced as described in Figure 24. The non-infarcted region was dissected from the infarcted region. Whole tissue protein lysates were isolated and these samples were subjected to SDS-PAGE and Western blotting for α-SMA (the marker for myofibroblasts). Above are representative Western blots, and below are summary graphs including at least n=4 for each condition. *Indicates statistical significance versus sham, p<0.05; #p<0.001 versus sham; †p<0.01 versus non-infarcted region.

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Shams Shams Inf Non-Inf Inf Inf Non-Inf Inf Non-Inf A. Non-Inf B. 3D 7D

6 ###† 6 5 5 4 4 3 3 * 2 2

1 1 Fold Change Fold Fold Change Fold 0 0 3Day Sham 3D Non-Inf 3D Inf 7D Sham 7D Non-Inf 7D Inf

Shams Non-Inf Inf Non-Inf C. Inf 14D

1 Fold Change 0 14D Sham 14D Non-Inf 14D Inf

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significantly increased in the infarcted region 16 weeks post-MI (Figure 29D). The pattern of expression of this particular integrin receptor is biphasic and similar to, but preceding that of myofibroblast appearance in the remodeling myocardium.

DISCUSSION

The goal of this study was to investigate the changes in type VI collagen and myofibroblast content within two weeks following MI, as well as to identify changes in the levels of integrin receptors that interact with type VI collagen. Concurrent elevation of type VI collagen and myofibroblast content would suggest a potential role for type VI collagen in promoting in vivo myofibroblast differentiation. MI induces cardiac remodeling which causes changes in the ECM composition, with the historical focus being on types I and III collagen. In normal adult myocardium, types I and III compose most of the collagen network, with types IV, V, and VI collagen making up the remainder

(Borg et al., 1982; Eghbali et al., 1989; Weber et al., 1994; Agocha et al., 1997;

Heeneman et al., 2003). Subsequent to MI, collagens I and III increase and are responsible for interstitial, perivascular, and scar fibrosis (Manabe et al., 2002). Although collagen VI has previously been thought of as a minor ECM component in the myocardium, a few studies have suggested an important role for type VI collagen in cardiac pathologies. Type VI collagen is elevated in hypertensive and diabetic rat models

(Spiro and Crowley, 1993), as well as in human hypertrophic cardiomyopathy (Kitamura et al., 2001). We have previously demonstrated that type VI collagen is significantly increased 20 weeks post-MI in the rat (Naugle et al., 2006). The current study

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Figure 27. αv integrin receptor subunit levels do not change within two weeks post-MI. MI in rats was induced as described in Figure 24. The non-infarcted region was dissected from the infarcted region. Whole tissue protein lysates were isolated and these samples were subjected to SDS-PAGE and Western blotting for αv integrin. Panels A, B, and C show representative Western blots from 3, 7, and 14 days post-MI, respectively. The lower band represents the αv subunit alone, whereas the higher band represents αv complexed with most likely a β subunit.

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Shams Shams A B Inf Non-Inf Non-Inf Non-Inf Inf Inf Inf Non-Inf 3 D αv complex 7 D αv complex

αv subunit αv subunit

Shams Non-Inf Inf

C Inf Non-Inf 14 D αv complex

αv subunit

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demonstrates that collagen VI is significantly elevated at both 7 and 14 days post-MI.

Likewise, myofibroblast content is significantly elevated 7 days following MI, but in contrast to type VI collagen returns to control levels by day 14. Cardiac fibroblasts have been reported to express the α3 integrin receptor (Thibault et al., 2001), and our cross- linking data demonstrates that it interacts with types III and VI collagen. This receptor is significantly increased 3 days following MI and returns to control levels by day 14. α3 integrin is elevated again at 16 weeks post-MI along with collagen VI and myofibroblast content, as previously demonstrated (Naugle et al., 2006). Thus, type VI collagen is consistently expressed during remodeling, whereas there is a biphasic pattern of appearance for myofibroblasts—an initial appearance one week post-MI, regression by two weeks, and re-appearance at longer remodeling time-points of 16 and 20 weeks.

Similarly, α3 integrin follows this biphasic pattern of appearance, with the initial elevation occurring by 3 days (see Figure 30).

We have previously demonstrated that collagen VI is a potent inducer of cardiac myofibroblast differentiation in vitro (Naugle et al., 2006), and this cell type is largely responsible for secreting the excess ECM which causes cardiac fibrosis. Myofibroblasts are the key cellular component of the wound healing process, and are essential for repair of the infarcted myocardium. It has been shown that in rats, by utilizing immunohistochemical techniques, myofibroblasts appear by day 3 following MI and remain in the myocardium through day 28. In this study, sham operated rat pericardial tissue also contained myofibroblasts at days 7, 14, and 28 post-MI (Sun et al., 2000). Our

Western blot studies revealed that myofibroblast content (α-SMA expression) was

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Figure 28. Collagen VI interacts with the α3 integrin receptor in focal adhesions. Cardiac fibroblasts were plated overnight on tissue culture plates pre-coated with 20 μg/ml of purified collagen I, III, or VI. Cells were washed three times with PBS and then incubated in 0.4 mM DTSSP (a cross-linker) for 10 minutes. The purpose of the cross- linker is to link extracellular matrix receptors to the matrix to which they are bound. Cardiac fibroblasts were rinsed once in 10 mM Tris-PBS for 2 minutes, rinsed two times with PBS and then incubated in 0.5 ml of lysis buffer for 10 minutes. After dissolving away the cells, lysis buffer was aspirated and cells rinsed with PBS two times. 30μL of lysis buffer was added to each 60 mm dish, and remaining protein (in focal adhesions) was scraped off the dish. Protein lysates were collected and subjected to SDS-PAGE and Western blot analysis. Blots were probed for α3 integrin to demonstrate interaction with the indicated collagens.

118 control type I collagen type III collagen type VI collagen

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significantly increased by day 7 post-MI, but returned to the sham control levels by day

14. The differences in experimental methods between studies are what most likely account for varying results. Western blotting is a more quantitative assay whereas immunohistochemistry is more qualitative. However, both studies indicate that myofibroblasts are abundant in the rat heart one week post-MI, and are a key cell type in the initial wound healing and remodeling process.

Integrin receptors in the heart are important mediators of ECM signaling in both myocytes and fibroblasts. Type VI collagen can potentially interact with the α1, α2, α3, and αv integrin receptors, the first three interact with native type VI collagen

(Jongewaard et al., 2002), whereas the latter binds to unfolded collagen VI (Davis, 1992).

The cross-linking studies performed on CFs in vitro indicated that α3 integrin was one key receptor that interacts with native type VI collagen. Our study indicates that while there are no changes in the αv integrin subunit within two weeks post-MI, the α3 integrin subunit was significantly elevated 3 days post-MI, returned to control levels by day 14, and was then increased again at 16 weeks. The post-MI α3 integrin receptor expression pattern is biphasic, similar to, but slightly preceeding that of myofibroblast appearance

(see Figure 30), which sets up the possibility that binding to the α3 integrin receptor could be promoting differentiation to myofibroblasts.

Overall, it is apparent that type VI collagen is elevated following MI, and the net accumulation of this collagen is still present 4-5 months post-MI. However, α-SMA and

α3 integrin exhibit a biphasic response to MI—and each phase could have distinct mechanisms of induction. The initial MI event induces a strong inflammatory response,

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Figure 29. α3 integrin is elevated 3 days and 16 weeks post-MI. MI in rats was induced as described in Figure 24. The non-infarcted region was dissected from the infarcted region and whole tissue protein lysates were isolated subjected to SDS-PAGE and Western blotting for α3 integrin. Above are representative Western blots, and below are summary graphs including at least n=4 for each condition. *Indicates statistical significance versus sham, p<0.05.

121

Shams Shams Inf Non-Inf Non-Inf Inf Non-Inf Inf A. Non-Inf Inf B. 3D 7D

3 3

* 2 2 *

1 1 Fold Change Fold Fold Change 0 0 3D Sham 3D Non-Inf 3D Inf 7D Sham 7D Non-Inf 7D Inf

Shams Shams Non-Inf Inf Inf Inf Non-Inf Non-Inf Non-Inf C. D. Inf 14D 16wk

4 * 3 3 2 2

1 1 Fold Change Fold

Fold Change Fold 0 0 16wk Con 16 wk Non-Inf 16 wk Inf 14D Sham 14D Non-Inf 14D Inf

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which includes elevation of the cytokine transforming growth factor-β (TGF-β) (Sun et al., 2000), which is a potent inducer of myofibroblast differentiation (Petrov et al., 2002).

TGF-β mRNA expression declines beginning 14 days post-MI (Sun et al., 2000), and likely does not play a significant role in cardiac remodeling 4-5 months post-MI. We hypothesize that the significant accumulation of type VI collagen by 4-5 months post-MI plays a potentially important role in the second phase of this biphasic response. Type VI collagen binds to the α3 integrin receptor, and has been shown to be a potent inducer of cardiac myofibroblast differentiation in vitro (Naugle et al., 2006). If type VI collagen plays a similar role in vivo, the continued presence of type VI collagen could be mediating the secondary phase of remodeling months following an MI, subsequent to the initial inflammatory response. This secondary phase could be potentially predisposing the heart to excess collagen production and fibrosis.

Determining the specific mechanisms involved in type VI collagen inducing myofibroblast differentiation post-MI is a critical part of understanding the remodeling process. Interactions among cardiac cells, the changing ECM composition, and the signaling that occurs between these cells and the ECM are all important targets for investigation and future therapies. Our data demonstrate a role for type VI collagen in short and long-term cardiac remodeling, as well as two distinct temporal phases of cardiac myofibroblast appearance. Taken together, we hypothesize that α3 integrin receptors in CFs bind to type VI collagen and potentially mediate responses to this and other collagens.

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Figure 30. Temporal changes in type VI collagen, myofibroblast content, and α3 integrin following MI. Understanding the remodeling changes that occur following MI is critical to gain insight into the progression of cardiac fibrosis. We’ve previously demonstrated that type VI collagen is elevated 20 weeks post-MI, and in this study we observe that collagen VI is elevated at 7 and 14 days post-MI as well. Myofibroblast content is increased by day 7 post-MI and was previously shown to be elevated 20 weeks post-MI. The α3 integrin receptor, which interacts with type VI collagen, is elevated at 3 days and 16 weeks post-MI.

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PROGRESSION OF CARDIAC FIBROSIS

↑ ↑

↑ ↑ ↑

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CHAPTER FIVE

DISCUSSION AND OVERALL CONCLUSIONS

Cardiovascular disease (CVD) is the number one killer in the United States and is responsible for one death every 35 seconds (Thom et al., 2006). Forms of CVD include hypertension, coronary artery disease, heart failure, and congenital defects. Hypertension and forms of coronary disease such as myocardial infarction (MI) typically lead to cardiac fibrosis, which is a secondary disease characterized by the accumulation of large amounts of ECM proteins, and can lead to eventual heart failure. Many therapies are aimed at alleviating symptoms of hypertension or MI, however, few are designed to target the fibrosis that predisposes the heart to failure. Medical therapies have been successful in prolonging human life during hypertension and following MI, however, understanding the longer-term effects of cardiac fibrosis is critical in treating heart failure.

Alterations in G-protein coupled receptor (GPCR) signaling occur in many diseases, including hypertension and heart failure. Our data demonstrate that ANG II enhances the β-AR signaling in CFs. Stimulation of the β-AR pathways leads to cAMP production, which has functional consequences of increasing proliferation (Kim et al.,

2002), decreasing collagen production (Ostrom et al., 2003), and inhibiting differentiation to myofibroblasts (Swaney et al., 2005). The augmentation of cAMP signaling by ANG II

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appears to have mostly anti-fibrotic effects via directly decreasing collagen secretion, and indirectly by inhibiting differentiation to the hypersecretory myofibroblast phenotype

(see Figure 31). Catecholamine levels become chronically elevated following an infarction and during heart failure, and this persistent activation of the β-ARs causes desensitization and downregulation of β-ARs in cardiac myocytes (Vatner et al., 1999;

Lamba and Abraham 2000). Whether or not this same paradigm of β-AR downregulation exists in CFs has yet to be determined, however, we have preliminary evidence suggesting that β-ARs downregulate in response to isoproterenol. In addition, infusion of

ANG II in rats can cause catecholamine production from cardiac sympathetic neurons, and this also promotes downregulation of cardiac β-ARs (Henegar et al., 1998). If these receptors are in fact downregulated in CFs, the ANG II-induced potentiation of the β-AR pathway and the anti-fibrotic effects of elevated cAMP would no longer be advantageous.

Thus, not only is the cellular signaling synergy important, but the microdomain within the cell in which the signaling takes place during cardiac pathologies also needs to be considered.

Chronic β-AR stimulation can be toxic to the myocardium, which is why there is an endogenous mechanism in place triggering downregulation of these receptors. β-AR antagonists such as carvedilol or metoprolol, have been successfully used to improve morbidity and mortality following infarction, as well as slowing the progression of heart failure. However, the longer term effects of these β-blockers on CFs have not been fully considered. The differences in β-AR subtype expression between cardiac myocytes and fibroblasts may be a key way to distinguish these cell types therapeutically, as myocytes

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express over 80% β1 receptors and the remainder are β2, whereas fibroblasts express predominantly β2 receptors (Lamba and Abraham, 2000; Turner et al., 2003). In general, it is thought that activation of β1 receptors leads to myocytes apoptosis, while stimulation of β2 receptors inhibits myoctye apoptosis. This theory is supported by studies from β-

AR knock out mice: knocking out β2-ARs led to increases in myocyte apoptosis, and knocking out β1-ARs enhanced myocyte viability (Fajardo et al., 2006). CF specific β-

AR stimulation via β2 receptors might impart anti-fibrotic effects, while simultaneous blockade of the β1 receptor in myocytes would limit muscle necrosis and lower the metabolic demands of the myocardium (Vatner et al., 1999; Zheng et al., 2004; Fajardo et al., 2006). Limiting collagen accumulation by regulating CF activation could also be achieved by overexpressing adenylyl cylase (AC) in CFs. In cardiac myocytes, AC5 and

6 are the predominant isoforms of this enzyme, acting to elevate cAMP, which results in positive inotropic effects. AC6 has been successfully overexpressed in cardiac myocytes using cardiac-directed expression in a model of cardiac hypertrophy (Gαq overexpressed mice). In this model, overexpression of AC6 restored myocyte responsiveness to β-AR stimulation by enhancing the production of cAMP, and led to enhanced cardiac function and increased lifespan of these animals. (Roth et al., 1999). Overexpression of AC could be achieved in CFs in vitro using an adenoviral delivery system (Ostrom et al., 2003), however, to test this hypothesis in vivo would most likely require a transgenic mouse overexpression model (Du et al., 2000) or an intracoronary adenoviral-mediated delivery system (Shah et al., 2000). By overexpressing AC3 (a Ca2+/calmodulin stimulated isoform) in CFs, ANG II could still exert its synergistic effects by directly stimulating

128

AC, which could diminish the problem of downregulated β-ARs. The end result would still be inhibition of myofibroblast differentiation and decreased collagen production.

Examining subcellular locations of specific signaling molecules is critical to determine their physiological relevance. β2-ARs are localized to caveolae (CAV) in both myocytes and fibroblasts, whereas β1-ARs are distributed between both CAV and non-CAV domains in myocytes. In neonatal cardiac myocytes, β2-AR signaling is dependent on receptor localization in CAV domains (Xiang et al., 2002). If the same is true for β2 receptors in CFs, then targeting signaling molecules in this pathway might require targeting to the CAV domain for effective therapies.

Hormone stimulated intracellular signaling has significant effects on cardiac fibroblast function, specifically in mediating collagen production. CFs and myofibroblasts are the primary cells responsible for ECM turnover following MI. We have determined that myofibroblasts are significantly elevated in the infarcted region 7 days post-MI, but return to control levels by day 14. The cytokine TGF-β is elevated within days following MI, and is a well characterized, potent inducer of myofibroblast differentiation. The initial appearance of myofibroblasts 7 days post-MI that we observed is likely attributed to the elevation of TGF-β (and possibly other inflammatory cytokines); and the disappearance of myofibroblasts by 14 days could be due to the waning of these signals and subsequent apoptosis (Sun et al., 2000). This initial phase of myofibroblast infiltration is critical for repair of the infarcted region, and the disappearance of these cells by two weeks indicates that they have performed their repair duties and regressed in a timely manner.

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Extensive cardiac remodeling occurs following MI and several studies have shown that this involves increased deposition of collagens I and III (Weber and Brilla,

1991; Gonzalez et al., 2002; Koshy et al., 2003). In addition, we have demonstrated that collagen VI is significantly elevated post-MI and a concurrent increase in myofibroblast content also occurs (see Figure 31). Collagen VI appears to play less of a structural role than collagens I and III, and instead promotes signals that induce differentiation to myofibroblasts in vitro (Naugle et al., 2006). Type VI collagen likely plays a similar role in vivo, and we hypothesize that the accumulation of collagen VI post-MI is responsible for the reappearance of myofibroblasts 4-5 months following the initial insult. Collagen

VI potentially contributes both directly and indirectly (via promoting differentiation to myofibroblasts) to cardiac fibrosis, and therefore, is a possible target for anti-fibrotic therapies. Inhibiting specific intracellular signaling molecules (which have yet to be determined) that lead to the production of collagen VI, or the receptors that transmit these signals would be a potential therapeutic target. Collagen I and III induce CF proliferation and therefore could be a potential anti-fibrotic target as well. Targeting specific collagen types for degradation may prove to be fairly difficult in vivo, however, identifying the signaling molecules involved in the production of these ECM proteins may prove to be a much more tangible therapeutic approach to limiting cardiac fibrosis.

Collagens typically initiate cellular signaling via interaction with the classic collagen-binding integrin receptors: α1β1, α2β1, α10β1, and α11β1 (Vogel, 2001).

However, other integrin receptors have been shown to be important in associating with type VI collagen, and potentially play a role in the signaling of type VI collagen

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Figure 31. Factors effecting CF activation during the progression of cardiac fibrosis. During hypertension and following MI, CFs become activated via proliferation or differentiation to myofibroblasts, both of which lead to increased collagen production. This excess collagen deposition leads to cardiac fibrosis and if untreated, eventual heart failure. Types I and III collagen promote proliferation of CFs, and type VI collagen induces myofibroblast differentiation, possibly through interaction with the α3 integrin receptor (refer to dotted line). Elevation of cAMP via the β-AR pathway inhibits myofibroblast differentiation and decreases collagen production. It has been well demonstrated that during heart failure, the β-AR pathway is downregulated in the heart, and if this occurs specifically in the cardiac fibroblasts, then the apparent anti-fibrotic effects of cAMP would be removed and could possibly be a key contributor to fibrosis (refer to dashed line).

131

132

induction of myofibroblast differentiation. Collagen VI has been shown to interact with the α3 integrin receptor subunit, and we have demonstrated in a cross-linking assay that type VI collagen associates with α3 integrin in focal adhesions in CFs. This is a potential avenue by which type VI collagen could mediate its effects on myofibroblast differentiation in vitro, and could also play a significant role in vivo. We observed that following MI in the rat, a rise in α3 integrin levels occurs just prior to myofibroblast infiltration at 7 days post-MI. It is possible that upregulation of these integrin receptors is an important step in the process of differentiation to myofibroblasts, although clearly more work must be done to confirm this idea. The biphasic appearance of both myofibroblasts and the α3 integrin receptor subunit suggests a relationship between the two, with the α3 integrin receptors as the first step, increasing slightly prior to the differentiation process. If α3 integrin receptors do in fact mediate collagen VI-induced myofibroblast differentiation, then blocking these integrin receptors might be a beneficial therapy post-MI. By blocking the effects of type VI collagen on differentiation, the initial phase of myofibroblast infiltration, which is likely driven by inflammatory cytokines, would still initiate wound healing effects; however, the longer-term accumulation of collagen VI could potentially lessen the increase myofibroblast content weeks to months following the infarction.

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OVERALL SUMMARY

One of the goals of this dissertation has been to depict the dynamic nature of the cardiac remodeling process, and to point out the many factors that affect cardiac fibroblast function. ANG II, catecholamines, and even specific ECM substrates have significant effects on CF activation and direct relevance to disease. Altered cardiac remodeling often leads to cardiac fibrosis, which is a direct result of CF function.

Stimulation of the β-AR signaling pathway in CFs translates into anti-fibrotic effects via inhibition of myofibroblast differentiation and decreasing collagen production. During heart failure, the β-AR pathway is downregulated, therefore, its anti-fibrotic effects can actually become pro-fibrotic. Not only do hormones affect CF activation, but the ECM, specifically type VI collagen, has pro-fibrotic effects by promoting differentiation to the hypersecretory myofibroblast phenotype. The dramatic elevation of type VI collagen post-MI potentially plays a significant role in overall myocardial fibrosis by directly contributing to the overall collagen content, or indirectly by promoting differentiation to myofibroblasts. Targeting cardiac fibroblasts by limiting the production or blocking the effects of factors which promote fibrosis would be a key step in limiting the progression of cardiac fibrosis and prolonging longevity following hypertension or myocardial infarction.

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FUTURE DIRECTIONS

Additional questions have arisen following the conclusion of this project, and would be useful to investigate in future studies. Investigating chronic β-AR stimulation in cardiac fibroblasts and its long-term effects on collagen production and myofibroblast differentiation would allow us to gain insight about cardiac fibroblasts in a more physiological, disease-based setting. Another question left unanswered is what is the mechanism by which type VI collagen induces myofibroblast differentiation? Identifying the integrin receptors and intracellular signaling mechanisms involved in this process would allow us to pinpoint molecular targets implicated in promoting differentiation by type VI collagen. Finally, studying post-MI remodeling in type VI knock out mice could permit us to establish a causative relationship between type VI collagen and myofibroblasts in vivo, and possibly identify future therapeutic targets for prevention or treatment of fibrotic diseases in the heart as well as other tissues.

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