Intracellular signaling of cardiac fibroblasts.

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Intracellular Signaling of Cardiac Fibroblasts Patricia L. Roche,1 Krista L. Filomeno,1 Rushita A. Bagchi,1 and Michael P. Czubryt*1 ABSTRACT Long regarded as a mere accessory cell for the cardiomyocyte, the cardiac fibroblast is now recognized as a critical determinant of cardiac function in health and disease. A recent renaissance in fibroblast-centered research has fostered a better understanding than ever before of the biology of fibroblasts and their contractile counterparts, myofibroblasts. While advanced methodological approaches, including transgenics, lineage fate mapping, and improved cell marker identification have helped to facilitate this new work, the primary driver is arguably the contribution of myofibroblasts to cardiac pathophysiology including fibrosis and arrhythmogenesis. Fibrosis is a natural sequel to numerous common cardiac pathologies including myocardial infarction and hypertension, and typically exacerbates cardiovascular disease and progression to heart failure, yet no therapies currently exist to specifically target fibrosis. The regulatory processes and intracellular signaling pathways governing fibroblast and myofibroblast behavior thus represent important points of inquiry for the development of antifibrotic treatments. While steady progress is being made in uncovering the signaling pathways specific for cardiac fibroblast function (including proliferation, phenotype conversion, and matrix synthesis), much of what is currently known of fibroblast signaling mechanisms is derived from noncardiac fibroblast populations. Given the heterogeneity of fibroblasts across tissues, this dearth of information further underscores the need for progress in cardiac fibroblast biological research. © 2015 American Physiological Society. Compr Physiol 5:721-760, 2015.

Introduction Throughout the body, fibroblasts play a valuable role in the synthesis, maintenance and breakdown of the extracellular matrix (ECM; see Table 1 for a list of abbreviations). Fibroblasts furthermore act as a source of intercellular signals to surrounding stromal cells, resulting in alterations in cell and tissue behavior. However, the mechanical requirements of different tissues vary widely, as do the physical forces impinging on these tissues, thus it is not surprising that the characteristics of fibroblasts can vary depending on their location within the body. Cardiac fibroblasts represent a unique cellular niche— they are exposed to constant periodic swings in physical forces transmitted via the ECM on a beat-to-beat basis; they signal to and receive signals from surrounding cardiomyocytes, a highly unique cell type within the body; and they can be significant contributors to cardiac pathology when their synthesis of ECM increases in the pathogenesis of cardiac fibrosis. The following review describes the current state of knowledge of the major signaling pathways active within cardiac fibroblasts and their phenotypic cousins, myofibroblasts, with due consideration of external signals including growth factors and physical force which act upon these cells. Key biological processes undertaken by fibroblasts, including matrix attachment, force sensing and mechanotransduction, migration, proliferation, and phenotype conversion are discussed in the context of the key signaling mechanisms responsible where known. The focus is on cardiac fibroblasts, but where deficiencies in the current literature are present, an

Volume 5, April 2015

overview of intracellular signaling in fibroblasts from other tissue sources is provided.

Cardiac Fibroblasts and the Extracellular Matrix Extracellular matrix production Fibroblasts are the most numerous individual cell type within the heart, accounting for over 50% of the cell population (a number that may vary widely between species), yet taking up a surprisingly low volume within the myocardium owing to their small physical size in comparison to cardiomyocytes (139, 399). During development, fibroblasts arise within the proepicardial organ from a precursor cell population dependent upon expression of the basic helix-loop-helix transcription factor TCF21: deletion of TCF21 abrogates cardiac fibroblast specification, resulting in late gestational lethality (2, 291). Fibroblasts are recognized as having a high degree of heterogeneity between organs and tissue types, with the expression of unique protein/mRNA signatures. This * Correspondence

to [email protected] Boniface Hospital Research Centre, University of Manitoba, Winnipeg, Manitoba, Canada 1 St.

P. L. Roche and K. L. Filomeno contributed equally to this work. Published online, April 2015 (comprehensivephysiology.com) DOI: 10.1002/cphy.c140044 Copyright © American Physiological Society.

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Intracellular Signaling of Cardiac Fibroblasts

Table 1 ACE

Abbreviations

Comprehensive Physiology

Table 1

(Continued)

Angiotensin converting enzyme

Meox2

Mesenchyme homeobox 2

5’ AMP-activated protein kinase

MI

Myocardial infarction

AngII

Angiotensin II

MLC

Myosin light chain

ANP

Atrial natriuretic peptide

MLCK

MLC kinase

AP-1

Activator protein-1

MMP

Matrix metalloproteinase

αSMA

α-Smooth muscle actin

MRL

Mig-10/RIAM/lamellipodin

bFGF

Basic fibroblast growth factor/FGF2

MRTF-A

Myocardin-related transcription factor-A Membrane-type MMP Nuclear factor of activated T cells

AMPK

Brain natriuretic peptide

MT-MMP

Cas

Crk-associated substrate

NFAT

Cdc42

Cell division control protein 42 homolog

NF-κB

Nuclear factor κB

CDK

Cyclin-dependent kinase

Nox4

NADPH oxidase 4

CTGF

Connective tissue growth factor

p70S6K

p70S6 kinase p21-activated serine-threonine kinase

BNP

Diacylglycerol

PAK

DDR1

Discoidin domain receptor 1

PDGF

Platelet-derived growth factor

Dok1

Docking protein 1

PI3K

Phosphatidylinositol 3-kinase

ECM

Extracellular matrix

PIP2

Phosphatidylinositol(4,5)-bisphosphate

Epidermal growth factor receptor

PIP3

Phosphatidylinositol-3,4,5-trisphosphate

Extracellular signal-regulated kinase

PIX

PAK-interacting exchange factor

ET1

Endothelin 1

PKA

Protein kinase A

FA

Focal adhesion

PKC

Protein kinase C

Focal adhesion kinase

PKG

Protein kinase G

Protein 4.1, ezrin, radixin, and moesin homology

PLC

Phospholipase C

Filamin A

PTB

Phosphotyrosine binding

FOXO3a

Forkhead box O 3a

PYK2

Proline-rich tyrosine kinase 2

FSP1

Fibroblast-specific protein-1

Rb

Retinoblastoma protein

GEF

Guanine exchange factor

RIAM

Rap1-GTP-interacting adaptor molecule protein

G-protein-coupled receptor kinase-interacting protein 1

ROCK

Rho-associated kinase

Grb2

Growth factor receptor-bound protein 2

ROS

Reactive oxygen species

HA

Hyaluronic acid

RTK

Receptor tyrosine kinase

Has

Hyaluronan synthase

SF

Stress fiber

Hepatocyte growth factor

SFK

Src-family kinase

Hypoxia-inducible factor-1α

SLC

Small latent complex

Heparan sulfate proteoglycan

Sp1

Specificity protein 1

ICAP-1

Integrin cytoplasmic domain-associated protein 1

SRE

SRF response element

IGF-1

Insulin-like growth factor-1

SRF

Serum response factor

Interleukin-1

SYF

Src-Yes-Fyn

ILK

Integrin-linked kinase

Syn4

Syndecan-4

IP3

Inositol 1,4,5-trisphosphate

TAK1

TGFβ-activated kinase 1

JNK

c-Jun N-terminal kinase

TGFβ

Transforming growth factor β

LAP

Latency-associated protein

TGFβR1

TGFβ receptor 1

Leukemia-associated Rho guanine nucleotide exchange factor

TIMP

Tissue inhibitor of metalloproteinases

TNFα

Tumor necrosis factor α

LIM

Lin11, Isl-1, and Mec-3

TRPC

Transient receptor potential channel

LLC

Large latent complex

VASP

Vasodilator-stimulated protein

LTBP

Latent TGF-binding protein

VEGF

Vascular endothelial growth factor

MAPK

Mitogen-activated protein kinase

WASP

Wiskott-Aldrich Syndrome protein

mDia1

Mammalian diaphanous-1

WAVE

WASP-family verprolin-homologous protein

MEF

Mouse embryonic fibroblast

DAG

EGFR ERK

FAK FERM FLNa

GIT1

HGF HIF1α HSPG

IL-1

LARG

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heterogeneity extends to the heart as well (82). Atrial fibroblasts have been shown to express higher levels of genes implicated in matrix formation, proliferation, cellular structure, and metabolism; however, the biological significance of these variations is not fully understood (64). For example, plateletderived growth factor (PDGF) and its receptor are more highly expressed in atrial fibroblasts, and it has been speculated that these chamber-specific differences may be due to unique hemodynamic, neurohumoral, and structural characteristics between these two regions of the heart, resulting in increased fibrotic responses in the atrium (64). In the healthy adult myocardium, cardiac fibroblasts function primarily to maintain homeostasis via synthesis and secretion of ECM constituents and remodeling enzymes such as matrix metalloproteinases (MMPs; Fig. 1) (391, 425). Fibroblasts are characterized by an interior volume extensively occupied by endoplasmic reticulum and Golgi apparatus—features indicative of the elaborate synthetic and

Mechanical tension PDGF ED-A fibronectin

secretory machinery required for the key role these cells play in producing and depositing ECM components. The cardiac ECM provides a dynamic physical scaffold for the heart and plays a crucial role in initiating biochemical and mechanical cues for cell growth, development and differentiation. It is comprised of a myriad of constituents including fibrillar collagens, elastins, fibronectin, laminin, thrombospondin, proteoglycans, periostin, hyaluronan, and MMPs (45, 110, 149, 211). Synthesis of these ECM components by cardiac fibroblasts is regulated by a complex network of signaling pathways downstream of various effector molecules such as transforming growth factor β (TGFβ), PDGF, basic fibroblast growth factor (bFGF), and others, in both healthy and diseased states of the heart (66, 148). Cardiac fibroblasts also synthesize and secrete many bioactive molecules including interleukins, tumor necrosis factor α (TNFα), TGFβ, angiotensin II (AngII), endothelin 1 (ET1), natriuretic peptides, and vascular endothelial growth factor (VEGF) (395).

Mechanical tension TGF-β angiotensin II CTGF

Fibroblast

Proto-myofibroblast

Myofibroblast

ECM homeostasis Proliferation Migration Nascent adhesions

Collagen synthesis α-SMA expression ED-A fibronectin expression α-SMA-negative stress fibers Contractility Focal adhesions

Collagen cross-linking α-SMA-positive stress fibers Increased contractility Mature focal adhesions

Figure 1

Cardiac fibroblast activation and phenotype conversion. In the healthy myocardium, cardiac fibroblasts proliferate relatively slowly and maintain extracellular matrix homeostasis by continual synthesis of matrix constituents (e.g., collagens) and remodeling enzymes (e.g., matrix metalloproteinases/MMPs) at low basal levels. In response to stressors such as myocardial infarction, related proinflammatory and profibrotic cytokines, and/or increased matrix stiffness, cardiac fibroblasts become activated and initiate the wound healing process through matrix degradation (via increased expression of MMPs), proliferation, and migration to the site of injury, where they facilitate cardiac remodeling. In vitro work with fibroblasts has revealed a transient intermediate phenotype—the proto-myofibroblast, which shares features of both fibroblasts and myofibroblasts. Proto-myofibroblasts synthesize increased amounts of fibrillar collagens type I and III, and begin to express α-smooth muscle actin (αSMA) and the ED-A splice variant of fibronectin. Additionally, adhesions with the matrix are strengthened in proto-myofibroblasts as are intracellular actin filaments, imparting contractility. As of yet, it remains unclear whether proto-myofibroblasts exist as discrete entities in vivo. Fully phenotype converted cardiac myofibroblasts are characterized by reduced proliferation and migration, and in comparison to their precursors, are hypersynthetic for fibrillar collagens and other matrix components. Cardiac myofibroblasts also express increased levels of ED-A fibronectin and αSMA, the latter of which is also incorporated into stress fibers that are stronger and thicker than their αSMA-negative counterparts in proto-myofibroblasts. In conjunction with these fibers, the maturation and strengthening of focal adhesions imparts greater contractile ability upon cardiac myofibroblasts. Contraction of surrounding scar tissue and collagen crosslinking performed by myofibroblasts is necessary for scar maturation, though their persistence in the healed myocardium has also been implicated in the development of cardiac fibrosis.


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Fibrillar collagen production Fibrillar collagens are the major component of the cardiac ECM architecture and play a central role in maintaining the structural integrity of the heart (27). The collagen fibrils provide a scaffold for cell attachment, as well as sequestration of signaling molecules such as TGFβ, thereby contributing to biomechanical homeostasis of the myocardium. Of the more than 25 documented types of collagen, two are predominant in the heart: type I fibrillar collagen, which constitutes approximately 80% of myocardial collagen, and type III fibrillar collagen, which comprises about 10% (27, 124, 395). The remainder are a mixture of various fibrillar and nonfibrillar collagens such as type IV, V, and VI (37, 321, 449). Fibrillar collagens are produced and secreted into the interstitial space as procollagen molecules, which are then processed into smaller, mature collagen molecules by enzyme-mediated cleavage of the terminal propeptide domains. Mature collagen molecules can then be assembled and cross-linked to form structural collagen fibers. A disintegrin and metalloproteinase with thrombospondin motifs 2 (ADAMTS2) cleaves the amino (N)-terminal propeptide, while bone morphogenetic protein 1 cleaves the carboxy (C)terminal propeptide (106, 247). Upon cleavage by N- and C-proteinases, these collagen propeptides are released into the circulation (547). Serum levels of the 100 kDa C-terminal propeptide are positively correlated with diastolic dysfunction, and this is the most commonly used biomarker for quantitative determination of type I collagen synthesis (318). Increased serum levels of the 42 kDa N-terminal propeptide of type III collagen have been positively correlated with heart failure and mortality (103). The N-terminal propeptide is a prominent marker of type III collagen biosynthesis (428). Although measurements of these propeptides are indicative of overall fibrillar collagen synthesis, the organization and thickness of collagen type I and III fibrils can vary between not only healthy and diseased myocardium, but also within compartments of the heart itself such as the atria and ventricles (36).

Canonical TGF𝛽 signaling Collagen synthesis in the heart is regulated primarily through the TGFβ signaling cascade, initiated by activation of the heterotetrameric TGFβ receptor composed of type I and II subunit dimers (92,201). There is substantial evidence demonstrating that the canonical Smad effector protein signaling pathway, downstream of TGFβ, is responsible for increased collagen deposition in the cardiac ECM following injury (57). The binding of TGFβ to its receptor facilitates the phosphorylation of R-Smad2 and/or R-Smad3 by the receptor’s intrinsic serine-threonine kinase activity. Phosphorylated R-Smads can then associate with Co-Smad4, and the resulting RSmad2/3-Smad4 complex translocates to the nucleus to act as a transcription factor and drive the expression of target genes such as type I collagens (Fig. 2) (350,563). Smad3 is required for the induction of matrix gene expression. Expression of

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TGF-β

TGF-β

Mechanical strain

LAP

LTBP-1

TGF-β receptor

Plasma membrane

Smad2/3

II I Smad2/3 P

Smad3 P

Smad4 Smad7

Scx

SMURF

Smad7

Smad2 P

sm Cytopla

SMURF

Nucleus

Scx

Smad3

Smad7

Smad4

P

Smad2 P

Ski Myofibroblast genes

COL1A2 Smad4

Scx

Smad3 P

Smad2 P

Figure 2

Canonical transforming growth factor-β (TGFβ) signaling and cardiac fibroblast gene expression. TGFβ is secreted by cardiac fibroblasts in an inactive form, bound noncovalently to latencyassociated peptide (LAP). LAP binds covalently to latent-transforming growth factor-β-bound protein-1 (LTBP-1) within the normal extracellular matrix, and these three components form the large latent complex, or LLC. Increased tension within the matrix causes a conformational change in the LLC that allows rapid release and activation of TGFβ. Such forces can be transmitted to the LLC either indirectly (via increased fibronectin fibril extension within the matrix), directly (through LLC-bound integrins on the surface of contractile myofibroblasts), or both. Active TGFβ can then bind to its receptor and induce heterotetramerization of TGFβ receptor subunits I and II. The serine/threonine kinase activity of TGFβ receptor subunit II is activated by ligand binding, in turn phosphorylating subunit I, which then phosphorylates receptor Smads 2 and 3 to allow recruitment of co-Smads such as Smad4. The Smad complex then translocates into the nucleus to activate transcription of target genes such as αSMA and fibrillar collagens. The transcription factor Ski attenuates this process in cardiac myofibroblasts, abrogating phenotype conversion. TGFβ-activated Smad3 increases expression of the transcription factor scleraxis (Scx), which acts synergistically with Smad3 to transactivate the collagen 1α2 gene. TGFβ receptor activation also activates a negative feedback loop via the expression of inhibitory Smad7, which suppresses Scx expression and forms a complex with Smurf in the nucleus that translocates to the cytoplasm to repress phosphorylation and activation of receptor Smads.


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fibrosis-related genes such as type I collagen and connective tissue growth factor (CTGF/CCN2) is interrupted in fibroblasts lacking the Smad3 gene (88, 137, 527). The inhibitory Smads, I-Smad6 and I-Smad7, prevent R-Smad activation through competitive binding for Smad2 and Smad3 to TGFβ receptor 1 (TGFβR1/ALK5) and increased receptor degradation (349, 362). The role of canonical signaling downstream of TGFβ has been extensively demonstrated in cardiac fibroblasts.

Noncanonical TGF𝛽 signaling Besides Smad-mediated regulation of collagen, TGFβ binding to its receptor also activates other noncanonical signaling cascades to regulate collagen synthesis, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), TGFβ-activated kinase 1 (TAK1), and p38 mitogenactivated protein kinase (p38 MAPK) pathways (57, 278). Experimental data indicates that Ras, ERK, and JNK are rapidly activated within minutes of TGFβ treatment in several cell types, supporting the existence of additional Smadindependent TGFβ signaling pathways (169, 170, 343). The Leask laboratory has demonstrated that JNK activation in response to TGFβ in normal fibroblasts depends on focal adhesion kinase (FAK) (295). Recently they also reported that the absence of TAK1 impairs TGFβ-induced JNK phosphorylation: in contrast to wild-type fibroblasts, TAK1-null fibroblasts failed to exhibit JNK activation in response to TGFβ, and failed to upregulate the expression of the myofibroblast marker α-smooth muscle actin (αSMA) and CTGF (458). Activation of the Ras/MEK/ERK noncanonical TGFβ signaling cascade involves the heparan sulfate proteoglycan (HSPG) syndecan-4 (Syn4). In Syn4-null fibroblasts, TGFβ was unable to phosphorylate ERK or induce cell contraction (91). Syn4 can act directly as a coreceptor for TGFβ or indirectly by modulating the expression of genes required for TGFβ-induced activation of ERK. In dermal and cardiac fibroblasts, ERK activation is required for the expression of type I collagen and CTGF (282, 296). In turn, CTGF is an effector of TGFβ-induced myofibroblast phenotype conversion and ECM synthesis. Smad-independent responses to TGFβ appear to be mediated, at least partially, by activation of p38 MAPK. A TGFβ receptor subunit I mutant defective for Smad activation has been shown to activate p38 MAPK signaling in response to TGFβ (576). Betaglycan, another member of the HSPG family, has been shown to bind TGFβ by its core protein compartment (155). It controls access of TGFβ to TGFβ receptor subunit II, thereby affecting intracellular TGFβ activity (299). This HSPG is capable of activating the p38 MAPK pathway independently of the R-Smads and TGFβ ligand, consequently affecting target gene expression (424). These multiple and overlapping collagen synthesis pathways explain the potent profibrotic effect of TGFβ. However, the mechanisms of TGFβ-mediated activation of noncanonical pathways involving ERK, JNK, or MAPK, and the functional consequences of


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activation of these pathways, are incompletely characterized in cardiac fibroblasts, in contrast to other cells such as dermal fibroblasts.

Renin-angiotensin-aldosterone system signaling Regulation of collagen synthesis by cardiac fibroblasts has also been associated with the renin-angiotensin-aldosterone system. Adult rat cardiac fibroblasts treated with AngII or aldosterone demonstrated a significant increase in collagen synthesis, which is abolished by pharmacological blockade of angiotensin receptors (49). AngII acts via two receptors (AT1 and AT2) that belong to the G protein-coupled receptor superfamily (135). The majority of the known physiological effects of AngII occur following binding to the AT1 receptor, inducing well-defined G protein-linked signaling pathways including phospholipase C (PLC) activation, which in turn causes the release of Ca2+ and subsequent activation of calmodulin kinase and protein kinase C (PKC) (309). AngII induces upregulation of TGFβ1 expression in cardiac fibroblasts through AT1, as well as increased collagen expression via TGFβ and ERK signaling (74, 175, 570). It was recently reported that AngII-induced ERK activation in cardiac fibroblasts occurs via a pathway which includes the Gβγ subunit of Gi and tyrosine kinases (e.g., Src, Ras, and Raf) (593). AngII also causes Smad2 phosphorylation, nuclear translocation of phospho-Smad2/4 and increased binding of the Smad complex to DNA, an effect blocked by the AT1 blocker losartan (414). AngII-treated cardiac fibroblasts exhibit αSMA-positive stress fibers (SFs) and contractile function in the presence or absence of TGFβ receptor subunit 1, suggesting that AngII can also stimulate myofibroblast phenotype conversion independently of TGFβ (122). Downregulation of the transcription factor serum response factor (SRF) in cardiac fibroblasts via RNA interference attenuates AngII-mediated myofibroblast phenotype conversion (122). In turn, TGFβ1 stimulates AT1 receptor expression directly via Smads 2/3 and TGFβ receptor subunit I/ALK5, providing evidence of autocrine regulatory cross-talk between the TGFβ and AngII signaling pathways (316). The stimulatory effect of TGFβ on AT1 receptor expression involves p38 MAPK, JNK, and phosphatidylinositol 3-kinase (PI3K) signaling (316). The renin-angiotensin-aldosterone system is arguably the second most characterized mechanism of ECM regulation by cardiac fibroblasts after the TGFβ signaling pathway.

Non-collagen matrix components The ECM protein fibronectin plays key roles in the developing heart and in the injured myocardium. Neonatal hearts synthesize more fibronectin than adult hearts (42, 514). More specifically, the ED-A splice variant of fibronectin is implicated in phenotype conversion of fibroblasts, as well as the inflammatory response to cardiac injury. The insertion of the ED-A module into fibronectin causes a conformational

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change which increases its cell adhesive properties (310). ED-A fibronectin regulates cell adhesion and proliferation by binding integrin α4/CD49d (290, 448). The accumulation of other ECM components, including type I collagen, depends upon polymerization of fibronectin within the ECM (474). In a model of myocardial infarction (MI) in mice lacking ED-A fibronectin, there was reduced synthesis and deposition of collagen in cardiac tissue during the remodeling process (16). ED-A fibronectin polymerization is required for TGFβ1 -induced fibroblast activation and phenotype conversion to myofibroblasts, preceding αSMA expression following TGFβ1 treatment of cultured cells, as well as during granulation tissue development in vivo (448). ED-A fibronectin induces proinflammatory gene expression by nuclear factorκB (NF-κB) activation and acts as an endogenous ligand for TLR2 and TLR4 (181,303). Fibronectin has also been identified as an in vivo substrate of MMP9 using proteomic analysis as demonstrated by Zamilpa et al. (581). ED-A fibronectin has been investigated primarily in fibroblasts of noncardiac origin, but has recently been recognized as an important marker of phenotype switching in cardiac myofibroblasts (425). Hyaluronan (hyaluronic acid, HA) is a critical component of the cardiac ECM due to its contribution to several functions such as providing a hydrated environment for cell migration and proliferation, interaction with ECM proteoglycans such as versican and aggrecan, and its association with surface receptors like CD44 to regulate cell behavior (510). Early studies showed that a HA-rich environment was necessary for proper development of cardiac valve precursor cushions (33). Hyaluronan synthase (Has) enzymes appear to play a critical role in the cardiac ECM, since mice with genetic disruption of Has2 exhibit deleterious cardiovascular defects (73). However, investigation into the role of HA and its regulation by cardiac fibroblasts is presently very limited, though HA secretion has been closely associated with maintenance of the noncardiac myofibroblast phenotype (324,546). Blockade of HA synthesis inhibits the upregulation of αSMA expression that occurs during myofibroblast phenotype conversion induced by TGFβ (545). Interaction of HA with its receptor CD44 triggers intracellular signaling via Ras, Rac, and PI3K Table 2

MMPs and matrix remodeling The cardiac ECM is a dynamic structure requiring a precise balance between synthesis and degradation of its components, particularly the collagens. Interference with this equilibrium can lead to significant disruption of matrix organization in the myocardium. This is evident in the failing heart, when collagen synthesis outpaces degradation, leading to excessive deposition of collagen, myocardial stiffness, and fibrosis. Conversely, excessive degradation may weaken the ECM and contribute to chamber dilatation. ECM degradation may occur as a result of physical damage, but is also dynamically regulated by the activity of a family of proteolytic enzymes, the MMPs (Table 2). Secreted by various cardiac cell types including fibroblasts, myocytes, and endothelial cells, as well as inflammatory cells such as macrophages, the MMPs are zinc-dependent remodeling endopeptidases capable of collectively degrading the majority of the ECM components (45, 294, 401, 530). These enzymes are synthesized as relatively inactive zymogens or pro-MMPs, which are activated by cleavage of an amino-terminal propeptide, exposing the enzyme’s catalytic domain. More than 25 MMPs have been cloned and characterized to date. Of these, MMP1, MMP2, MMP3, MMP8, MMP9, MMP12, MMP13, MMP28, and membrane-type MMPs (MT1-MMP/MMP14) are involved in myocardial remodeling (246, 302, 312, 475). MMP1 (collagenase I) targets collagen types I, II, and III, as well as basement membrane proteins. MMP1 activity is induced by stimuli such as interleukin-1 (IL-1), brain natriuretic peptide (BNP), PDGF, and hypoxia-reoxygenation, but is reduced in response to AngII treatment (49, 85, 375, 516, 571). MMP2 and MMP9, also known as gelatinases, can process collagen types I, IV, and V, while MMP2 can also degrade collagen type III (482). Furthermore, MMP2 and MMP9 play an active role in the development of fibrosis—both MMPs can release ECM-bound latent TGFβ, thereby inducing collagen synthesis via the canonical Smad and noncanonical MAPK

Matrix Metalloproteinases Secreted by Cardiac Fibroblasts, and Their Target Molecules

Group Collagenase

MMP

Name

Substrates

1

Interstitial collagenase

Fibrillar collagens, aggrecan, proteoglycans, versican

8

Neutrophil collagenase

13 Gelatinase

Stromelysins

Membrane-type MMP

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(161, 233, 363). It may be through these pathways that HA contributes to myofibroblast development and maintenance.

Collagenase 3

2

Gelatinase A

9

Gelatinase B

3

Stromelysin 1

7

Matrilysin

14

MT1-MMP

Denatured fibrillar collagens, collagen type IV, fibronectin, laminin, proteoglycans Collagens I, III, IV, fibronectin, proteoglycans

Fibrillar collagens, perlecan, versican


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signaling pathways (577). Cardiac fibroblasts constitutively secrete MMP2 and its expression is significantly upregulated by TGFβ, TNFα, IL-1β, BNP, mechanical stress, and oxidative stress (32, 54, 189, 220, 308, 337, 468, 469, 480, 516). IL-1 and BNP are also capable of inducing MMP3 (stromelysin1) secretion by cardiac fibroblasts (468, 516). Under basal conditions, cardiac fibroblasts express very low amounts of MMP9. However, this increases dramatically when these cells are exposed to proinflammatory cytokines and oxidative stress (396, 468, 469). AngII appears to elicit variable, species-dependent responses in MMP9 secretion from cardiac fibroblasts (375, 477). MMP8 and MMP13 also degrade type I, II, and III collagen, whereas MMP12 specifically targets elastin. The membrane-type MT1-MMP, which is likely associated with the cell surface (rather than being secreted into the matrix), is capable of cleaving ECM proteins such as fibronectin, laminin, and type I collagen (26, 314, 454). Overall, the activity of most MMPs is generally upregulated by oxidative stress, mechanical stretch, BNP, and proinflammatory cytokines with varying effects caused by AngII. These modulators have also been shown to induce transcription of tissue inhibitors of metalloproteinases (TIMPs) in the myocardium, which oppose and control the proteolytic activity of MMPs. TIMPs are the principal inhibitors of MMPs in the heart, and are produced primarily by cardiac fibroblasts (338,522). Of the four TIMPs that have been cloned and characterized to date, TIMP2, TIMP3, and TIMP4 are expressed in the healthy myocardium (530). The expression of TIMP1 in the healthy heart is quite low but it is significantly upregulated in diseased cardiac tissue (204,234,288). TIMP1 expression is regulated by TNFα and IL-1β, and TIMP2 expression is similarly regulated by these cytokines as well as BNP (130, 516). In the healthy myocardium, MMPs and TIMPs are coexpressed and strictly controlled to maintain homeostasis of the cardiac matrix, while dysregulation of this balanced system resulting in net changes in proteolytic activity contributes to myocardial remodeling and development of heart failure (476,523). A number of clinical studies have revealed that the activity of MMPs is increased while TIMPs are decreased in the failing heart (287, 503, 521).

Cell-Matrix Adhesions Adhesions formed between cardiac fibroblasts and their surrounding ECM allow these cells to “mechanosense” their environment, that is, to detect and respond to changes in matrix structure and stiffness. Changes in matrix composition can thus result in alterations in gene expression, providing a mechanism to maintain tissue homeostasis. Growth of cardiac fibroblasts and other mesenchymal cells is highly reliant upon their adhesion to ECM or a similar substrate, as cells in suspension or on nonadhesive substrates fail to proliferate, even in the presence of serum or growth factors (403). During contraction and migration—critical processes in cardiac wound healing and tissue remodeling—fibroblasts



also exert forces on the surrounding matrix. Transmission of inward and outward forces by fibroblasts relies on the form and function of cell-matrix adhesions. These cell-matrix adhesions are composed of multiple proteins including adaptor proteins talin, α-actinin, vinculin, zyxin, and paxillin; scaffolding proteins such as p130Cas; and associated FAK and Src Family Kinases (SFKs), which associate directly with β-integrins (97,158,221). These adhesions are linked directly to F-actin filaments (in fibroblasts) and Stress Fibers (SFs) (in myofibroblasts), and mediate force transmission to the cytoskeleton and associated signaling pathways. Focal complexes and adhesions are highly variable in their size and composition, and signaling through such structures has been implicated in a number of cellular processes including cellcycle regulation and migration (432).

Formation of adhesions In general, the process of formation of cell-matrix adhesion is initiated with the formation of weak, nascent adhesions during attachment (called focal complexes or contacts), followed by cell spreading, and eventually the formation of stronger Focal Adhesions (FAs) and SFs (163). After formation of focal complexes upon fibroblast interaction with its substrate, contacts can mature into stronger, larger FAs. Both types of adhesions contain integrins as the major transmembrane protein, which interact with ECM components during attachment to increase cell surface contact with the matrix and facilitate cell spreading.

Roles of Rho and Rac Focal complexes are less than 1 μm in diameter, are highly dynamic, and utilize integrin αvβ3 as their major integrin, which recognizes matrix proteins by their RGD motifs (97). The formation of focal complexes is dependent upon activation of the RhoA pathway, which has a large number of downstream effectors involved in not only adhesion formation, but also maturation and association with SFs (in myofibroblasts), among other cellular processes (418). A number of studies have shown that RhoA/Rho-associated kinase (ROCK)-mediated, myosin II-generated contractility of the actin cytoskeleton is required for the development and maturation of focal complexes in fibroblasts (67, 412, 418). However, early adhesion involves a transient downregulation of RhoA, as well as phosphorylation and activation of the RhoA inhibitor p190RhoGAP through a c-Src-dependent, integrin-mediated mechanism (17, 18, 128). Rac itself may contribute to RhoA inhibition, as it has been shown to activate p190RhoGAP, both directly and indirectly via reactive oxygen species (ROS) production (65, 355).

Role of FAK The formation of a FAK-p120RasGAP-p190RhoGAP complex during focal complex formation in fibroblasts implies that FAK also plays a role in downregulating RhoA activity

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during adhesion formation (507). Additionally, Rac1 activation and localization to focal contacts is higher in FAKexpressing fibroblasts than in FAK-null cells (81). FAK autophosphorylation at Y397 creates a Src-binding site that facilitates further FAK phosphorylation by Src and full FAK activation (78,435,439). FAK Y397 autophosphorylation also facilitates the recruitment of adaptor proteins Shc and p130Cas to focal complexes, permitting the formation of a Src, p130Cas and Dock180 complex in fibroblasts. The formation of this FAK-Src-Cas-Dock180 complex is associated with Rac and JNK elevation, suggesting its involvement in transient RhoA downregulation during adhesion formation (215). Both Src and FAK directly mediate p130Cas and paxillin tyrosine phosphorylation, leading to the recruitment of adaptor proteins Crk and Nck, and signaling complex assembly at FAs (415, 437, 439, 518). Additionally, in FAK-null mouse embryonic fibroblasts (MEFs), the related tyrosine kinase Pyk2 was found to regulate p190RhoGAP and thus RhoA activity, suggesting a compensatory role of these two similar tyrosine kinases in adhesion formation (293).

Role of mDia1 Though RhoA and Rac signaling counteract each other in focal complex formation, mammalian diaphanous-1 (mDia1), involved in cytoskeletal actin polymerization, acts upstream of both factors during adhesion formation. Rho-dependent Rac activation is mediated by mDia1-induced phosphorylation of p130Cas, and is antagonized by ROCK (downstream of RhoA) (515). However, even in the absence of RhoA activity, constitutively active mDia1 is capable of inducing the formation of focal complexes in NIH-3T3 fibroblasts (418). Thus, during focal complex formation, the balance in RhoA signaling appears to be tipped in the direction of mDia1, reinforcing Rac activity and thus its own inhibition. Clearly, the formation of adhesions between fibroblasts and their surrounding matrix is a highly coordinated process involving complex regulatory networks of the Rac and RhoA pathways.

Maturation of focal complexes to focal adhesions Gradual Rac inhibition is accompanied by RhoA activation in the maturation of focal complexes to stronger and larger FAs (several micrometers in diameter) characteristic of cardiac myofibroblasts (184). The establishment of mechanically stable contacts between integrins and their ECM ligands precedes the maturation of focal complexes into FAs, a process which involves coclustering of different integrins. One such clustering involves the vitronectin receptor integrin αvβ3, found in focal complexes, with the fibronectin receptor integrin α5β1. It is during maturation that adaptor proteins including talin, vinculin, paxillin and α-actinin are recruited, allowing attachment of FAs to the SFs that develop during phenotype conversion of cardiac fibroblasts to myofibroblasts (97).

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The importance of RhoA activation in FA maturation lies within the establishment of cytoskeletal tension and is characterized by the recruitment of talin, paxillin, vinculin, αactinin, and tensin, accompanied by an increase in membranecytoskeleton interactions (128, 176). As in focal complex formation, actomyosin contractility also appears to be involved in FA maturation, as blebbistatin treatment of MEFs results in reduced FAK autophosphorylation (Y397) and impaired FA maturation (587). Two major effector pathways are implicated in the development of RhoA-mediated tension force within the fibroblast: ROCK and mDia1. Further upstream, guanine exchange factors (GEFs) enhance RhoA activation by facilitating the exchange of GDP for GTP (44). Downstream of Fyn, a tyrosine kinase implicated in SF development, RhoA activation is stimulated by the GEFs p115RhoGEF and p190RhoGEF, as well as leukemiaassociated Rho guanine nucleotide exchange factor (LARG) (141, 213, 565). Phosphorylation and activation of LARG can be achieved by FAK (95). However, in MEFs subjected to force, LARG is activated even with blockade of either FAK or MAPK/ERK, but is attenuated in Src-Yes-Fyn (SYF)deficient cells. Thus, LARG activates RhoA in this pathway in response to phosphorylation by Fyn, and is potentially amplified by FAK activity (185). The transmembrane proteoglycan Syn4 may also aid in FA maturation in cardiac fibroblasts. Syn4 binds different domains in matrix proteins via heparan sulfate side chains, and interacts with the actin cytoskeleton via paxillin and α-actinin (76, 129). Syn4 has been shown to localize with integrins in fibroblast FAs, and even in the absence of integrin binding is capable of recruiting adaptor proteins (30). Mechanical deformation of Syn4-binding sites induces activation of the MAPK/ERK pathway, and thus may contribute to FA maturation in concert with FAK (30). In the myocardium, force also appears to induce RhoA activation by a parallel FAK-dependent Ras-Raf-MAPK-ERK pathway during integrin-mediated adhesion maturation (588). In the absence of FAK, force-induced activation of Ras and ERK is completely abolished (185). In addition, force induces MEK activation and causes phosphorylation of GEF-H1, inducing RhoA activation (185). Inhibition of either FAK or MEK prevents activation of GEF-H1, which has been shown to be phosphorylated by ERK (171, 232). This additional pathway (FAK-Ras-Raf-MAPK-ERK-GEFH1-RhoA) does not appear to be dependent upon Src, as Src inhibition did not affect activation of FAK or Ras (185). Thus both pathways likely converge in force-induced RhoA-mediated adhesion maturation, at least in MEFs, and may play redundant/ compensatory roles in cardiac fibroblasts as well.

Regulation of cell-matrix adhesions Intracellular regulators The structure and function of cell-matrix adhesions is regulated by various factors, including extracellular forces (e.g., substrate rigidity and mechanical strain), integrin-ligand


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binding and activation, and the cytoplasmic environment of cardiac fibroblasts. By far, the cytoplasmic component of cell-matrix adhesions is the most complex, comprised of a vast number of proteins. Two major groups of cytoplasmic proteins interact directly with β-integrin domains in FAs, namely, structural and regulatory proteins. Structural proteins such as talin, α-actinin, and Filamin-A (FLNa) participate in FA assembly and adhesion-mediated fibroblast migration, whereas regulatory proteins (FAK, vinculin, and paxillin) modulate adhesion formation and dynamics. FLNa and talin are actin-binding proteins that associate with the cytoplasmic tail of β1-integrins (250). Though some studies indicate that FLNa inhibits integrin-mediated adhesion by competing with talin for integrin tail binding, more recent evidence suggests that FLNa may be required for β1 integrin activation (via interaction with vimentin and PKCε) and adhesion to collagen substrate by human gingival fibroblasts and mouse NIH-3T3 fibroblasts (249, 251, 252). In cultured NIH-3T3 cells, FLNa functions to assist in the preservation of talin-integrin interactions in FAs, and also appears to play a mechano-protective role by preventing force-induced apoptosis through the Rac/Pak/p38 MAPK signaling pathway (392, 459). Though talin is not required for initial spreading of fibroblasts, it is critical for adhesion stabilization. Talin depletion of fibroblasts results in cell lifting and rounding (586). Recent work in MEFs has shown that α-actinin and talin may in fact compete for binding to β3 integrins in focal complexes, though they interact cooperatively in binding β1 integrins (413). α-Actinin provides a link between integrins and the actin cytoskeleton and is involved in cross-linking of actin filaments, in turn regulating FA maturation, as well as the development of intracellular tension (369). Increased tension in adhesion-associated α-actinin coincides with FA growth and maturation, downstream of RhoA activation. Inhibition of Rho-ROCK signaling results in decreased α-actinin tension and dissociation of FAs in Madin-Darby canine kidney cells (573). α-Actinin expression is necessary for FA maturation— depletion of α-actinin in MEFs decreases FA maturation and subsequent mechanotransduction (413). Knockdown of the type 4 α-actinin isoform ACTN4 in murine lung fibroblasts also impairs FA maturation and cell motility, though cell spreading is increased (450). Thus, it appears α-actinin may be directly involved in regulating FA maturation, though its involvement in initial adhesion formation may be negligible, and studies of its effect in cardiac fibroblasts are currently lacking. Vinculin is a cytoskeletal protein composed of three primary regions—the N-terminal head, a proline-rich and flexible neck region with hinge action, and a C-terminal tail (150). Vinculin in its inactive form exists in a folded conformation, and upon force-induced activation and recruitment to FAs, switches to an open conformation (24, 185). Conformational activation and recruitment of vinculin to FAs via interaction with various FA proteins is dependent upon sustained actomyosin-mediated forces (77). The head



region of vinculin regulates integrin dynamics and clustering in FAs, and participates in FA formation and maturation, independently of vinculin tail region interactions (146, 218). In contrast, the full-length (and activated) molecule is necessary for the development of vinculin-dependent tensile force within MEFs. The head region of vinculin interacts with talin and α-actinin, allowing attachment to membrane integrins (77). Interaction of vinculin with talin is critical to the formation and maturation (increased size and strength) of FAs in fibroblasts (210, 218). The vinculin neck region interacts with signaling proteins including vinexin, vasodilator-stimulated protein (VASP), ponsin, and the Arp2/3 complex (77). VASP localizes to FAs where it binds with Arp2/3, talin, and Rap1-GTP-interacting adaptor molecule protein (RIAM), a member of the Mig10/RIAM/lamellipodin (MRL) family of proteins (285, 446). Though the role of VASP and Arp2/3 in fibroblast adhesion dynamics has not yet been examined, VASP appears to modulate F-actin nucleation by Arp2/3 and enhance actin turnover rates (via integrin β1 activation), potentially contributing to adhesion-cytoskeleton interactions (446,560). RIAM is a critical downstream mediator of Rap1-dependent fibroblast adhesion, and via interaction by its N-terminus with talin, also promotes talin-induced integrin activation (285). Knockdown of RIAM reduces adhesion, integrin activation, and cellular F-actin (192, 267, 272). The tail region of vinculin interacts with phosphatidylinositol(4,5)-bisphosphate (PIP2 ), paxillin, and actin to link FAs to the actin cytoskeleton (77, 218, 591). Linking of F-actin with FAs causes an inward pull, inducing conformational changes in vinculin and its subsequent activation, which further increases adhesion growth and stabilization (77). Paxillin is a cytoskeletal protein localized to FAs which is heavily tyrosine-phosphorylated during fibroblast phenotype conversion and adhesion formation. Paxillin is not necessary for the formation of FAs, and appears to be recruited either downstream or independently of vinculin (218). In fibroblasts, paxillin recruitment to FAs occurs independently of interactions with vinculin and/or FAK and appears to rely on its Lin11, Isl-1 and Mec-3 (LIM) domains (52). In vitro, paxillin binds to vinculin as well as c-Src (via its SH3 domain) and Crk (via its SH2 domain). The N-terminal half of paxillin also contains a putative binding region for the FA tyrosine kinase pp125Fak, though this interaction has not been observed in cardiac fibroblasts (520).

Integrins The major transmembrane components in adhesions are integrins, which mediate fibroblast adherence to ECM components such as fibronectin, collagen, and vitronectin. Additionally, integrins may participate in the formation of cell-cell adhesions through binding of adhesion molecules like vascular cell adhesion molecule and intercellular adhesion molecule (386). Integrins are heterodimeric transmembrane receptors

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essential to cell-matrix adhesions that function primarily in mechanotransduction (179, 222, 564). They possess a large extracellular domain to which various ECM components bind, a single transmembrane segment, and a short cytoplasmic tail that interacts with cytoplasmic adapter proteins that link integrins to the actin cytoskeleton (176, 219, 580). Integrin heterodimers are composed of one α and one β subunit, resulting in at least 24 possible combinations. Each integrin dimer is capable of binding various extracellular ligands, and each ligand is typically able to bind various integrins, resulting in tremendous diversity and potential for functional redundancy (83, 224, 377, 445, 466). Cardiac fibroblasts have been shown to express specific integrins including α5β1 (which binds fibronectin and osteopontin), αvβ1, αvβ3, and αvβ5 (which binds fibronectin, osteopontin, and vitronectin as well as latent TGFβ) (331). Additionally, expression of α1, α2 (which are collagen-specific), α5 (which specifically binds fibronectin), β1, and β2 integrin subunits has been demonstrated in primary cardiac fibroblasts (59, 315). Integrins function bidirectionally across the plasma membrane. In the absence of external stress, integrins exist in a “bent,” low affinity conformation, and require activation to take on an extended high-affinity conformation (572). Integrin activation can occur by the binding of ECM ligands (“outside-in” signaling), or by the binding of its cytoplasmic tail to intracellular activators (“inside-out” signaling) (453). Binding of the major cytoskeletal protein talin to βintegrin tails has been shown by numerous lines of evidence to be a common final step in activation of β1, β2, and β3 integrins—at least in vitro (15, 68, 71, 351, 465, 489). Interaction of talins with β-integrins occurs through binding of a high-affinity phosphotyrosine-binding (PTB) domain found in the FERM (protein 4.1, ezrin, radixin, and moesin homology) domain of the N-terminal head of talin to the first NPXY motif in the β-integrin cytoplasmic tail (12, 70, 387, 489). Other regions of the N-terminal head have been shown to enhance integrin activation by the PTB domain (46). Additional proteins containing PTB domains have been shown to bind integrins without activation (69). Activation by talin requires an additional interaction of the N-terminal head of talin with the membrane-proximal region of the integrin β3 cytoplasmic tail (69, 524, 551). This additional interaction mediates formation of a stable helix that spans the transmembrane region of β-integrin and forms a salt bridge with an aspartate residue on the β3-integrin cytoplasmic tail, disrupting the interaction of this residue with an arginine on the α-integrin component of the heterodimer (13,551). The alteration of β-integrin tilt angle at its transmembrane segment may explain why talin is critical for integrin activation (342). Studies in various nonfibroblast cells have identified other FERM domain proteins called kindlins as coactivators in talinmediated integrin activation (231, 336). In dermal fibroblasts, kindlin-2 was essential for activation of β1 integrin, as well as stabilization and maturation of FAs and SFs, but it is unclear whether a similar mechanism occurs in cardiac fibroblasts (199).

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The question remains as to how talins (and perhaps kindlins) are recruited to integrins, but there is evidence that FAK may fulfill this role. In wild-type MEFs, talin, and paxillin colocalize with integrins in nascent adhesions (276). However, in FAK-null MEFs, talin was absent from paxillinpositive nascent adhesions, yet present in mature adhesions. Interestingly, β1 integrin was observed even in talin-null adhesions in these cells, and in mammary epithelial cells depleted of talin 1 (539). Additionally, talin colocalized with FAK in early adhesions in MEFs with a mutation (Y783A) in the talin-binding region of β1-integrin tails. Together, these results suggest that FAK may recruit talin to β-integrin tails in nascent adhesions, and is also capable of shuttling talin to adhesion sites in the absence of integrin binding. However, these studies also imply that talin is not required for β1-integrin activation, though this may not be the case with other β-integrin types, and involvement of talin in integrin activation may also be cell type specific. The mechanism by which FAK itself is recruited to integrins remains unknown, but may involve p190RhoGEF, which binds FAK (327). Another potential candidate in talin recruitment to integrins involves the Ras GTPase subfamily member Rap1, which has been shown to stimulate integrin activation in epithelial and endothelial cells (40, 43, 264). MRL proteins have been shown to act as scaffolds that link Rap1 to talin and facilitate the recruitment of talin to β3 integrin tails at the plasma membrane of CHO cells (285). Short N-terminal sequences in the MRL proteins RIAM and lamellipodin both bind directly to talin, and in the case of RIAM this interaction is required for activation of αIIbβ3 integrin (285). In T cells, RIAM was shown to act downstream of Rap1 in β1 and β3 integrin activation, and was necessary for Rap1-induced cell adhesion. RIAM also interacts with profilin and VASP, proteins involved in regulation of actin dynamics. Not surprisingly, RIAM knockdown not only displaced Rap1 from the plasma membrane of these cells, but also impaired adhesion and cytoskeletal actin polymerization (272). Regulation of talin binding to β-integrin tails and subsequent integrin activation is regulated by several potential mechanisms. PTB domain-containing proteins, which have also been shown to bind the NPXY motif of β-integrin tails, may compete with talin for binding and inhibit integrin activation through “inside-out” signaling. In vitro binding assays have identified potential competitors, including Numb (a Notch signaling inhibitor), EPS8 (a regulator of Rac signaling), tensin (a FA protein), integrin cytoplasmic domainassociated protein 1 (ICAP-1), and docking protein 1 (Dok1, a downstream target of integrin signaling), which bind to various classes of β-integrins (69). Though in most cases the binding affinity is low, and thus not competitive with talin, in some cases binding affinity can be increased by tyrosine phosphorylation of these competitors by enzymes like SFK (12, 370). For example, in the absence of SFK, Dok1 binding is low and does not cause a decrease in integrin activation (12, 370). However in the presence of SFK, Dok1-binding affinity for the NPXY domain is increased and competition occurs.


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Other mechanosensitive pathways have been linked to force-induced integrin activation, such as activation of FLNa by mechanical force application on beads bound to β1integrins. Force application by this method was found to induce association of p38 MAPK with the transcription factor specificity protein 1 (Sp1) and subsequent Sp1 bindingmediated FLNa activation (117). In addition to integrin activation, association of the talin N-terminal head with β-integrin cytoplasmic tails is also implicated in integrin clustering, an important step in mechanosensory outside-in signaling (202, 421). Clustering of integrins at the cell membrane of fibroblasts is linked to the localization of FAK at the cell periphery and its activation by autophosphorylation at Y397, leading to downstream activation of Rac1 and the formation of lamellipodia (378). In mouse and human gingival fibroblasts, as well as NIH-3T3 fibroblasts, discoidin domain receptor 1 (DDR1) regulates interaction of β1 integrins with fibrillar extracellular collagen (386, 479). Overexpression of DDR1 resulted in increased number, length, and size of FAs, as well as increased β1integrin activation (479). Clearly, the regulation of FA formation, maturation, and association with the actin cytoskeleton involves a complex temporal and spatial interplay of a variety of intracellular and transmembrane players. Additionally, extracellular forces sensed via mechanotransduction play an important role in cell-matrix adhesion dynamics, cytoskeletal organization, and gene expression.

Force Transduction in Cardiac Fibroblasts Mechanosensing of extracellular forces As in fibroblasts of the skin and tendons, cardiac fibroblasts respond to mechanical cues and physical forces deriving from the cardiac ECM. In a fibroblast firmly attached to its substrate, the external and internal forces are in equilibrium; however, a disturbance of this balance in either direction leads to changes in cell form and function. Transmission of forces between fibroblasts and the ECM occurs through adhesion contacts and attached cytoskeletal components. Active “pulling” on such components by the cell provides information about the mechanical state of the surrounding matrix, and in turn, mechanosensing alters the dynamics of cell adhesions and attached cytoskeleton components. In the normal myocardium, cardiac fibroblasts are exposed to a low degree of tissue stiffness, typically in the range of 4 to 18.5 kPa (34, 35, 180, 554). In the fibrotic heart, however, the increase in interstitial collagen results in increased tissue stiffness, in the range of 20 to 100 kPa (208). In response to increased stiffness, cardiac fibroblasts convert to the myofibroblast phenotype, increasing αSMA expression and forming actin SFs that terminate at FAs, which increase in size, number, and strength (208). This phenotype conversion event is capable of occurring in response to rigid substrates without any additional factors


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present, thus a mechanism must exist to permit cells to identify physical changes in their extracellular environment and initiate the appropriate intracellular responses. A number of different mechanisms have been proposed in fibroblast force mechanotransduction. One candidate is the activation of mechanosensitive cell membrane channels such as transient receptor potential cation channels (TRPCs), which induce signaling by facilitating the movement of ions such as calcium into the cell, resulting in activation of calcineurin/NFAT signaling (381). Another candidate mechanism is the activation of a critical step in RhoA signaling in a force-dependent manner. FAK may play the role of the critical step in this mechanism, as its activation is induced by integrin-mediated force in skin fibrosis, activating inflammatory signals such as monocyte chemoattractant protein-1 (559). FAK is also induced by cyclic strain in MEFs (378). Tensile strain on matrix adhesions in MEFs results in autophosphorylation (on Y397) and activation of FAK, which is blocked by RhoA/ROCK inhibitors (511). Additionally, it has been hypothesized that mechanotransduction may occur via direct transmission of force (either external or cell-generated) to transcriptional regulators such as Yap and Taz. These factors are elements of the growthmodulating Hippo pathway, and have been found to be involved in stiffness-mediated transcriptional changes. In epithelial cells on rigid substrates, Yap/Taz is localized to the nucleus, whereas on softer substrates, Yap/Taz is found in the cytoplasm or reduced altogether (228). In this model, changes in the shape of the nucleus and alterations of chromatin organization result in transmission (rather than transduction) of mechanical signals through a mechanically-integrated cytoskeleton. The linker of nucleoskeleton and cytoskeleton complex (LINC) is capable of transmitting such forces to the nuclear matrix or the lamin network underlying the nuclear membrane, resulting in structural changes in the nucleus and chromatin that could alter chromatin folding, protein association, and thus regulate transcription by direct force transmission (228). However, the latter mechanism has not yet been examined in the context of the cardiac fibroblast.

Force-activated ion channels Tension applied to the plasma membrane of cells on stiff substrates may increase cation influx (e.g., calcium) through stretch-activated channels, disrupting intracellular equilibrium. For example, the enzyme proline-rich tyrosine kinase 2 (PYK2) is only weakly activated by integrin activation in response to fibronectin attachment (whereas this causes strong FAK activation), but is highly sensitive to intracellular calcium signals (292). PYK2 and FAK have similar domain structures (their domains include FERM, kinase, proline-rich, and FAT), possess common autophosphorylation sites, and act as scaffolding proteins in the transmission of signals from G protein-coupled receptors to downstream signaling pathways such as MAPK (506). Despite these similarities however, FAK and PYK2 possess distinct signaling roles due to differential

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binding of targets to their FERM or FAT domains, respectively. In addition, FAK is localized to FAs whereas PYK2 displays a perinuclear distribution (258). Additionally, cardiac fibroblast TRPCs including TRPM7, TRPC3, TRPC6, and TRPV4 regulate Ca2+ influx necessary for myofibroblast phenotype conversion (122, 140, 194, 502, 579). Although all four TRP channels exhibit mechanosensitivity in other cell types, only TRPV4 has been specifically studied in the context of mechanosensing by cardiac fibroblasts (359,380). TRPV4 inhibition in cardiac fibroblasts attenuated the combined phenotype conversion effects of substrate stiffness and TGFβ treatment, as measured by incorporation of αSMA into SFs (3).


The most well-defined responses to increased substrate stiffness include increased actomyosin contraction, activation of RhoA, increased tyrosine phosphorylation of various cytoplasmic proteins, and activation of FAK. The modality through which such signals are produced involves physical forces being exerted on cell-matrix adhesions consisting of transmembrane integrins, adaptor/scaffold proteins (talin, paxillin, vinculin, zyxin, and p130Cas), associated kinases (Src kinase and FAK) and the actin cytoskeleton (F-actin, α-actinin, and myosin II). Intracellular signals transmitted through these complexes result in alterations in cell morphology, migration, contractility, phenotype conversion, and other functions (315,383). Integrins are critical components of the mechanotransduction apparatus in cardiac fibroblasts, and their activation is involved in stimulating downstream kinases (such as FAK or integrin-linked kinase/ILK), small GTPases (Rho and Rac), and signaling pathways such as those involving Akt, Raf, PI3K, and the MAPK/ERK pathways (Fig. 3). Expression of ILK is necessary for force-induced stimulation of RhoA signaling (307).

complexes (40, 272, 431, 587). Conversely, ROCK activity downstream of RhoA antagonizes this pathway (515). Inhibition of ROCK by the compound Y27632 suppresses tyrosine phosphorylation of FAK and paxillin, but not p130Cas (515). Though p130Cas has been placed downstream of kinase activation, it appears to have mechanosensory capabilities. In response to stretch of either the cytoskeleton or individual Cas molecules, the CasSD becomes exposed, facilitating its phosphorylation, Cas activation, and downstream signaling events (431, 495). Exposure of the CasSD involves separation of the C-terminus, which binds SFK, and the N-terminus, which contains an SH3 domain with which proline-rich sequences of its critical binding partner FAK interact (162, 348, 422). Colocalization of FAK and Cas, as well as high levels of Cas phosphorylation at adhesion sites, is lost in fibroblasts with a Cas binding-deficient version of FAK (712-715 P>A) (587). However, since blocking FAK kinase activity and Y397 autophosphorylation does not affect CasSD Y165 phosphorylation, it appears as though FAK may act as a docking molecule for Cas in adhesions, but is not necessary for activation of its downstream signaling pathways. Activation of Cas appears to be adhesion dependent, as suspended fibroblasts treated with RGD peptide (a soluble integrin ligand) do not exhibit increased CasSD phosphorylation (587). During initial spreading of MEFs on fibronectin-coated substrate, p130Cas phosphorylation was found to be catalyzed by F-actin polymerization and enhanced by integrin engagement and anchorage to fibronectin. Independently of its kinase activity, FAK linked p130Cas to an N-WASP (ubiquitous WASP homolog)/actin polymerization complex. When p130Cas expression was silenced via shRNA, MEFs showed reduced cell spreading on fibronectin and cell-matrix adhesions failed to mature (587). In addition to cell-matrix adhesions, N-cadherins are able to act as force and stiffness sensors in fibroblasts (174, 259). N-cadherins associate with α-/β-catenin complexes that are capable of binding vinculin, α-actinin, and F-actin, and direct activation of cyclin D1 by the β-catenin component (260,499, 575).

P130Cas

Focal adhesion kinase

The Crk-associated substrate (Cas) family consists of multidomain scaffolding proteins activated by phosphorylation. Src and Ab1 family kinases target a motif consisting of 15 YxxP repeats in the central substrate domain (CasSD) of p130Cas (125). Phosphorylation at the CasSD by FAK produces docking sites for SH2 domain proteins such as Crk (162, 257, 422, 533). In turn, Crk recruits GEFs to Rac1 and Rap1, activators of lamellipodium protrusion and cell-matrix adhesion formation (62, 192, 431, 451, 495). Additionally, p130Cas mediates adhesion-induced integrin signaling in an SFK-dependent manner (360, 452, 587). Phosphorylation of p130Cas induces downstream activation of the small GTPase Rap1, which then activates RIAM, subsequently causing activation of talin and integrins, and the formation of focal

FAK is the principal downstream target of integrin activation in cardiac fibroblast mechanosensing (315). Integrindependent FAK phosphorylation at Y397 promotes SH2 domain binding of SFK and other SH2 domain-containing proteins, such as PLC and growth factor receptor-bound protein 2 (Grb2) (436, 585). Downstream of FAK activation and SFK binding, adhesion dynamics are affected by activation of the Ras and ERK/MAPK pathways, as well as myosin light chain kinase (MLCK) (331). Additionally, force-induced transcriptional activation of αSMA expression [via myocardin-related transcription factor-A (MRTF-A)] in NIH-3T3 fibroblasts required FAK, gelsolin (a key regulator of actin filament assembly), and type-I phosphatidylinositol 4-phosphate 5 kinase-γ (involved in uncapping gelsolin from

Effector Signaling Pathways in Mechanosensing

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ECM ligands (FN, OPN, VTN)

Activated integrin heterodimer

Inactive integrin heterodimer Plasma membrane

α β

Talin

αβ

FAK Rap1

Talin

WASP

FAK

SFK

RIAM Dok1 and others

p130Cas

SFK RhoA F-act in

Ras

mDia1

ROCK

Raf

MAPK

MRTF-A

MRTF-A

F-actin formation and contraction

ERK

sm Cytopla

SRF

Nucleus

α SMA MRTF-A

SRF

c-fos, egr-1

Sap

Figure 3

Mechanosensing through integrins. Integrin activation and downstream signaling occurs in response to both outside-in and inside-out signaling, both of which evoke conformational changes in integrin structure. The inside-out pathway involves binding of talin to the β-integrin NXPY motif. Various mechanisms have been proposed in the regulation of talin binding-mediated integrin activation through competing factors such as the Src-family kinase (SFK)-activated docking protein 1 (Dok1), among others. Outside-in signaling requires interaction of extracellular matrix (ECM) ligands with integrins, a mechanism that is sensitive to the degree of extracellular tension or force. Ligands expressed by cardiac fibroblasts and found in the ECM include fibronectin (FN), osteopontin (OPN), and vitronectin (VTN). Similar to inside-out signaling, recruitment of talin to integrins is a key step. Putative candidates in talin recruitment in cardiac fibroblasts include focal adhesion kinase (FAK) and Rap1-GTP-interacting adaptor molecule (RIAM) downstream of Rap1 activation. Force-activated integrins mediate phosphorylation of SFK and FAK, the latter of which forms a complex linking the mechanosensory protein p130Cas to Wiskott-Aldrich Syndrome protein (WASP) and cytoskeletal F-actin filaments. Force-induced FAK activation upregulates the small GTPase RhoA, activating downstream mammalian diaphanous-1 (mDia1) and Rho-associated kinase (ROCK), inducing subsequent actin filament polymerization and myosin II-mediated contraction. ROCK activation also induces the release of myocardinrelated transcription factor-A (MRTF-A), which forms a complex with serum response factor (SRF) and directly activates α-smooth muscle actin (αSMA) transcription. Activation of FAK and SFK also results in downstream activation of the Ras-Raf-MAPK-ERK pathway, inducing SRF activation, which in turn activates the transcription factor Sap to upregulate downstream target genes.


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actin filaments), all of which colocalized at sites of force application (79).

RhoA/ROCK pathway Mechanosensing between cardiac fibroblasts and their ECM requires “pulling” on adhesions and therefore is reliant upon actomyosin contractility of the cell’s cytoskeleton, which is largely regulated by the RhoA pathway. RhoA is activated in cardiac fibroblasts and numerous other mesenchymal cell types by mechanical stretch or when cells are cultured on rigid substrates (379, 562). Though initially decreased in response to integrin-mediated adhesion to a rigid substrate, sustained adhesion activates several GEFs, which in turn induce an increase in RhoA activity (18, 141, 293, 405). Applying tension directly to integrins (isometric tension) in cells including cardiac fibroblasts causes RhoA activation, confirming its mechanosensory role (185, 588). Numerous lines of evidence suggest that RhoA activation and subsequent actin polymerization is mediated by two pathways (see section on Stress fibers, below), that is, the ROCK-cofilin-LIM Kinase pathway, and via the effector mDia1 (228, 588).

Cell cycle genes The downstream effects of mechanosensing are not limited to changes in cell motility, shape, or adhesion, but may also affect cell survival by altering the expression of cell cycle genes. In floating collagen gels, human foreskin fibroblasts responded to decreased stiffness of their environment by increasing their expression of p27Kip1, while activation of the MAPK pathway mediator ERK was reduced, as was cyclin D1. In MEFs exposed to increased substrate stiffness, autophosphorylation of FAK was increased, leading to Rac activation and Rac-dependent cyclin D1 transcriptional activation (256). Cyclin D1 is implicated in the suppression of adhesion and induction of fibroblast migration. Cyclin D1null MEFs exhibit increased adhesion and reduced motility, and these cells show increased RhoA and ROCK activity, accompanied by increased phosphorylation of LIM Kinase, cofilin, and MLCK (289).

Immediate early genes SRF has been shown to be particularly sensitive to mechanical changes in the ECM (561). SRF is a transcription factor that binds CArG (C AT-rich G) box elements (or SRF response elements, SREs) of its target genes. The SRE contains two sites that are critical to transcriptional activation, including the ternary complex factor (TCF) site, which confers fibroblast responses to varying degrees of cell-matrix adhesions. With increased substrate rigidity, expression of SRF increases, stimulating JNK-mediated phosphorylation of the transcription factor Sap (561). Sap then binds the TCF, initiating transcription of SRF-regulated genes such as Fos and Egr-1. Conversely, on low-rigidity substrate, SRF

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expression is inhibited via p38-dependent phosphorylation of the transcriptional repressor Net, which binds the TCF region of SRF target genes and inhibits their transcriptional activation by this pathway (561).

MRTF-A Myocardin and its related transcription factors MRTF-A and -B are expressed in numerous cell types and act during mechanosensing to convey stimulatory signals from RhoA and the actin cytoskeleton (329). In the nonstressed fibroblast, MRTF-A associates with and sequesters G-actin monomers, preventing assembly into F-actin filaments (329). In response to mechanical stress and RhoA/ROCK activation, MRTFA is released from actin filaments and actin polymerization proceeds (20, 177, 217, 301, 307, 427, 588). Additionally, MRTF-A is translocated into the nucleus where it binds SRE CArG boxes of mechanoresponsive genes including SRF and αSMA, thereby regulating major components of fibroblast phenotype conversion (19, 80, 329, 561). Upon activation of SRF, MRTF-A forms a complex with SRF, which in turn binds and transactivates the αSMA promoter (217, 329, 588). In NIH-3T3 cells subjected to mechanical force, FAK was necessary for MRTF-A-induced αSMA activation (Fig. 3) (79). Evidence indicating αSMA is regulated by various forceinduced pathways suggests it plays a central role in mechanotransduction and subsequent morphological and functional changes in cardiac fibroblasts (537).

Cardiac Injury and Repair Following injury such as MI, extensive myocardial cell death triggers a wound healing process consisting of three phases: an initial inflammatory response, a proliferative phase marked by fibroblast proliferation and migration to the site of injury, and a remodeling phase that largely overlaps with the proliferative phase, and which is marked by extensive cardiac ECM remodeling, fibroblast-to-myofibroblast phenotype conversion and de novo ECM synthesis (104, 116). These responses occur over the minutes, hours and days following the injury, and serve to mitigate further exacerbation of cardiac damage as well as to prevent aneurysm. The beginning of the inflammatory phase is marked by activation of the innate immune system in response to cardiomyocyte necrosis in the injured area. A myriad of injury signals, including cytokines, growth factors, cellular debris, and matrix fragments activate the inflammasome, a multiprotein complex located within the cytosol of resident cardiac fibroblasts. The inflammasome contains apoptosis-associated speck-like protein containing a CARD, which recruits caspase-1. In turn, caspase-1 cleaves pro-IL-1β to produce IL1β as well as IL-18. This initial cytokine production by fibroblasts facilitates the infiltration of neutrophils, leukocytes, and mononuclear cells that mediate the inflammatory response, of which IL-1β is a major mediator (164, 165, 244, 317, 319).


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In cardiac fibroblasts, IL-1β inhibits fibroblast phenotype conversion to avoid premature wound healing (433). The main mechanism by which IL-1β produces this effect appears to be through inhibition of TGFβ signaling, abrogating TGFβ-induced αSMA expression. Two mechanisms of IL-1β repression of TGFβ signaling in cardiac fibroblasts have been elucidated: upregulation of BAMBI, a transmembrane molecule that inhibits TGFβ receptor heterotetramerization; and downregulation of endoglin, a TGFβ coreceptor (367, 433). A recent study reported that BAMBI overexpression protected mice from pressure overload by restraining TGFβ signaling, while BAMBI deletion resulted in an increase in TGFβ-mediated profibrotic effects in isolated cardiac fibroblasts (529). IL-1β also stimulates cardiac fibroblasts to produce MMP3 and MMP8, facilitating clearance of existing ECM and infiltration of inflammatory cells, while inhibiting cardiac fibroblast proliferation. Through negative regulation of cyclins and cyclin-dependent kinases (CDKs), IL-1β prevents fibrotic responses from occurring before the dead or injured tissue has been cleared from the site of cardiac injury (265, 374, 433). At the end of the inflammation phase, macrophages phagocytose neutrophils and other inflammatory mediators and secrete TGFβ and IL-10 (89,447). Expression of the IL-1β receptor IL-1R1 is attenuated, helping to bring the inflammatory phase to an end (433). The proliferative phase, which is initiated after the inflammatory phase has begun but prior to its resolution, is characterized by proliferation of fibroblasts and migration of these cells to the site of injury. The remodeling phase may occur shortly thereafter or concomitantly, and features conversion of fibroblasts into myofibroblasts, the primary matrix-producing cells within the infarct scar (Fig. 1) (105, 555). Leukotrienes, IL-1, CT-1, TGFβ, and FGF all participate in attracting fibroblasts to the infarcted region (38,137,164,167,330). There, they are responsible for inducing synthesis and secretion of ECM and contractile proteins for scar formation (105, 166). Macrophage-produced IL-23 has been found to be an important mediator in wound healing, and its receptor is upregulated within the infarcted area of the myocardium (528). In cardiac fibroblasts, IL-23 signals through STAT3 to mediate interstitial matrix deposition and capillary growth (205, 430). STAT3 activation also aids in fibroblast phenotype conversion in a process mediated by oncostatin M (347). IL-23 knockout animals exhibit decreased collagen synthesis, αSMA expression and activation of STAT3 and ERK1/2. The importance of this cytokine in mediating scar formation is underscored by the observation that IL-23 deficiency following MI ultimately results in fatal ventricular rupture (430). Following MI, scar tissue is created from cross-linked collagen deposited by myofibroblasts during the proliferative phase of wound healing, and thins during subsequent maturation. There is a substantial increase in collagen type I and type III production and accumulation in the infarct scar (105). AngII, which increases in response to various types of cardiac injury, plays an active role in profibrotic signaling within the myofibroblast where it signals through AT1 receptors to


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induce TGFβ1 expression and to enhance TIMP-1 production, promoting collagen accumulation and scar formation within the affected area (74, 102). After the mature scar is formed, growth factors and profibrotic regulatory proteins are cleared from the area and some myofibroblasts undergo apoptosis, resulting in their clearance from the infarct scar area (100, 164, 493). However, it has been shown that myofibroblasts can persist in the infarct scar within the myocardium for years, contributing substantially to cardiac fibrosis (555,583). Different types of fibrosis can occur within the heart, depending on the affected area and the specific pathological circumstances. Interstitial and perivascular fibrosis, the latter of which occurs in the adventitia of the coronary arteries, do not involve cardiomyocyte necrosis and can be triggered in the absence of cardiac events such as MI (48, 548). Conversely, focal fibrosis, which is sometimes referred to as reparative or replacement fibrosis, occurs as a result of cardiomyocyte necrosis and is usually preceded by a cardiac event (434, 549). The remodeling performed by myofibroblasts that resist apoptosis and persist for prolonged periods of time contributes to detrimental changes in systolic and diastolic function and decreases overall compliance of the myocardium (51, 108, 550). Increased active tension with a decrease in relaxation time has also been found in the hearts of spontaneously hypertensive rats with cardiac fibrosis (108). These changes in heart function contribute to impaired contraction and ultimately heart failure (322).

Cardiac Fibroblast Migration Migration of cardiac fibroblasts to sites of myocardial injury is a critical step in the early reparative remodeling process. External cues such as mechanical strain and biochemical stress mediators stimulate this coordinated cell movement, which involves the assembly and disassembly of the actin cytoskeleton, accompanied by turnover of cell-matrix adhesions. The dynamic nature of the actin cytoskeleton links changes in cell architecture at the leading edge of the cell to the formation, maturation, and degradation of cell-matrix adhesions. Factors involved in regulating directed cardiac fibroblast migration include the topography and stiffness of the ECM, cell polarity, receptor activation including integrins and their clustering, as well as cytoskeletal contraction. Migration of cardiac fibroblasts is induced primarily by injury-related proinflammatory cytokines (Fig. 4). The matrix component fibronectin and its truncated form (called migration stimulating factor) are capable of inducing fibroblast spreading and migration (154, 442). AngII appears to induce cardiac fibroblast migration through a NADPH oxidase 4 (Nox4)-ROS-dependent ERK pathway leading to activation of transcription factors NF-κB, Activator Protein-1 (AP-1) and Sp1, which induce MMP activation and downregulation of the adhesion protein REversion-inducing-Cysteine-rich protein with Kazal motifs/RECK (460, 491, 542). IL-8 stimulates a similar pathway via Nox4 and ROS with a

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Signaling pathways governing cardiac fibroblast migration. Fibroblast migration requires both intracellular actin remodeling to drive motility, and the extracellular release of remodeling enzymes to facilitate cell movement through the surrounding stroma. A host of extracellular ligands act on cardiac fibroblasts to induce migration, acting via distinct pathways to govern both activities. Adipocyte-released cytokines leptin and adiponectin act on their respective receptors in cardiac fibroblasts to induce migration. Leptin-induced signaling activates the RhoA-RhoGTPase-associated kinase (ROCK)-cofilin pathway. Cofilin severs actin filaments, inducing actin remodeling and reducing F-actin content. Leptin signaling also causes activation and relocation of type-I transmembrane matrix metalloproteinase (MT1-MMP) to the cell surface, where it can cleave and activate pro-MMP2, causing degradation of extracellular matrix components and increased migration. Adiponectin also activates MT1-MMP downstream of the adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing-1 (APPL1)-AMP-activated protein kinase (AMPK) signaling cascade. Binding of angiotensin II (AngII) and interleukin-8 and -18 to their respective transmembrane receptors causes activation of NADPH oxidase-4 (Nox4), resulting in increased reactive oxygen species (ROS) production. AngII-induced ROS production activates the transcription factors Nuclear Factor κB (NF-κB), activator protein-1 (AP-1), and specificity protein-1 (Sp1) through extracellular signalregulated kinase (ERK) signaling. Through this pathway, NF-κB and AP-1 induce activation of various MMPs, and both Sp1 and AP-1 repress activity of the adhesion protein reversion-inducing-cysteine-rich protein with kazal motifs (RECK). Thus, AngII signaling induces cardiac fibroblast migration by increasing matrix degradation and reducing cell adhesion. Interleukin-8 and -18 also utilize Nox4-ROS signaling, but with a tumor necrosis factor receptor-associated-3-interacting protein-2 (TRAF3IP2) intermediate. Activation of AP-1 and NF-κB downstream of these interleukins induces MMP activity, increasing cardiac fibroblast migration. It is likely that additional pathways identified in noncardiac fibroblasts are also involved in cardiac fibroblast migration, however they remain to be characterized.

TRAF3-interacting protein-2/TRAF3IP2 intermediate (147). IL-18 also utilizes Nox4 signaling, resulting in NF-κB/AP1-induced MMP activation (525). In kidney myofibroblast activation, RhoA/ROCK signaling has been shown to act

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upstream of Nox4 and ROS production (311). IL-17 also stimulates MMP activation and cardiac fibroblast migration, though this appears to occur through Akt/miR-101/MKP1-dependent activation of the p38 MAPK/ERK pathway,


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causing downstream activation of NF-κB, AP-1, and CCAAT enhancer-binding protein (111, 526). Interleukins have also been shown to stimulate migration in fibroblasts from other tissues through these and other nuclear proteins (248, 345). In a rat model of MI, expression of the cytokine cardiotrophin-1 was observed to advance from the border zone alone at 1 day post-MI to the infarct zone within 4 days, concomitant with rapid phosphorylation of Janus kinase, Jak2, STAT1, STAT3, p42/44 MAPK, and Akt (168). Adipocyte cytokines are also capable of stimulating cardiac fibroblast migration. The metabolic regulator adiponectin induces cardiac fibroblast migration facilitated by enhanced MMP activity through Adaptor protein, Phosphotyrosine interaction, PH domain and Leucine zipper-containing 1/APPL1mediated phosphorylation of 5’ AMP-activated protein kinase (AMPK). AMPK activation results in secretion and activation of MMP14 (MT1-MMP) (119). Similarly, leptin induces cardiac fibroblast migration by increasing expression and cell surface localization of MMP14 via RhoA/ROCK-dependent reorganization of the actin cytoskeleton involving the actin disassembly protein cofilin (443, 444).

Cell polarization In the absence of stimuli, cardiac fibroblasts on tissue culture plates exhibit a stellate shape with an evenly distributed actin meshwork and cell-matrix adhesions. For efficient migration, cells must become polarized, developing a leading edge (lamellipodia extending in the direction of migration) and a trailing edge (retracted by retrograde actin flow). In addition to chemical factors, fibroblast polarization is induced by stiff substrates in a protein tyrosine kinase-regulated manner (398). In preparation for movement, the polarization process involves asymmetric distribution of various signaling proteins and actin cytoskeleton reorganization. Besides actin filaments, microtubules comprised of polymerized tubulin monomers play an important role in fibroblast polarization. In fibroblasts, the activity of cell division control protein 42 homolog (Cdc42) is highest at the tip of the leading edge, and Rac1 activity is highest immediately behind the leading edge (172). Rac and Cdc42 regulate the formation of FA complexes at the lamellipodia, and in fibroblasts Cdc42 has been implicated in rapid activation of Rac1, followed by RhoA activation (358). Rac1 is necessary for the formation of lamellipodia, and Cdc42 is similarly necessary for the formation of filopodia, the initial protrusions in directed fibroblast migration (266,410,481). Interaction between Cdc42 and Rac is involved in the integration of these membrane protrusions and coordinated movement (358). Cdc42 and its downstream target N-WASP are both necessary to establish and maintain fibroblast cell polarity, the former acting specifically in migrating primary rat embryonic fibroblasts to regulate lamellipodial activity and reorientation of the Golgi apparatus (325, 357). N-WASP is an activator of actin polymerization and regulates actin-based processes, including cell protrusion, at least


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in part by activation of the Arp2/3 complex (331). FAK, NWASP, and the mechanosensory protein p130Cas form a complex at the distal edge of protruding lamellipodia in spreading fibroblasts (587). The activity of Src-family kinases, including Src, Yes, and Fyn, is induced by fibronectin-activated integrins in the plasma membrane. Subsequently, Src-family kinases phosphorylate p130Cas within the Cas-FAK-N-WASP complex, inducing downstream activation of Rac1 and actin polymerization within the lamellipodium (451). Microtubule stabilization in migrating fibroblasts occurs, at least in the case of lysophosphatidic acid-induced motility, via RhoA and its downstream effector mDia1, though these proteins are not restricted to the lamellipodia, indicating the need for additional regulatory factors (109, 372, 373). The Arp2/3 complex facilitates actin polymerization at the leading edge of migrating fibroblasts. Studies with fibroblasts lacking the ARPC3 gene, which encodes the p21 subunit of this complex, have provided evidence of a critical role for Arp2/3 in polarization and lamellipodia protrusion (487). ARPC3−/− fibroblasts are unable to extend lamellipodia, generating leading edges primarily composed of filopodia exhibiting concentrated mDia1 at their tips. Additionally, these cells lack the ability to coordinate protrusion and directional migration, indicating a role for Arp2/3 (and potentially mDia1) in establishing and maintaining cell polarity. In fibroblasts expressing a dominant negative form of FAK, cell spreading is impaired and cells lack a continuous lamellipodium. The dominant negative FAK also causes reduced phosphorylation events in FAK at Y397 and p130Cas at Y165 (587). Studies in various fibroblast lines have provided evidence that FAK, but not Cas, Src, Fyn, or Yes, is necessary for microtubule stabilization and acts upstream of RhoA and mDia1 to support this function (373). Although some cells utilize mechanisms such as blebbing for extending their membranes, the primary mode by which mesenchymal cells migrate involves the formation of membrane extensions including lamellipodia and filopodia (also referred to as membrane ruffling). Membrane ruffling in NIH-3T3 fibroblasts is inhibited by a dominant-negative mDia1 mutant (515). Inhibition of p130Cas phosphorylation induces membrane ruffling, though there is evidence for FAK C-terminal binding of p130Cas via a SH3 domain, promoting migration through coordinated Rac activation in the lamellipodia (331,515). This suggests FAK may act to sequester and prevent activation of p130Cas, which is involved in stabilization of adhesions and may be suppressed during cardiac fibroblast migration. Part of the polarization process involves the development of lipid rafts at what will be the leading edge of the cell, enabling Rac1 recruitment to the lamellipodia. The development of lipid rafts is regulated by FAK and/or integrin signaling (126). Using the lipid raft marker ganglioside GM1 , Palazzo et al. demonstrated a lack of lipid rafts in fibroblasts null for FAK, but not for SYF or Cas (373). Thus it appears that an integrin-FAK pathway enables the formation of a lipidrich leading edge, as well as signaling by RhoA and downstream mDia1, resulting in microtubule stabilization during

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polarization of migrating fibroblasts. Recent work by Carisey et al. has also indicated vinculin, interacting through its tail region with the actin cytoskeleton, is necessary for polarization in migrating MEFs (77).

Lamellipodia protrusion and adhesion turnover Lamellipodia are sheet-like structures composed of an actin meshwork that terminates on focal complexes, and filopodia are finger-like protrusions with thin parallel actin bundles (358). Just behind the lamellipodia lies the lamellum, containing SF-like actin filaments and transverse arcs which, along with microtubule growth, provide the driving force for lamellipodial protrusion (471, 558). Downregulation of the Rho-kinase pathway is involved in adhesion turnover during fibroblast migration. Blocking the Rho pathway in NIH-3T3 fibroblasts causes dissolution of existing FAs and stimulates the formation of new complexes, in addition to stimulating membrane ruffling (418). These processes are associated with advancement of the cell front during migration. The GTPase Rac1 antagonizes RhoA activity in most situations, and its activation appears to be a critical component in adhesion turnover during cardiac fibroblast migration. Rac1 activation is induced by microtubule growth, and its activity is required for lamellipodial protrusion (558). Additionally, Rac can activate Rho, inducing the formation of new adhesion sites and the assembly of contractile filaments within the advancing lamellipodium (358). The cellular pathways by which Rac activity makes a switch from RhoA suppression to RhoA activation remain unknown, but likely involve signaling crosstalk. The p21-activated serine-threonine kinase (PAK)interacting exchange factor PIX is a GEF enriched in Cdc42/Rac1-induced adhesions, acting upon Rac1 to enhance its activity (81, 498). PIX associates with and is required for recruitment of PAK to sites of adhesions (313). In CHO cells, the localization of PAK at the plasma membrane of migrating cells is critical to cytoskeletal reorganization involved in directed motility (53). In fibroblasts, there is evidence of Cdc42-induced activation of a PAK/PIX complex, leading to Rac1 activation and adhesion turnover (313). Additionally, PAK interacts with the adaptor protein noncatalytic region of tyrosine kinase adaptor protein 1 (Nck1) (313,589). PIX also binds to the G-protein-coupled receptor kinaseinteracting protein GIT1, which can promote adhesion disassembly even in the absence of actin-myosin contractility (589). The C-terminal domain of GIT1 interacts directly with paxillin, and GIT1 overexpression results in delocalization of paxillin from cell-matrix adhesions (589). In turn, paxillin has been shown to mediate actin redistribution by recruiting the PAK/PIX complex, via interactions with p95PKL (paxillin-kinase linker), to nascent focal complexes (519). Moreover, the process of PAK/PIX-mediated Rac1 activation and adhesion turnover during fibroblast migration appears to also involve FAK. GIT has been shown to directly bind FAK via a conserved Spa2 homology domain, and FAK (but not

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SFKs) directly associates with and tyrosine phosphorylates PIX (81, 589).

Focal adhesion kinase in migration FAK is a tyrosine kinase expressed in many cell types including cardiac fibroblasts, and is recruited to and involved in formation of cell-matrix adhesions (331). However, its primary role is in the disassembly (or “turnover”) of FAs, a process required for cell motility and the production of tensile forces (462). FAK-null fibroblasts demonstrate excessive adhesion formation and reduced migration in response to chemotactic stimuli (223). Activation of FAK is induced by autophosphorylation at Y397, in response to either integrin clustering or force applied to matrix adhesions (47, 378). In the latter, RhoA/ROCK signaling appears necessary, as its blockade prevents phosphorylation of Y397 and thus prevents FAK activation (511). FAK is composed of an N-terminal FERM domain, a central kinase domain, numerous proline-rich regions, and a C-terminal FA-targeting (FAT) domain (331). The FERM domain has been shown to link signals from receptor tyrosine kinases (RTKs) such as epidermal growth factor receptor (EGFR) and PDGF Receptor, and its overexpression blocks activation of FAK and inhibits cell migration (196, 461, 485). Two proline-rich regions in the C-terminal domain of FAK contain Src-homology 3 (SH3) domain binding sites that allow its interaction with adaptor proteins (331). Though SH3-mediated binding of GTPase regulator associated with FAK (GRAF) and Arf GTPase-activating protein containing SH3, ankyrin repeat and pleckstrin homology (PH) domains1 (ASAP1) proteins to the FAK C-terminal domain plays a role in mediating actin cytoskeleton dynamics and FA assembly, the downstream interactions of these two proteins remain unknown (378). The C-terminal domain also contains the FAT region, which mediates colocalization of FAK and integrins at FAs (331). Initially, it was presumed that FAK bound directly to the cytoplasmic tail of integrins, however more recent evidence indicates that FAK associates with cell-matrix adhesions indirectly through complex formation with paxillin and talin (331). The ability of integrins to induce FAK phosphorylation is dependent upon interaction between the FAT domain of FAK and talin (84). FAK phosphorylation (Y925) induces recruitment of Grb2 and promotes activation of the Ras/Raf/MEK/ERK pathway (438,440). In turn, Ras regulates turnover of adhesions and actin filaments essential for fibroblast motility (357). ERK and MEK are downstream modulators of Ras activity and blockade of Ras in wounded rat embryonic fibroblasts results in persistent cell-matrix adhesions and lack of motility, indicating that Ras is required for adhesion turnover during fibroblast migration (357).

Retraction of the trailing edge With the protrusion of the lamellipodia and the formation of new attachments at the leading edge, tension is generated


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within the cell, mediated at least in part by myosin II-induced contractility (6,67,334,411). The formation of integrin-based nascent adhesions and F-actin polymerization at the lamellipodium is necessary to cause retraction of the trailing edge of cardiac fibroblasts. The actin network in lamellipodia and lamella of migrating cardiac fibroblasts is far from stationary with respect to their substrate. In a process termed “retrograde flow,” the pressure of actin polymerization at the leading edge and contractility within the cell results in a net movement of the actin cytoskeleton away from the leading edge of the cell (6, 113). In the trailing edge, actin filament disassembly provides the majority of the force required for retraction (113). Combined retrograde actin flow and myosin II activity appear to be important in the development of new adhesions in fibroblast lamella regions (10). In addition to F-actin, adhesion proteins such as zyxin and VASP also undergo retrograde flow at regions of cell-matrix adhesion in the lamella of migrating fibroblasts (107, 188). The lamellipodium is characterized by more rapid actin turnover and retrograde flow (which involves the Arp2/3 complex and downstream actin depolymerizing factor cofilin) than the more central lamellum, which contains more mature adhesions and harbors contractility components such as tropomyosin and myosin II (6, 216, 261, 394). Activation of Rac at the leading edge induces lamellipodia formation, and Rac directly activates WASP-family verprolin-homogolous protein (WAVE), which in turn causes Arp2/3 activation (326, 371, 494). WASP and WAVE proteins bind to the actin monomer-recruiting protein profilin; however, studies of these interactions have not yet been completed in cardiac fibroblasts (376). The inhibitory protein Arpin also acts at the lamellipodium downstream of Rac to inhibit Arp2/3 and control directionality during fibroblast migration (120). Thus, a balance appears to exist between Rac-Arpin-Arp2/3 inhibition signaling and Rac-WAVE-Arp2/3 activation signaling, though how these pathways are regulated in the cardiac fibroblast remains unknown. Vinculin, a protein found in focal complexes and adhesions, also appears to regulate retrograde actin flow by coupling the actin cytoskeleton to cell-matrix adhesions and acting as a molecular clutch. Vinculin expression in MEFs slows F-actin flow and promotes the development of nascent adhesions, while inhibiting their maturation, within the lamellipodium (501). In cells with disrupted vinculin-actin binding, establishment of the lamellipodium-lamellum border and generation of traction forces are inhibited.

Cardiac Fibroblast Proliferation The numerous players involved in cell proliferation and progression of the cell cycle have been well characterized in general, and are beyond the scope of this review. However, excessive proliferation of cardiac fibroblasts, coupled with failure to remove fibroblasts and/or myofibroblasts from healing sites of injury, contributes to fibrosis; thus a brief overview


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is appropriate. In eukaryotes, the cell cycle consists of four phases separated by a system of checkpoints to govern cell progression to the next phase. If the cell is damaged, for example, it will undergo cell cycle arrest at a specific checkpoint, and will not proceed further through the cycle (152,193,354). Along with the system of checkpoints, the cell cycle is also mediated by serine/threonine kinases called CDKs, which are activated through the binding of cyclin proteins and participate in signaling mechanisms that guide the cell through to the next phase of the cycle. CDKs are also regulated by phosphorylation, subcellular localization within the nucleus, and CDK inhibitors (339-341, 456). The cell cycle begins in the first gap phase (G1 )—the longest phase within the cycle. While the cell is in G1 , the activated CDK4/Cyclin D complex phosphorylates the retinoblastoma protein (Rb) to form phospho-Rb. Late in G1 , the CDK2/Cyclin E complex further phosphorylates phosphoRb, which is then able to activate E2F transcription factors to induce the expression of genes required for the next phase (151, 193, 300, 352, 353, 455, 456, 553). The cell then enters the S phase and DNA synthesis commences. In S phase, the CDK2/Cyclin A complex is activated, leading the cell to the second gap phase G2 (193, 483, 567). During the G2 phase, the Cdc2/Cyclin B complex becomes active and permits progression to the mitotic phase and cell division (193, 255). While this general mechanism holds true for most eukaryotic cells, different cells may employ different regulatory mechanisms. In cardiac fibroblasts, several proteins and factors have been found to play regulatory roles in the cell cycle and cellular proliferation. For example, ERK 1/2 is activated by growth factor-mediated intracellular signaling, and mediates proliferation, differentiation and apoptosis of cardiac fibroblasts (323, 368, 568). ERK 1/2 has been shown to enhance proliferation by indirect downregulation of the CDK inhibitor p27 in response to mitogen signals (127). ERK 1/2 upregulates S-phase kinase-associated protein 2 (Skp2), which negatively regulates p27 through post-translational mechanisms, and also downregulates forkhead box O 3a (FOXO3a), which normally enhances transcription of p27. Through this reciprocal regulation of Skp2 and FOXO3a, ERK1/2 promotes cardiac fibroblast proliferation (399). AngII induces proliferation of adult cardiac fibroblasts via activation of ERK1/2. AngII binds to the AT1 receptor, activating PLCγ which then cleaves membrane bound phosphatidylinositol-3,4-bisphosphate to produce inositol 1,4,5-trisphosphate (IP3 ) and diacylglycerol (DAG). IP3 facilitates the mobilization of intracellular calcium stores while DAG activates PKCδ (96,112,365,419). AngII signals specifically through the PKCδ isoform. Both of these pathways operate in parallel to activate ERK1/2. Inhibition of both pathways simultaneously is needed to significantly attenuate ERK1/2 activation, illustrating the importance of this convergence (365). AngII-induced ERK1/2 activation is not PI3Kdependent and does not involve EGFR transactivation or c-Src within the signaling pathways. This further illustrates how different adult cardiac fibroblasts are from other cell types

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including neonatal cardiac fibroblasts, which utilize EGFR transactivation in their AngII-induced proliferation pathway (365). PDGF is an important inducer of proliferation in cardiac fibroblasts. The PDGF-BB isoform stimulates the MAPK signaling cascade through a PKC-dependent pathway to regulate DNA synthesis and thus proliferation. PKC can either activate Raf1 directly through phosphorylation or indirectly via activation of Ras. Raf1 in turn phosphorylates ERK1/2 to induce proliferation (39, 382). Despite the widespread heterogeneity of fibroblasts across tissues, experiments to confirm the details of known cell signaling mechanisms specifically in cardiac fibroblasts are relatively uncommon. However, both PDGF-AA and PDGF-BB have been shown to induce early genes such as c-myc and c-fos, indicators of proliferation induction, through the activation of MAPK (464). A number of growth factors such as PDGF and FGF bind to RTKs to induce proliferation through Akt in myriad cell types. RTKs recruit the p85 and p110 PI3K subunits to form the active enzyme, which in turn catalyzes the conversion of PIP2 to phosphatidylinositol-3,4,5-trisphosphate (PIP3 ) (230, 298,472). PIP3 recruits Akt to the plasma membrane enabling phosphorylation by PDK1, resulting in both Akt activation and translocation into the nucleus (5, 566). In the nucleus, p70S6 Kinase (p70S6K) is activated downstream of phosphoAkt. p70S6K then phosphorylates the S6 protein in the 40S ribosomal subunit to mediate cell growth and proliferation (Fig. 5) (58, 464). PI3K may also associate with the AT1 receptor, allowing AngII to promote Akt phosphorylation as well (538). Hepatocyte growth factor (HGF) is one of several factors that act as an antifibrotic cytokine within the heart. HGF binds within the Sema domain of its receptor c-Met. Ligand binding induces dimerization of two receptor molecules, leading to its activation and phosphorylation (235, 263, 478). In cardiac fibroblasts, HGF appears to negatively regulate proliferation through induction of the c-Met-Akt-TGIF pathway: TGIF, a Smad transcriptional corepressor downstream of c-Met, inhibits proliferation by blocking the nuclear translocation of phospho-Smad2/3 to attenuate TGFβ signaling (297,574). Loss-of-function studies have supported this mechanism in cardiac fibroblasts. Knockdown of HGF induces expression of Proliferating Cell Nuclear Antigen, Ki67 and Cyclin D1, which increases the overall TGFβ-induced proliferative response (574). HGF downregulation also has an inhibitory effect on the phosphorylation of c-Met and Akt (574). Cardiac fibroblasts can also respond in an autocrine fashion to exogenous signals to induce proliferation. For example, evidence of the cAMP-adenosine pathway has been found in cardiac fibroblasts through the use of specific enzyme inhibitors (142). This pathway is induced by exogenous factors such as hormones, resulting in stimulation of adenylyl cyclase to produce cAMP which can itself signal or be converted intracellularly by phosphodiesterase to AMP, which is then converted into adenosine by 5′ -nucleotidase. Adenosine can be transported into the extracellular space via facilitated

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transport mechanisms, where it activates A2 receptors in an autocrine fashion (41, 142). The overall effect of A2 receptor activation is antimitogenic, attenuating cellular growth and proliferation (143, 144). This pathway may also take place more efficiently in the extracellular space catalyzed by ectophosphodiesterase and ecto-5′ -nucleotidase, since adenylate kinase competes for AMP within the cytosol (142). Negative regulators of proliferation in cardiac fibroblasts may serve a cardioprotective role at times of myocardial injury or hypertrophy, protecting against pathological persistence of fibroblasts within the heart (142). Conversely, dysregulation of this finely tuned, intricate web of proliferative signals in cardiac fibroblasts may contribute to fibrotic disease progression. It is also important to consider, however, that fibroblast proliferation represents an important step of the reparative process in the infarct region after MI. Thus, attempts to regulate fibrosis by targeting fibroblast proliferation must consider the nature of the underlying disease and reparative processes. Even in the case of MI, modulation of fibroblast proliferation may be viable since distal fibrosis typically occurs after the infarct scar has formed (116).

Fibroblast-to-Myofibroblast Phenotype Conversion Fibroblasts are not only responsible for the secretion and maintenance of ECM components, but also in regulating the transmission of mechanical and electrical stimuli within the healthy working heart (72, 425). Upon myocardial injury, in response to stress including hypoxia or cell stretch, or following stimulation by profibrotic growth factors such as TGFβ, fibroblasts undergo a phenotype conversion to myofibroblasts (Fig. 1) (116). While this conversion is frequently referred to in the literature as a differentiation process, this nomenclature is difficult to rationalize since fibroblasts already clearly exhibit signs of differentiation such as SFs, and it would be difficult to argue that fibroblasts are undifferentiated. Rather, this conversion may better be considered as a “fine-tuning” of the cell phenotype to meet the changing demands of the surrounding tissue. There are multiple factors and interconnecting pathways that modulate fibroblast-to-myofibroblast phenotype conversion. The dysregulation of any one of these regulatory pathways may result in either the overaccumulation of collagens or impaired healing of the injured heart, making phenotype conversion a double-edged sword. Although myofibroblasts are required to heal the injured myocardium following MI, they are also the primary drivers of tissue fibrosis. Thus, these pathways have been the subject of extensive research, and while our picture of the phenotype conversion process is more complete than ever, significant gaps in our understanding remain. For example, there is debate as to whether an intermediate cell type, called the proto-myofibroblast, arises during fibroblast phenotype conversion in vivo, although a number of laboratories have reported the existence of these cells in vitro


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GSK3

Cyclin D1 P70S6 Kinase

40S subunit

P S6 protein

Cell growth and proliferation

Figure 5 Akt-mediated regulation of cardiac fibroblast proliferation. Growth factors such as PDGF and FGF bind to their cognate receptors, resulting in associated receptor tyrosine kinase (RTK) activation. Upon ligand binding, PI3K subunits p85 and p110 are recruited to the receptor to form the activated PI3K enzyme, catalyzing the conversion of PIP2 to PIP3 . PIP3 recruits Akt to the plasma membrane, where it is phosphorylated by PDK1 thereby promoting its translocation into the nucleus. p70S6 Kinase is activated downstream of phospho-Akt and in turn phosphorylates the S6 protein within the 40S ribosomal subunit, positively regulating cell growth and proliferation. Phospho-Akt further promotes cell proliferation by phosphorylating and thereby deactivating GSK3. GSK3 flags Cyclin D1 for translocation into the cytoplasm of the cell, make it unavailable to regulate the cell cycle and resulting in cell-cycle arrest.

(139, 209, 508). The proto-myofibroblast is characterized by increased expression of FA proteins, and the first stages of SF formation. Proto-myofibroblast SFs are composed of βand γ-actins but specifically lack αSMA, despite the fact that αSMA expression is increased in these cells (139, 508).


Additionally, proto-myofibroblasts show TGFβ-induced activation of Rho-GTPase and ROCK, facilitating polymerization of G-actin into F-actin. However, while proto-myofibroblasts can be readily detected in vitro, the many technical challenges of studying cell adhesions in vivo leave the existence of

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proto-myofibroblasts during tissue pathogenesis in doubt (209). Myofibroblasts represent an activated form of fibroblast, and are the primary mediators of cardiac wound healing (116). In contrast to fibroblasts, myofibroblasts exhibit mature SFs containing actin and myosin, which unlike protomyofibroblast SFs, also have αSMA incorporated. These SFs exert tension on the ECM and facilitate the production of contractile force by the myofibroblast, yet at the same time oppose retractile force, aiding the physical remodeling of the ECM (72, 121, 145, 425, 508). Phenotype conversion of fibroblasts occurs very quickly and is mainly induced by mechanical tension, deposition of the ED-A splice variant of fibronectin, and TGFβ stimulation, although many other factors have been found to affect this process both positively and negatively (see below) (416, 448, 484). Indeed, the conversion process is easily activated in vitro, providing a challenge for the study of primary fibroblasts once isolated from cardiac tissue. Tissue culture plates that are most commonly used for cell culture are highly rigid, with stiffness several orders of magnitude higher (within the MPa range) than even highly fibrotic tissue (207). As a result, by the time isolated fibroblasts have been expanded in culture prior to experimentation, they have already begun expressing αSMA, and thus have already begun converting to proto-myofibroblasts or myofibroblasts. This can occur even before the very first cell passage, and makes studying these phenotypes challenging (209, 425).

Positive regulation of phenotype conversion In cardiac fibroblasts, TGFβ1 stimulation induces an elevation in collagen production and αSMA expression in a dosedependent manner (66, 132, 388). TGFβ1 induces cardiac fibroblast phenotype conversion to myofibroblasts through canonical Smad signaling (157). There is also evidence to suggest that TGFβ1 signaling converges with the AngII pathway to induce the myofibroblast phenotype. It has been shown that TGFβ1 induces expression of the angiotensin converting enzyme (ACE) protein, which is essential for the production of AngII (389). Pharmacological suppression of AngII by resveratrol or losartan results in the prevention of TGFβ1 induced fibroblast phenotype conversion and the reduction of Smad2 and Smad4 expression, respectively (136, 364). AngII has also been shown to induce TGFβ1 expression in cardiac fibroblasts (74, 284). This cross-talk may behave as a positive feedback mechanism, in which TGFβ1 induces more TGFβ1 production in an autocrine fashion through mediation of ACE. Although the effects of these cytokines on cardiac fibroblasts are well characterized, the precise molecular mechanisms remain to be elucidated. TGFβ has many origins including secretion both from resident cardiac cells and infiltrating inflammatory cells, as well as inactive stores within the tissue itself, emphasizing the importance of this cytokine in many organs including the heart. Generally, TGFβ is synthesized and secreted in a homodimeric protein pro form, consisting of a TGFβ

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molecule covalently linked to latency-associated protein (LAP). The resulting small latent complex (SLC) prevents TGFβ from associating with its receptor (11). TGFβ can also be secreted as a large latent complex (LLC), composed of a SLC linked to latent TGF-binding protein (LTBP) by a disulfide bond (420). LTBPs bind to ECM proteins such as fibrillin-1, vitronectin and fibronectin, creating a TGFβ store within the ECM (11, 441, 490, 497). Latent TGFβ is released as an active cytokine following proteolytic cleavage by enzymes that degrade ECM, including the MMP family (11, 118, 305, 577). Glycosidases release active TGFβ by removing carbohydrates from the SLC, resulting in a conformational change of the complex and TGFβ release (332). Thrombospondin-1 also releases TGFβ from the SLC or LLC through the disruption of the bond between LAP and TGFβ (346). Aside from latent TGFβ already present within the ECM and interstitium of the tissue, TGFβ may also be secreted by activated macrophages during wound healing (21). Neonatal cardiac fibroblasts have also been shown to secrete TGFβ1 and TGFβ2 in response to norepinephrine and AngII stimulation, working in an autocrine and paracrine fashion (160). The endothelin family of biopeptides consists of endothelins 1-3 (ET1-3). The majority of ET1 is generated by endothelial cells and is produced in the heart, kidney, pituitary, and central nervous system (225, 226). ET1 is a potent vasoconstrictor and is known to increase blood pressure and vascular tone (4). ET1 is also involved in wound healing postmyocardial injury, and leads to phenotype conversion of cardiac fibroblasts to myofibroblasts (356). One laboratory has reported that overexpression of TRPC6 or constitutively activated NFAT in cardiac fibroblasts attenuated the effect of ET1 and reduced fibroblast-to-myofibroblast conversion (270). In sharp contrast, however, more recent studies have implicated TRPC6 as a profibrotic factor: deletion of the TRPC6 gene impaired both cardiac and dermal wound healing, and TRPC6 was sufficient to increase type I collagen expression and fibroblast phenotype conversion via induction of calcineurin, the Ca2+ -dependent phosphatase that activates NFAT via dephosphorylation (122). Haploinsufficiency of the TGFβ coreceptor endoglin attenuated activation of the calcineurin/TRPC6 pathway and reduced expression of αSMA (236). Other TRP channels such as TRPC3 and TRPM7 have also been implicated in the phenotype conversion of fibroblasts to myofibroblasts and in atrial fibrosis, and the common mode of action of these channels appears to be facilitating entry of Ca2+ to activate calcineurin/NFAT signaling (502, 578). The remodeled matrix itself also contributes to fibroblast phenotype conversion. As the ECM is remodeled and disrupted, fibroblast protection from mechanical stress is reduced, resulting in fibroblast phenotype conversion through the Rho/ROCK signaling cascade (508, 588). Through this pathway, mechanical force may induce αSMA expression and ultimately induce fibroblast to myofibroblast phenotype conversion.


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There is growing evidence that microRNAs can play a role in fibroblast phenotype conversion. MicroRNAs suppress protein expression by either facilitating mRNA degradation or by inhibiting translation (29, 50). One such example is miR145, which has been reported to induce the myofibroblast phenotype similar to TGFβ. miR-145 overexpression upregulated the myofibroblast markers fibronectin, vimentin, and αSMA as well as connexin 43, and downregulated the fibroblast marker DDR2 in cultured fibroblasts, indicative of a shift toward the myofibroblast phenotype. The cells also formed highly organized SFs and F-actin bundles, providing contractility, as well as enhanced deposition of mature collagen type I (543). It has been shown in vascular smooth muscle cells that miR-145 regulates cellular phenotype through the negative regulation of KLF5, which itself is a negative regulator of myocardin (94). Wang et al. reported that this pathway exists in cardiac fibroblasts. At 3 days following MI, miR-145 was downregulated, alleviating its effect on KLF5 expression and resulting in decreased myocardin expression. At 7 days post-MI, miR-145 expression returns to basal levels, reducing KLF5 expression and in turn upregulating myocardin (94,543). In contrast, however, recent work has suggested that myocardin is not endogenously expressed by cardiac fibroblasts (470). This discrepancy could be explained by the fact that myocardin and MRTFs are very closely related and share homology in multiple functional domains [as reviewed by Pipes et al. (393)], thus it may be possible for MRTFs to be detected using supposedly myocardin-specific primers. Both proteins are associated with SRF (535). Myocardin is primarily localized within the nucleus whereas MRTFs are bound to G-actin via their N-terminal RPEL domain (269, 470). As described above, ROCK signaling reorganizes actins within the cytoplasm and releases the MRTFs, making them available to translocate into the nucleus where they enhance smooth muscle gene expression (304, 329, 588). Small et al. found a 50% reduction in scar size within the hearts of MRTF-A null mice (470). This was accompanied by attenuated upregulation of SM22, αSMA, Col1α1, Col1α2, Col3α1, and elastin post-MI, consistent with a diminished myofibroblast phenotype. However, the MRTF-A null mice did manage to create sufficient scar to prevent cardiac rupture. This research group also found that inhibition of ROCK signaling with Y27632 partially attenuated MRTF-A translocation and TGFβ1 -mediated collagen synthesis, providing evidence of a new potential treatment option to prevent excessive fibroblast differentiation without risking cardiac rupture.

Negative regulation of phenotype conversion TGFβ signaling is negatively regulated by inhibitory I-Smad7, which both interferes with phosphorylation of RSmad2/3 by the TGFβ receptor and facilitates degradation of the type I TGFβ receptor subunit through the recruitment of Smurf2 (242). The proto-oncoprotein Ski has also been shown to negatively regulate cardiac fibroblast phenotype conversion


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(115). Ski inhibits Smad signaling by binding to DNA-bound R-Smad2 and repressing the transcription of Smad-activated genes (488). Though research to date has specifically focused on R-Smad2, it has been hypothesized that Ski negatively regulates both R-Smads (Fig. 2) (115). Through interference with Smad-mediated gene regulation, Ski downregulates the production of Zeb2 (Smad interacting protein-1) (114). Zeb2 acts as a negative regulator of mesenchyme homeobox 2 (Meox2), which has been shown to inhibit conversion to the myofibroblast phenotype by downregulating αSMA and ED-A fibronectin expression (90, 114). The attenuation of Smad signaling and Zeb2 expression by Ski is alleviated following its translocation to the cytoplasm, resulting in induction of the profibrotic gene expression program (114, 115). It has been reported that Ski is upregulated within the cardiac infarct scar, but is sequestered in the cytoplasm, leaving Zeb2 to repress Meox2 and resulting in upregulation of αSMA, ED-A fibronectin, and the myofibroblast marker smooth muscle embryonic myosin heavy chain (114, 115). Atrial natriuretic peptide (ANP)/cGMP/protein kinase G (PKG) signaling may serve a cardio-protective role by interfering with TGFβ signaling (286). ANP induces upregulation of cGMP, which in turn activates PKG (404). Activated PKG phosphorylates Smad3, but this occurs at different sites than those phosphorylated by the TGFβ receptor kinase, thus inhibiting the translocation of the Smad-complex to the nucleus and subsequently inhibiting collagen synthesis, fibroblast proliferation, and phenotype conversion (286). Elevated BNP and nitric oxide levels similarly attenuate TGFβ expression through the activation of cGMP (281, 417). Another factor regulating fibroblast phenotype is bFGF, which besides being a potent mitogen has been shown to prevent cardiac fibroblast phenotype conversion and to act as an antifibrotic factor. In cardiac fibroblasts from hypertensive rat hearts, bFGF downregulated αSMA and collagen types I and III expression (254). It also downregulated expression of MMP2, MMP9, and TIMP2 while upregulating TIMP1. This effectively attenuated both remodeling of the ECM and collagen production.

The Cardiac Myofibroblast Myofibroblasts can arise from a number of different sources. Besides conversion from resident fibroblasts within the myocardial interstitium, myofibroblasts may also arise from differentiation or phenotype conversion from other cells such as circulating myeloid or bone marrow-derived cells, pericytes, fibrocytes, and epithelial cells from the epicardium (1, 335, 402, 582). A recent study of kidney fibrosis found that approximately 50% of myofibroblasts arose from resident fibroblasts, with 35% from differentiation of bone marrow-derived cells and the remainder from epithelial- or endothelial-to-mesenchymal transition (283). It is unclear, however, whether myofibroblasts in the fibrotic or healing heart arise from similar sources, or in similar ratios.

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Stress fibers SFs are the major contractile structure in numerous nonmuscle cell types, with the same basic macromolecular architecture as the contractile apparatus in smooth muscle cells. SFs are comprised of large bundles of actin filaments that often span a cell’s diameter, and are anchored at one or both ends by FAs (178). They contain actin-myosin-based contractile proteins such as tropomyosin and filamin. Cross-linking between SFs is achieved via periodically distributed α-actinin, alternating with myosin II. The presence of myosin filaments is responsible for the contractility of SFs observed in fibroblastic cells of many tissue sources, and can be used to regulate isometric tension within the cell (60, 240, 268, 385). Studies have demonstrated that central SFs are mostly regulated by the RhoA pathway, whereas peripheral SFs appear to be regulated primarily by MLCK activity (237, 238, 240). Two types of contraction are observed in cultured cardiac fibroblasts: that which is calcium-dependent, and regulated via a calmodulin/MLCK signaling system; and calcium-independent contraction, regulated by ROCK2 activation (8, 9). Generally, calcium-dependent contraction tends to be short-lived and rapid, whereas RhoA-mediated contraction is sustained by flexible control of myosin phosphatase activity (through ROCK; Fig. 6) (513). Cardiac myofibroblasts are characterized by the formation of SFs that incorporate αSMA. Migrating fibroblasts and those cultured on soft substrates do not develop SFs, and they are rarely observed in situ, although they have been identified in wounded dermis (60, 173, 203, 384, 508). On immobilized collagen gels, isometric tension induces the development of SFs in fibroblasts. However, when gels are released, contraction occurs and SFs are disassembled (183, 191, 333, 509). Three different manifestations of SFs have been identified in myofibroblasts, that is, ventral SFs, dorsal SFs, and transverse arcs. Most common usage of the term “SF” refers to ventral SFs, which are the most commonly observed and which are anchored at both ends by FAs, often spanning the length of the cell. Dorsal, or radial, SFs are anchored at only one end by a focal complex or adhesion, and are likely precursors to ventral SFs. In migrating cells, dorsal SFs form at the leading edge. In contrast to these two types of SFs, transverse arcs are not anchored by FAs and are generally much smaller and convex, developing at the base of lamellipodia in spreading and migrating NIH-3T3 fibroblasts (200,473,541). Although they are not considered SFs by traditional definitions, transverse arcs may also give rise to ventral SFs (214). SFs anchored by one or both ends by FAs are composed of F-actin cross-linked by a periodic pattern alternating between α-actinin and myosin II (61). Both RhoA and ROCK have been found to bind SFs, and Rho-associated GTPases control the formation and disassembly of SFs (331, 408, 409). In line with the development of SFs, culture of fibroblasts on rigid substrates induces RhoA activation, in turn activating downstream effectors such as ROCK/ROCK2 (379, 562). Activation of ROCK2 mediates

744

Mechanical strain

Calcium ion influx

Ca2+

Integrin

Plasma membrane

TRPC

Cytoplasm

Ca2+

RhoA Ca2+

Calmodulin

ROCK MP MLCK

P

F-actin

MLC

Slow, sustained contraction

MLC

MLC

Rapid, transient contraction

Figure 6

Myofibroblast contractile response. Contraction of stress fibers occurs via calcium-dependent and -independent signaling pathways in cardiac fibroblasts. Calcium-dependent contraction is typically rapid and occurs primarily in peripheral stress fibers. Through receptors such as transient receptor potential cation (TRPC) channels, calciumdependent contraction occurs in response to an influx of Ca2+ ions, which form a complex with and activate calmodulin, resulting in myosin light chain kinase (MLCK) activation and subsequent phosphorylation of myosin light chain (MLC) bound to stress fibers. However, contraction is not sustained due to the activity of myosin phosphatase (MP) within the cell, which dephosphorylates and inactivates actin-bound MLC. Conversely, calcium-independent contraction is relatively slow and acts primarily on central stress fibers in sustained contractile events, usually in response to mechanical strain. Calcium-independent contraction occurs primarily through integrin activation and downstream RhoARho-GTPase-associated kinase (ROCK) signaling, which inhibits MP to maintain phosphorylation of MLC, allowing for prolonged contractile events.

the organization of SFs and FAs and its inhibition with Y-27632 causes disassembly of centrally located ventral SFs (7-9, 237, 241, 409). The RhoA/ROCK2 complex is localized on SFs via myosin phosphatase-RhoA interacting protein/ M-RIP (407). ROCK2 then phosphorylates MLC in a calcium-independent manner either directly or indirectly. ROCK2-dependent phosphorylation of MLC is essential for


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fibroblast ventral SF formation, and occurs by T697 phosphorylation of the 130 kDa myosin-binding subunit of myosin phosphatase (159, 227, 238, 245, 253, 344, 406, 512, 556). Thus, myosin phosphatase activity upon MLC is inhibited, which also is necessary for sustained contraction. Additionally, RhoA and ROCK activation induces LIM kinase activation, which inhibits cofilin phosphorylation, rendering it inactive (306). Because cofilin mediates F-actin severing, ROCK activation of LIM kinase also contributes to increased F-actin content and RhoA-induced ventral SF assembly (14, 25, 63, 275, 500). Cofilin severing of actin filaments (and subsequent SF disassembly) is also inhibited by cellular tension—less cofilin is bound to actin filaments with tension than in fibroblasts “at rest” (198). In dorsal SFs, RhoA activation of the effector molecule mDia1 drives actin polymerization (214, 361). Nonetheless, both arms of the RhoA pathway (i.e., ROCK and mDia1) are necessary for normal SF patterning in myofibroblasts. Watanabe et al. determined that in the absence of RhoA and ROCK, mDia1-mediated SF formation resulted in sheet-like filaments with no discernible “bundles” of F-actin. In the absence of both RhoA and mDia1, ROCK-induced SFs were increased in size and appeared in a star conformation rather than longitudinally along the cell (544). The ability of SFs to cause cardiac myofibroblast contraction lies in their architecture. Rather than being composed of unidirectional F-actin filaments, SFs have unipolar filaments that are oppositely oriented at each myosin bridge. That is, a unipolar filament is formed with actin polymerization and SF formation, but requires cleavage and capping, followed by actin nucleation at the other side of the “cap” to generate filaments polarized in the opposite direction, which are eventually joined by myosin II (63). Though the proteins involved in these cleavage and capping functions have not been identified in cardiac fibroblasts, it is possible that the actin-severing protein formin may fulfill cleavage, capping, and elongation functions (195). Recruitment of myosin II is likely dependent on tropomyosin, an actin-binding protein recruited to various actin filament populations (186, 187). In the absence of tropomyosin, SFs spontaneously disassemble, possibly through elevation of cAMP, which has also been shown to cause SF disassembly (23, 55, 190, 273). cAMP elevation results in phosphorylation of RhoA [dependent upon protein kinase A (PKA) activity] which causes RhoA sequestration by enhancing its binding to Rho guanine nucleotide dissociation inhibitor (153,274,429). Additionally, cAMP elevation induces PKA phosphorylation of MLC kinase, in turn inhibiting its kinase activity in phosphorylating MLC (273).

Contraction Unlike contraction in muscle cells, which can be nearly instantaneous and involves actin-myosin cross-bridge cycling, contraction of cardiac myofibroblasts is slower, often more sustained, and depends upon the phosphorylation state of the


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light chain of myosin II bound to SFs. In myofibroblasts with a myosin II mutation resulting in disrupted motor activity, actin binding capability remained although the cells were unable to generate SF-induced contractile force (99). SFs isolated from fibroblasts with Triton X-100 detergent extraction contract in response to Mg2+ -ATP treatment in the presence of calcium ions, a process caused by phosphorylation of MLC via calmodulin and MLCK signaling, the same mechanism by which smooth muscle contraction occurs (239, 240). Treatment of fibroblasts with RhoA activators (e.g., lysophosphatidic acid and bombesin) induces SF reorganization and contraction (408, 409). ROCK activation is calcium independent and relies on upstream RhoA activation (238, 513). ROCK interacts with SFs by binding directly to myosin II (243). ROCK activation induces SF contraction via phosphorylation of MLC, both directly by phosphorylation (S854/T697) and indirectly through inhibition of myosin phosphatase (Fig. 6) (8, 9, 238, 513).

Cardiac myofibroblasts and migration A growing body of evidence produced by studying fibroblast to myofibroblast phenotype conversion indicates that myofibroblasts are less motile than their precursors (425). This is likely attributable to the increased size and strength of FAs in myofibroblasts. Decreased migration of cardiac myofibroblasts logically follows increased adhesion, synthesis of matrix components, reduced MMP activity, and increased contractility. Although the reduction in motility observed in myofibroblasts can likely be attributed primarily to increased adhesions and synthesis, there is the potential for the involvement of distinct signaling pathways, though these remain undetermined. Wnt-Frizzled signaling is implicated in phenotype conversion, and reduces migration in telomerase-immortalized cardiac fibroblasts (271). However, dishevelled-1, a signaling component of the Wnt-Frizzled pathway, appears to be associated with migration of cardiac fibroblasts during post-MI wound healing, appearing in the border zone of the infarct 1 day post-MI and in both the border zone and infarct scar after 4 days (86). Additionally, inhibition of MMPs results in increased density of the matrix and may also downregulate migration. For example, TIMP-4 is capable of inhibiting myofibroblast motility (517). Conversely, some studies report increased migration by myofibroblasts compared to fibroblasts. On gelatin-coated substrates, TGFβ induced migration of myofibroblasts, and Smad3-null MEFs have shown a decrease in migratory ability (137, 592). Another study found that TGFβ1 induced MMP activity in cardiac fibroblasts, facilitating their motility, through amplification of the activity of the convertase furin, which acts to activate TGFβ1 , and downstream MMP4 activation (480). Thus, although the majority of the evidence is supportive of decreased myofibroblast motility, controversy remains regarding not only the migratory capacity

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of these cells, but also the definition of “fibroblast” and “myofibroblast” in vitro. Two issues should be considered in resolving this paradox: first, the heterogeneity of cells in culture derived from primary tissues introduces the possibility that, at the time of assay, some cells are myofibroblasts while others, presumably with higher motility, retain their fibroblast character, and it is these latter cells that actually migrate (e.g., in scratch assays). The second possibility is that fibroblasts migrate initially, followed by conversion to the myofibroblast phenotype. Careful experimental design, such as the performance of mobility assays followed immediately by fixation and immunostaining for fibroblast- and myofibroblast-enriched markers, may help to address this issue.

Increased matrix production by myofibroblasts Myofibroblasts are characterized by hyper-secretion of ECM components such as type I collagen, periostin, fibronectin, and ED-A fibronectin (448, 508). The healthy myocardium is largely devoid of myofibroblasts; however, these cells become abundant within a few days after myocardial injury such as MI (486). Myofibroblasts are also defined by the de novo expression of smooth muscle cell genes including caldesmon, SM22, and αSMA, the latter being incorporated into SFs and increasing cell contractility as noted above (131, 209, 277, 400, 426). Excessive production and remodeling of ECM components by myofibroblasts directly leads to cardiac fibrosis. As mentioned previously, TGFβ plays a prime role in the phenotypic conversion of fibroblasts to myofibroblasts (206, 280, 534). TGFβ stimulation of either cell type induces ECM gene expression and deposition of ECM components, simultaneously with alterations in expression of MMPs and TIMPs. This paradigm is not limited to the heart but extends to most tissues in the body (122, 390, 534, 584). The canonical TGFβ-Smad2/3 signaling pathway directly transactivates ECM genes including Col1α1 and Col1α2, as well as MMPs and TIMPS (22, 156, 527). Hearts from Smad3 null mice exhibit attenuated collagen production and deposition, and reduced fibrotic remodeling following MI (137). Additionally, the transcription factor scleraxis has been shown to tightly coordinate the regulation of type I collagen expression in synergy with R-Smad3 in primary isolated cardiac fibroblasts and myofibroblasts (22, 156). The noncanonical signaling pathway initiated by TGFβ binding to TGFβ receptor subunit II induces recruitment of TAK1 and TAK1 binding protein/TAB followed by activation of MAPK signaling cascades including JNK and p38 MAPK (123). Targeted inhibition of noncanonical TGFβ signaling using TGFβ receptor subunit II KO mice resulted in reduced fibrosis and remodeling in the pressureoverloaded myocardium (262). Inhibition of p38 signaling can also block TGFβ-induced myofibroblast traits including expression of type I collagen, fibronectin, αSMA-positive SF formation, and collagen gel contraction (122). Collectively, these studies identify TGFβ signaling pathways as critical drivers of myofibroblast-mediated fibrosis.

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AngII is elevated in fibrotic pressure overloaded hearts and appears to positively affect myofibroblast activity and fibrosis including increased ECM production (197, 212). AngII treatment of cardiac fibroblasts leads to induction of TGFβ expression through the AT1 receptor causing upregulation of downstream fibrotic genes including type I collagen, an effect that could be blocked by the administration of losartan (74, 284). Activation of the Smad2/3 and MAPK signaling pathways occurs in response to AngII treatment in cardiac myofibroblasts (175,570). AngII thus appears to be a crucial regulator of the myofibroblast phenotype through multiple mechanisms. As described above, significant crosstalk also appears to exist between TGFβ and AngII signaling pathways, resulting in reinforcing and synergistic effects on fibroblast phenotype and function. ET1 production during cardiac injury induces myofibroblast conversion from primary cardiac fibroblasts in vitro (356). ET1 also strongly induced ECM production in fibroblasts (279). AngII is capable of increasing ET1 expression in cardiac fibroblasts, thus driving them to a myofibroblast phenotype (93). This effect can be blocked using losartan. TGFβ also works together with ET1 in driving the myofibroblast phenotype (457). Thus it appears that ET1 is a downstream synergistic mediator of TGFβ and AngII signaling, promoting fibroblast activation, maintenance of the myofibroblast phenotype and consequent tissue fibrosis (278, 280).

Senescence and cell death In dermal wounding, myofibroblasts arise as part of the healing process, but are lost through apoptosis once healing is completed (116). In contrast, cardiac myofibroblasts may persist for years, thus the discovery of an “off-switch” to remove myofibroblasts without adversely affecting proper maintenance of the ECM and the structural integrity of the heart would represent a therapeutic boon. To this end, understanding the factors governing myofibroblast senescence and death is of key concern. Cellular senescence refers to permanent cell cycle arrest, which occurs in most higher eukaryotic cells as they reach the end of their life cycle (75, 328). When a cell reaches senescence naturally at the end of its life cycle, it loses the ability to respond to mitogenic factors (28). Replicative senescence appears to serve as an excellent model for age-related senescence, since cultured cardiac fibroblasts at higher passages show similar signs of cellular senescence as primary fibroblasts taken from the hearts of older animals (134, 540). Senescence of cardiac fibroblasts remains a poorly understood area. Some groups have suggested that senescence is a way of limiting the fibrotic response, while others contend that senescent fibroblasts add to the problem, reporting that fibroblasts become larger, cease growth, and secrete more collagen (496, 540, 590). PDGF-AA is a strong mitogenic factor for cardiac fibroblasts (463). In older fibroblasts, PDGF-AA is less efficient at stimulating a mitogenic response and also showed attenuated


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expression of Akt, a required component in the signaling cascade for PDGF to stimulate proliferation; conversely, Akt overexpression in older fibroblasts partially restored responsiveness to PDGF (134). In other cell lines it has been shown that PIP3 recruits the serine/threonine kinase Akt to the plasma membrane, where it can be phosphorylated by PDK1 (5). Phospho-Akt in turn phosphorylates GSK3, thereby deactivating it and preventing GSK3-mediated phosphorylation of cyclinD1 which would result in cyclinD1 translocation into the cytoplasm, making it unavailable to participate in cell cycle signaling cascades. The phosphorylation of cyclinD1 thus ultimately results in cell cycle arrest (133). This pathway may be one of many involved in aging-related cellular senescence due to attenuated Akt expression. As fibroblasts age, F-actin fibers become disorganized and there is an upregulation of 4-hydroxynonenal, an aging marker, and β-catenin, suggestive of dysregulation in the Wnt/β-Catenin pathway (540, 557). This is accompanied by downregulation of p70S6K, Cdc42, Mdm2, LOX-1, and MSR1 (540). LOX-1, the receptor for oxidized low-density lipoprotein, is partially responsible for the downregulation of these other proteins (87, 540). The overexpression of LOX-1 in older fibroblasts increased the expression of Mdm2, pAkt, Cdc42 and p70S6K, although protein levels were still lower than normal basal levels (540). Cdc42 promotes cell cycle progression into the S phase (366). Changes in the expression of microRNAs may also play a role in age-related cellular senescence in cardiac fibroblasts. As the heart ages, miR-22 is upregulated in cardiac fibroblasts (229). miR-22 negatively regulates expression of osteoglycin, which leads to the induction of senescence through the upregulation of p16. miR-21 is upregulated in cardiac fibroblasts during heart failure and induces hypertrophy and tissue fibrosis through the induction of bFGF/FGF2 secretion and inhibition of apoptosis (504, 505). Bcl-2, an antiapoptotic factor, is positively regulated by miR-21 (138, 320). miR-21 has also been shown to induce activation of the ERK/MAPK pathway, thereby enhancing proliferation of cardiac fibroblasts (505). This poses a possible mechanism for apoptosis resistance in cardiac myofibroblasts and the persistence of the profibrotic response following myocardial injury. Although many targets for miR21 have been proposed, the specific protein(s) it targets to impact FGF2 secretion, apoptosis and proliferation remains to be elucidated (138). Though cellular senescence occurs naturally in cardiac fibroblasts, it also occurs in response to myocardial injury. The tumor suppressor p53 regulates many genes including those involved in apoptosis, senescence and cell-cycle arrest (532). p53 activity increases following myocardial injury, resulting in limitation of collagen deposition and tissue fibrosis (532, 590). In cardiac fibroblasts, it has been proposed that p53 directly decreases Col1α1 expression, inhibits proliferation through the activation of p21 and attenuates TGFβinduced CTGF expression through unknown mechanisms (496). Fibroblast Specific Protein-1 (FSP1) regulates collagen


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production and proliferation via the inhibition of p53 (496). FSP1 undergoes a conformational change when it binds to calcium and becomes activated. It dimerizes and modulates the binding of p53 to DNA by directly binding the protein (31, 182, 423). FSP1 is upregulated in cardiac fibroblasts of hypertensive rats, and FSP1 knockout has been shown to significantly reduce the fibrotic response following myocardial injury (496). This mechanism may help explain resistance to cellular senescence in cardiac fibroblasts that results in maladaptive healing after injury. During ischemic conditions within the heart, ROS are widely produced concomitant with elevated intracellular calcium levels. Reperfusion following ischemia results in protein degradation and ultimately ischemia/reperfusion injury, leading to cell death via necrosis or apoptosis (56, 98, 397, 467). pERK1/2 levels decrease during ischemia but are replenished following reperfusion whereas pAkt levels remain at basal levels during ischemia and are upregulated during reperfusion (531). It has been proposed that insulin-like growth factor-1 (IGF-1) is produced by cardiac fibroblasts and could play a cardio-protective role following myocardial injury (492). IGF-1 resists pERK1/2 downregulation during ischemia, further upregulates pAkt during ischemia and reperfusion and also partially prevents cell death during reperfusion. Apoptosis induction is inhibited through the activation of both the MEK-ERK and PI3K/Akt pathways simultaneously (531). Apoptosis may also be induced by severe hypoxia (101). Hypoxia inducible factor-1α (HIF1α) protects against excessive apoptosis in response to hypoxia. HIF1α is the subunit of heterodimeric HIF1 that regulates gene expression and helps restore oxygen homeostasis (536, 552). The proapoptotic effects of hypoxia include the induction of Bax expression, increased intracellular calcium levels, caspase-3 activation, and downregulation of antiapoptotic Bcl-2 (569). Simultaneously, hypoxic conditions result in the upregulation of HIF1α within the nucleus, which not only inhibits the proapoptotic effects of hypoxia but also promotes increased fibroblast viability (569). Thus, although proapoptotic and antiapoptotic signals are being transduced simultaneously, it is the net effect of these two signals that will ultimately determine the cell fate.

Summary and Conclusions Cardiac fibroblasts and myofibroblasts are central players in the health and diseases of the heart, yet remain poorly understood in comparison to their myocyte neighbors. Concerted efforts over the past decade, by an ever-increasing tally of research laboratories, have finally begun to reveal the innermost workings of these intriguing cells (Table 3). This work has been driven forward not only by intellectual curiosity, but also by the burgeoning need to develop novel therapeutics against unexploited targets in the treatment of cardiovascular diseases. Attempts to therapeutically target pleiotrophic

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Table 3


Major Effectors and Signaling Pathways of Key Cardiac Fibroblast/Myofibroblast Biological Functions

Biological function

Cellular process

Major regulatory effectors and pathways

Extracellular matrix synthesis and degradation

Extracellular matrix production

AngII (RAAS), bFGF, CTGF, PDGF, TGFβ

Matrix remodeling (MMPs, TIMPs)

AngII, BNP, IL-1β, PDGF, TGFβ, TNFα

Phenotype conversion of fibroblasts to myofibroblasts

Cell-matrix adhesions

Migration

Myofibroblast hypersynthesis of ECM

AngII, ET1, TGFβ

Positive regulators

AngII, ET1, miR-145, MRTF-A, Rho-ROCK, TGFβ,TRPC3, TRPC6, TRPM7

Negative regulators

ANP/BNP, bFGF, I-Smad7, Meox2, NO, Ski, Smurf2

Adhesion formation

FAK, mDia1, Rac, RhoA

Adhesion maturation

Ras-Raf-MAPK-ERK, RhoA, Syn 4

Mechanosensing/mechanotransduction

FAK, integrins, p130Cas, RhoA-ROCK, Yap/Taz

Cell polarization

FAK, Rac1, RhoA

Adhesion turnover

Rac1

Retraction of trailing edge

Rac, vinculin

Cardiac injury response

Reparative processes

AngII, Caspase-1, CT-1, FGF, IL-1β, IL-10, IL-18, IL-23, MMP3, MMP8, TIMP1, TGFβ

Cell cycle and proliferation

Proliferation

Adenylyl cyclase, AngII, CDK2, CDK4, Cyclin A, Cyclin B, Cyclin D, Cyclin E, ERK1/2, FGF, FSP1, HGF, miR-21, PDGF-AA, PDGF-BB, PI3K, Skp2

Senescence and cell death

4-Hydroxynonenal, β-catenin, HIF1α, LOX-1, miR-22, p53, PDGF-AA, PIP3, ROS

Stress fiber formation

Rho-associated GTPases, ROCK

Contraction

MLCK, RhoA-ROCK

Contraction

regulators of fibrosis, including growth factors like TGFβ and AngII or general signaling pathways like ERK, are unlikely to be fruitful given the likelihood of off-target effects such as has been observed with oncogenesis and TGFβ inhibition. Instead, a greater focus on targeting one or a combination of factors that appear to be much more specific to the processes of ECM formation and fibrosis may improve specificity of treatment, such as the potential for targeting the function of scleraxis, or for augmenting or blocking the function of specific miRNAs to attenuate expression of downstream target genes like collagens. Approximately one-third of individuals in the developed world will die as a result of cardiovascular diseases, and cardiac fibrosis will be a contributory factor for many of these patients given its association with such common pathologies as MI, hypertension, diabetes, valve disease, cardiomyopathies, and simple aging, particularly as our population collectively gets older. The development of new noninvasive technologies such as late gadolinium enhancement of magnetic resonance imaging for identifying and measuring fibrosis in cardiac patients promises to provide a much clearer picture of exactly how big the clinical problem is. Precise dissection of the intracellular signaling pathways governing fibroblast/myofibroblast proliferation, migration, and ECM synthesis offers the promise of identifying the means to

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specifically attenuate one or more of these processes, leading to the development of therapeutic relief just as it is most needed.

Acknowledgements This work was supported by a Manitoba Health Research Council Manitoba Partnership Program grant and an Open Operating Grant from the Canadian Institutes of Health Research to MPC (MOP 106671). K. L. Filomeno was supported through a grant from the Heart & Stroke Foundation of Canada. P. L. Roche was the recipient of a graduate studentship from MHRC. R. A. Bagchi was the recipient of a doctoral fellowship from CIHR/MHRC.

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Cardiac fibroblasts as sentinel cells in cardiac tissue: Receptors, signaling pathways and cellular functions.

Origins of cardiac fibroblasts.

Dynamic mass redistribution analysis of endogenous β-adrenergic receptor signaling in neonatal rat cardiac fibroblasts.

Sirtuin-6 inhibits cardiac fibroblasts differentiation into myofibroblasts via inactivation of nuclear factor κB signaling.

Androgen attenuates cardiac fibroblasts activations through modulations of transforming growth factor-β and angiotensin II signaling.

Circadian rhythm of intracellular protein synthesis signaling in rat cardiac and skeletal muscles.

SLC41A1 knockdown inhibits angiotensin II-induced cardiac fibrosis by preventing Mg(2+) efflux and Ca(2+) signaling in cardiac fibroblasts.

Adiponectin mediated APPL1-AMPK signaling induces cell migration, MMP activation, and collagen remodeling in cardiac fibroblasts.

Ca2+-calcineurin signaling is involved in norepinephrine-induced cardiac fibroblasts activation.

Intracellular signaling in the gonads.

INTRACELLULAR SIGNALING BY BILE ACIDS.

Mitogen-Activated Protein Kinase and Intracellular Polyamine Signaling Is Involved in TRPV1 Activation-Induced Cardiac Hypertrophy.

Control of protein synthesis in human fibroblasts by intracellular potassium.

Reversible and irreversible differentiation of cardiac fibroblasts.

Intracellular distributions of mechanochemical proteins in cultured fibroblasts.

Intracellular accumulation of azithromycin by cultured human fibroblasts.

Modeling the intracellular organization of calcium signaling.

Optogenetic control of intracellular signaling pathways.

MicroRNA: master controllers of intracellular signaling pathways.

Analysis of intracellular PTEN signaling and secretion.

Cystinotic fibroblasts accumulate cystine from intracellular protein degradation.

Targeting cardiac fibroblasts: the pressure is on.

Cardiac fibroblasts: more than mechanical support.

Extracellular and Intracellular Signaling for Neuronal Polarity.