INVITED REVIEW

Focal adhesion signaling in heart failure

Allen M. Samarel

Received: 26 December 2013 /Revised: 15 January 2014 /Accepted: 19 January 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract In this brief review, recent evidence is presented toindicate a role for specific components of the cardiomyocytecostamere (and its related structure the focal adhesion com-plex of cultured cardiomyocytes) in initiating and sustainingthe aberrant signal transduction that contributes to myocardialremodeling and the progression to heart failure (HF). Specialattention is devoted to the focal adhesion kinase family ofnonreceptor protein tyrosine kinases in bidirectional signaltransduction during cardiac remodeling and HF progression.Finally, some speculations and directions for future study areprovided for this rapidly developing field of research.

Keywords Costameres . Focal adhesion kinase . PYK2 .

Talin . Vinculin

Introduction

Heart failure (HF) results from the inability of the heart topump blood forward at a sufficient rate to meet the metabolicdemands of the body or the ability to do so only at the expenseof abnormally high filling pressures. Even before the onset ofovert HF, reduced cardiac performance leads to profoundmechanical and neurohormonal stresses on the remainingfunctional myocardium. These stresses trigger signal trans-duction pathways that ultimately result in additional structural

changes in the cardiomyocytes and nonmuscle cells of theheart (“myocardial remodeling”) which contribute to furtherfunctional deterioration of the already diseased myocardium[7]. The downward spiral in cardiac performance during car-diac remodeling may be initiated by specific signaling mole-cules that function to integrate both neurohormonal and me-chanical signals in order to elicit compensatory responses inan attempt to counteract the functional deterioration. Howev-er, some of these compensatory responses ultimately provedetrimental. In this brief review, I will focus on recent evi-dence to indicate a role for specific components of thecostamere (and its related structure the focal adhesion com-plex) in initiating and sustaining the aberrant signal transduc-tion that contributes to myocardial remodeling and the pro-gression to HF.

Cardiomyocyte costameres are sites of attachment to theextracellular matrix (ECM) Cardiomyocyte costameres (andtheir focal adhesion counterparts in cultured cells) arecritical cytoskeletal elements involved in bidirectionalmechanotransduction [67]. The term “costamere” was firstused by Craig and colleagues [57, 58] to describe vinculin-containing, rib-like bands that encircle the cardiomyocyteperpendicular to its long axis. In addition to vinculin,costameres contain many other structural elements that coa-lesce beneath the sarcolemmal membrane and resemble themetal ribs of a wooden barrel. They flank the Z-disc andoverlying I-bands of sarcomeres that run just beneath thecardiomyocyte plasma membrane (Fig. 1). Immunoelectronand fluorescent microscopic analyses have revealed that car-diomyocyte costameres also form discrete physical attach-ments between the underlying, outer Z-discs of the musclecell and their surrounding, three-dimensional, stress-tolerantECM. Attachment is mediated in part by integrins, which arecell-surface receptors for a variety of ECM proteins, and thedystrophin-glycoprotein complex, which mediates attachment

A. M. Samarel (*)The Cardiovascular Institute and the Department of Medicine,Loyola University Chicago Stritch School of Medicine, Building110, Rm 5222, 2160 South First Avenue, Maywood, IL 60153, USAe-mail: asamare@lumc.edu

A. M. SamarelThe Cardiovascular Institute and the Department of Physiology,Loyola University Chicago Stritch School of Medicine, Building110, Rm 5222, 2160 South First Avenue, Maywood, IL 60153, USA

Pflugers Arch - Eur J PhysiolDOI 10.1007/s00424-014-1456-8

to the ECM protein laminin. It is through these attachmentsthat costameres directly transmit contractile forces generatedwithin the cardiomyocyte to the surrounding ECM and wherelongitudinal displacement of the ECM by adjacent musclecells is transmitted directly to the internal contractile machin-ery. Thus, both externally applied and intrinsically generatedmechanical loads are transmitted bidirectionally throughcostameres.

Focal adhes ion complexes in cu l tured card io -myocytes Costameres share many of the structural

features of cell-to-matrix adherens junctions and thus maybe considered striated muscle-specific elaborations of focaladhesions found in cultured nonmuscle cells [17]. Immunolo-calization studies have confirmed that many, if not all of thesame proteins that comprise the costamere in vivo, eventuallyreassemble within focal adhesions as isolated cardiomyocytesattach and spread in culture. Furthermore, culturedcardiomyocytes that retain contractile activity, or are stimulat-ed to contract in culture, reassemble their costameric proteinsalong the cell-substratum interface in register with the overly-ing Z-discs of their remodeled sarcomeres. These basal,costameric attachment sites, as well as the remodeled cell-to-cell adherens junctions derived from the remaining compo-nents of the intercalated disc, provide the major cell adhesionsites of both neonatal and adult cardiomyocytes in culture.

A detailed structural analysis of the various focal adhesioncomponents of cardiomyocyte costameres and focal adhesionshas not yet been obtained. However, using photoactivatablefusion proteins of several focal adhesion proteins and inter-ferometric photoactivated localization microscopy,Kanchanawong et al. [34] have described the three-dimensional organization of focal adhesion complexes in hu-man osteosarcoma and mouse embryonic fibroblast cells(Fig. 2). Their images reveal a highly organized, verticallylayered structure consisting of at least three strata: amembrane-apposed integrin signaling layer containingintegrin cytoplasmic tails, focal adhesion kinase (FAK), andthe adaptor protein paxillin; an intermediate force-transduction layer containing talin and vinculin; and an up-permost actin-regulatory layer containing zyxin, vasodilator-stimulated phosphoprotein (VASP), and α-actinin overlyingand attached to the actin cytoskeleton. The integrin cytoplas-mic domains and the subcortical actin layer were separated bya distance of approximately 40 nm, indicating an importantrole for talin, vinculin, and other intermediary proteins abovethe plasma membrane in bidirectional force transmission.

These images provide enormous detail in describing themolecular architecture of focal adhesions, but do not revealthe dynamic nature of focal adhesion formation and dissolu-tion. However, the rapid turnover of focal adhesion

Fig. 1 Cellular localization of costameres in striated muscle. a A sche-matic diagram is depicted, illustrating costameres as circumferentialelements that physically couple peripheral myofibrils to the sarcolemmain periodic register with the Z-disc. The protein composition ofcostameres includes integrins, a variety of cytoskeletal and adaptor pro-teins, and signaling kinases. b An inside-out sarcolemma that was me-chanically peeled from a single myofiber and stained with antibodies toγ-actin to reveal the costameric cytoskeleton. Bar=10 μm. The figurewas reproduced from [17] by copyright permission of The AmericanSociety for Biochemistry and Molecular Biology

Fig. 2 Nanoscale architecture offocal adhesions. Schematic modelof focal adhesion moleculararchitecture, depictingexperimentally determinedprotein positions. Note that themodel does not depict proteinstoichiometry. The figure wasreproduced from [33] bycopyright permission of theNature Publishing Group

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components plays a crucial role in cellular differentiation andmigration during cardiac development [18, 23, 24, 30, 90] andmay also be an important regulatory factor during new sarco-mere addition in response to hypertrophic stimuli [10, 47].Using fluorescence recovery after photobleaching (FRAP)and mathematical modeling, Ingber and colleagues [42]showed that various components of the focal adhesion com-plex display residence times that vary from as little as 1 s forvinculin and up to 111 s for talin. Sanger and co-workers [87]had previously observed a similar dynamic range of exchangebetween costamere/Z-disc proteins and the cytoplasm ofspreading skeletal muscle myotubes. Using FRAP, we subse-quently demonstrated that the phosphorylation of FAK, acritical component of the signaling layer of cardiomyocytefocal adhesions, regulates the stability of paxillin within car-diomyocyte focal adhesions and ultimately controls the rate ofcell spreading and myofibrillar organization of culturedcardiomyocytes in response to both static stretch and thehypertrophic agonist endothelin-1 [10]. Thus, the dynamicnature of cytoskeletal assembly and disassembly within focaladhesion complexes appears critical during the response ofcultured cardiomyocytes to neurohormonal and mechanicalstimuli.

Focal adhesion complexes assemble in response to mechani-cal overload in vivo In pressure-overloaded feline myocardi-um, Kuppuswamy and co-workers [39, 41] first demonstratedthe cytoskeletal assembly of c-Src and other signaling pro-teins, which was partially mimicked in vitro using adult felinecardiomyocytes embedded within a three-dimensional colla-gen matrix and stimulated with an integrin-binding Arg-Gly-Asp (RGD) peptide [41]. In subsequent elegant studies,Franchini and co-workers [15, 20, 86] analyzed the rapidassembly of focal adhesion complexes in response to pressureloading of the isolated perfused rat heart. The assembly offocal adhesion complexes was also an early response of cul-tured cardiomyocytes to a variety of neurohormonal and me-chanical stimuli that ultimately lead to cardiomyocyte hyper-trophy [16, 63, 77, 83, 85].

Clustering of β1-integrins at the sarcolemmal membraneand their attachment to ECM proteins were critical factors inthe regulation of focal adhesion assembly in these settings.Ross and colleagues [76] further demonstrated the importanceof β-integrins in costamere assembly. They used Crerecombinase driven by the myosin light chain-2 ventricular(MLC-2v) promoter to induce β1-integrin gene excision ex-clusively in ventricular cardiomyocytes. They found that β1-integrin knockout mice had significantly depressed contractileperformance with extensive cardiac fibrosis and were intoler-ant to pressure-overload produced by transverse aortic coarc-tation (TAC). Surviving mice developed spontaneous heartfailure by 6months of age, with abnormal myocyte membraneintegrity as determined by Evan’s blue dye staining. This

group subsequently described the structural and functionalconsequences of cardiomyocyte-specific excision of the β1-integrin gene in adult cardiomyocytes [43]. Using atamoxofen-inducible promoter to drive expression of Crerecombinase, even partial excision of the β1-integrin generesulted in multiple defects in mechanotransduction signalingwhen hearts were stressed by TAC. Adaptive hypertrophy wasblunted, and the response to adrenergic stimuli was alsomarkedly impaired. Thus, these results demonstrated a criticalfunction of β1-integrins in the postnatal myocardium and firstlinked defects in β1-integrin-dependent costamere assemblyto the development of cardiomyopathy.

In the majority of aforementioned studies, a hemodynamicstress in the form of an increase in ventricular afterload of theintact heart [20, 39, 41, 43, 76, 86] was used to stimulate focaladhesion assembly. However, Domingos et al. [15] showedthat increasing ventricular preload (by raising diastolic pres-sure from 0 to 15 mmHg in the beating, isolated perfused ratheart) also rapidly increased FAK tyrosine phosphorylationand binding of c-Src and Grb2 to FAK. This was paralleled byactivation and binding of ERK1/2 to the cardiomyocyte cyto-skeleton, indicating enhanced focal adhesion signaling inresponse to increased diastolic stress. Balloon inflation to raiseintraventricular pressure in hearts perfused with cardioplegicsolution also activated FAK and ERK1/2. In contrast, earlyinduction of volume overload 4 weeks after surgically inducedmitral regurgitation in the dog decreased focal adhesion sig-naling [66]. Similarly, rats with surgically induced aortocavalfistulae demonstrated increased ECM degradation, decreasedfocal adhesion signaling, and increased apoptosis [75]. Down-regulation of focal adhesion signaling was mediated by theactivation of PTEN, a dual lipid and protein phosphatase.Beta-adrenergic blockade prevented the PTEN activation,the downregulation of focal adhesion signaling, and the in-creased rate of apoptosis, but had little effect on ECM degra-dation. These data suggest that volume overload per se is arelatively weak stimulator of focal adhesion signaling as com-pared to pressure overload of the intact heart. Attempts tomodel the differential effects of pressure vs. volume loadingby subjecting cardiomyocytes to transverse vs. linear stretchsupport these observations [21, 74, 79].

β1-integrin cytoplasmic domains form cytoskeletal attach-ments through talin dimers Unlike growth factor receptors,the cytoplasmic tails of integrins do not possess intrinsiccatalytic activity. However, they interact with other cytoskel-etal proteins to transmit extrinsically applied and internallygenerated mechanical force. A major binding partner of theβ1-integrin cytoplasmic domain is the cytoskeletal proteintalin, which is a rod-shaped, multidomain protein involvedin bidirectional activation of integrins [71]. Cell-surfaceintegrins can exist in either low- or high-affinity states, andcellular modulation of integrin affinity is in part accomplished

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by reversible binding of the talin N-terminal, globular headdomain to the C-terminal, β1-integrin cytoplasmic tail [8].Cardiomyocytes predominantly express the muscle-specificβ1D-integrin isoform, which has the highest binding affinityfor talin of the various β-integrin cytoplasmic domains. Thisobservation suggests that integrin engagement to the cardiacECM favors a mechanically strong, activated integrin-bindingconformation [2].

The mammalian genome contains two genes for talinencoding two structurally similar proteins (talin-1 and talin-2) that share 74 % sequence identity [93]. The specific func-tion of each talin isoform is not known, but it appears that thetalin-2 isoform plays a unique role in muscle development anddisease. Localization of talin-2 is restricted to costameres andintercalated discs in striated muscle [73], but talin-2 appearsdispensable in the presence of talin-1 for normal costamereassembly. However, deletion of both genes produced a severedefect in sarcomere assembly in striated muscle, with pro-found defects in the assembly of focal adhesion complexesand sarcomeres by cultured myoblasts isolated from doubleknockout embryos [11]. The failure of normal sarcomeredevelopment in these immature muscle cells also highlightsthe potential importance of talin (and perhaps other cytoskel-etal linker proteins) in adaptation to hypertrophic stimuli.

Ross and colleagues [46] recently evaluated talin-1 andtalin-2 expression in the normal embryonic and adult mouseheart, as well as in control and failing human adult myocardi-um. Using isoform-specific antibodies and nonfailing humancardiac tissue, they showed that talin-1 was only weaklydetected in the cardiomyocyte plasma membrane and rarelyco-localized with dystrophin, a muscle-specific costamericprotein (Fig. 3). Talin-1 was also more readily detected in

nonmuscle cells of the cardiac interstitium. In contrast, talin-2was readily detected in a costameric pattern in cardiomyocytesas demonstrated by strong co-localization with dystrophin.Talin-1 function was then tested in the basal and mechanicallystressed myocardium after cardiomyocyte-specific excision ofthe talin-1 gene. They found that during embryogenesis, bothtalin isoforms are highly expressed, but talin-2 is the mainisoform expressed in adult mammalian cardiomyocytes,where it was predominantly localized to costameres. Howev-er, talin-1 expression was upregulated during cardiac hyper-trophy, suggesting that it plays an important role in the com-pensatory response of the heart to stress. In human failingheart, cardiomyocyte talin-1 was also increased as comparedto control samples from normal functioning myocardium.Talin-1 knockout mice showed normal basal cardiac structureand function, but when subjected to pressure overload, theseanimals showed blunted hypertrophy, less fibrosis, but im-proved cardiac function versus controls. Overall, these datasuggested that reduction of cardiomyocyte talin-1 expressionmight lead to improved cardiac remodeling following pressureoverload.

Exactly how talin isoforms mediate integrin-dependentsignal transduction in the heart remains unknown. Adhesionto ECM proteins via β1-integrins causes the recruitment oftalin dimers to the cytoplasmic face of the sarcolemmal mem-brane, leading to integrin transition to its high-affinity state.This process is an important, early step in “outside-in” signal-ing during cell attachment of cultured cardiomyocytes andmay play an important functional role in costamere assemblyduring the addition of new sarcomeres in vivo. Conversely,talin activation by a number of intracellular signaling path-ways causes the physical displacement of α-integrin subunits,

Fig. 3 Expression of talin-1(Tln1) and talin-2 (Tln2) in adulthuman cardiac tissue. Adulthuman cardiac tissue wasevaluated for expression of Tln1and Tln2. Tln1 was weaklydetected in the cardiomyocytemembrane, as shown by co-localization with dystrophin (Dys),a muscle-specific membranemarker. It was also detected innonmyocyte cells (asterisks). Tln2was detected in a costamericpattern in cardiomyocytes asdemonstrated by strong co-localization with Dys (arrows).DAPI staining (blue) shows theposition of cell nuclei. Scale bar=20 μm. The figure was reproducedfrom [45] by copyright permissionof The American Society forBiochemistry and MolecularBiology

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thereby allowing for high-affinity ECM engagement during“inside-out” signaling [2, 81]. Intracellular talin binding aloneis sufficient to alter the conformation of integrin extracellulardomains and promote their attachment to ECM proteins [91].The recruitment of talin involves the Src-dependent tyrosinephosphorylation of the β-integrin tail and its recognition bythe talin head region [1]. Thus, intracellular stimuli that causeSrc-dependent integrin phosphorylation promote the activa-tion of integrins via talin recruitment to costameres and stim-ulate costamere formation during inside-out signaling. Subse-quent recruitment of additional cytoplasmic linker proteins(such as paxillin and vinculin) to the costamere may also berequired for Z-disc assembly and premyofibril formation dur-ing myofibrillogenesis [13, 80].

FAK and cardiomyocyte mechanotransduction Talin localiza-tion during integrin engagement and clustering may initiateoutside-in signaling, but talin, like β1-integrin cytoplasmicdomains, has no intrinsic catalytic activity capable of relayingbiochemical signals into the cell interior. However, secondaryrecruitment and activation of protein tyrosine and serine/threonine kinases to the cytoplasmic adhesion plaque mayaccomplish this function. FAK is clearly one candidate en-zyme tha t i s respons ib le for in tegr in-media tedmechanotransduction within cardiomyocyte focal adhesionsand costameres. As indicated above, FAK is a nonreceptorprotein tyrosine kinase that functions as an “activatable scaf-fold” [70] in integrin-dependent signal transduction. FAKcontains a central kinase domain flanked by long N- and C-terminal extensions (Fig. 4). An autoinhibitory FERM do-main, located within the N-terminal region of FAK, associateswith the plasma membrane via its interaction with severaldifferent growth factor receptors. The C-terminal region ofFAK comprises the focal adhesion targeting (FAT) domain.The FAK-FAT domain binds directly to paxillin and talin,

which in turn bind to the cytoplasmic tail of β1-integrins atsites of integrin clustering. Once localized, FAK phosphory-lates itself at a single tyrosine residue (Y397). This autophos-phorylation site serves as a high-affinity binding site for theSH2 domain of Src-family protein tyrosine kinases [69]. Oncebound to FAK, active Src then phosphorylates FAK at resi-dues Y576 and Y577 within its catalytic domain (which aug-ments FAK kinase activity toward exogenous substrates) andat Y861 and Y925 near its C-terminus [19]. Phosphorylation ofthe Y861 site promotes the binding of p130Cas to FAK [45].The Y925 phosphorylation site promotes the binding of Grb2to FAK and other adapter proteins and kinases containing SH2domains. The FAK/Src complex can also phosphorylatepaxillin, p130Cas, and other cytoskeletal proteins involved incostamere and myofibrillar assembly.

FAK serine phosphorylation and sarcomere assembly In addi-tion to multiple tyrosine phosphorylation sites, FAK containsseveral serine residues (S722, S843, S846, and S910) that undergoreversible phosphorylation in response to hypertrophic stim-uli. These serine residues are in close proximity to criticalprotein-protein interaction sites within the C-terminal regionof FAK, such as the binding site for p130Cas, Grb2, and theadjacent FAT domain. The functional role of FAK serinephosphorylation in cardiomyocytes is largely unknown, butone report indicates that serine (and tyrosine) phosphorylationof FAK increases dramatically in hypertensive rats, with dif-ferent sites of phosphorylation appearing to regulate FAKsubcellular localization [92]. Our group [10] recently demon-strated that endothelin-1 and other hypertrophic factors in-duced a time- and dose-dependent increase in FAK-S910phosphorylation. Endothelin-induced FAK-S910 phosphory-lation required endothelin type A receptor-dependent activa-tion of protein kinase Cδ (PKCδ) and Src via parallel Raf-1→MEK1/2→ERK1/2 and MEK5→ERK5 signaling pathways.

Fig. 4 Structural domains of focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2). Proline-rich tyrosine kinase 2 (PYK2)shares a similar domain arrangement with focal adhesion kinase (FAK),with 60% sequence identity in the central kinase domain, conservation ofproline-rich regions (PRRs), and identical positions of four tyrosinephosphorylation sites. PYK2 tyrosines 402, 579, 580, and 881 correspond

to FAK tyrosines 397, 576, 577, and 925, respectively. FAK and PYK2both contain a C-terminal focal adhesion targeting (FAT) domain thatbinds to paxillin. However, PYK2 shows a perinuclear distribution and isnot strongly localized to focal contacts in many cells. The figure wasreproduced from [49] by copyright permission of the Nature PublishingGroup

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Replication-deficient adenoviruses expressing wild-type FAKand a nonphosphorylatable, S910A-FAK mutant were thenused to examine the functional significance of FAK-S910phosphorylation. Unlike wild-type FAK, S910A-FAK in-creased the half-life of GFP-tagged paxillin within costameres(as determined by total internal reflection fluorescence mi-croscopy and FRAP) and increased the steady-state FAK-p a x i l l i n i n t e r a c t i o n ( a s d e t e rm i n e d b y c o -immunoprecipitation and Western blotting). These alterationsresulted in reduced neonatal rat ventricular myocyte (NRVM)sarcomere reorganization and cell spreading. Finally, wefound that FAK was serine-phosphorylated at multiple sitesin nonfailing, human left ventricular tissue, and both FAK-S910 phosphorylation and ERK5 expression were dramatical-ly reduced in patients undergoing heart transplantation forend-stage dilated cardiomyopathy. These results suggest thatreduced FAK-S910 phosphorylation may contribute to sarco-mere disorganization that is frequently observed in end-stageheart failure patients [10].

The ERK-dependent, regulated exit of FAK from cardio-myocyte costameres [10] is reminiscent of the cyclic FAKtyrosine and serine phosphorylation that occurs during migra-tion of nonmuscle cells [52]. In this scenario, FAK/Src tyro-sine phosphorylation at Y397 and Y925 promotes the assemblyof new costameres in response to hypertrophic agonists ormechanical strain. Subsequent ERK1/2/5 activation then leadsto the serine phosphorylation of FAK at S910, which promotesFAK exit from newly formed costameres and its replacementby vinculin during premyofibril formation [68]. The dynamicnature of FAK entry and exit from focal adhesions andcostameres is consistent with the time required for new sarco-mere addition in response to longitudinal strain and its depen-dence on FAK localization and activation [47].

FAK knockout mice and the development of HF Studies ofglobal and cardiomyocyte-specific FAK knockout mice lendfurther support for an important role for FAK in cardiomyo-cyte mechanotransduction and myofibrillar assembly. GlobalFAK deletion led to lethality at embryonic day 8.5, and themutant embryos displayed a profound defect in the develop-ment of all mesodermal structures, including the heart andvasculature [31, 32]. Interestingly, the developmental defectsfound in FAK−/− embryos were phenotypically very similar intiming and phenotype to the morphological defects observedin fibronectin-null mice, suggesting an important relationshipbetween fibronectin- and FAK-dependent signaling, especial-ly with respect to development of the cardiovascular system[70]. In both cases, the developmental defects were attributedto the inability of mesodermal cells to migrate normally.Indeed, fibroblasts isolated from FAK−/− embryos displayedmarkedly reduced mobility and abnormally large focal adhe-sions, indicating a defect in focal adhesion turnover. However,cardiomyocyte-restricted FAK knockout mice have a variable

cardiac phenotype depending on precisely when during de-velopment FAK is deleted. Embryonic deletion of FAK incardiomyocytes caused perinatal lethality due to the presenceof large ventricular septal defects and abnormalities in outflowtract alignment, indicating again that FAK predominantlyregulates mammalian cardiomyocyte proliferation and migra-tion during early cardiac development [24, 61]. However,cardiomyocyte FAK deletion during later prenatal develop-ment was not associated with any congenital heart defects, butled to spontaneous dilated cardiomyopathy in aged animals[60], and a blunted hypertrophic response to angiotensin IIinfusion [60] or TAC [14] in the adult heart. The inability torespond normally to hypertrophic stimuli supported earliercell culture studies that demonstrated impaired hypertrophicresponses of cultured cardiomyocytes overexpressing FAK-related nonkinase (FRNK, a naturally occurring inhibitor ofFAK), Y397F-FAK (a FAK autophosphorylation mutant),FAK antisense RNA, or just the FAK-FAT domain [16, 27,38, 47, 53, 63, 85]. FAK also plays an important role inlimiting stress-induced cardiomyocyte apoptosis and improv-ing cardiomyocyte survival after ischemia-reperfusion injury[25, 88]. Furthermore, Vander Heide and co-workers recentlyshowed that the tissue-specific, inducible knockout of FAK inadult cardiomyocytes substantially reduced the precondition-ing response during ischemia-reperfusion injury [62]. Thus,there are substantial data indicating an important role for FAKand FAK-dependent signal transduction in preserving cardiacfunction and preventing disease progression and HF duringadaptation to hemodynamic, neurohormonal, and ischemicstress.

PYK2, the other member of the FAK family of protein tyrosinekinases The FAK family of protein tyrosine kinases actuallyconsists of two members [51], both of which are expressed incardiomyocytes. Proline-rich tyrosine kinase 2 (PYK2; alsoknown as FAK2, cell adhesion kinase-β (CAK-β); or celladhesion tyrosine kinase (CADTK)), is the other member ofthe family and shares ~45 % protein sequence homology withFAK. PYK2 has a similar domain structure as FAK (Fig. 4),and both kinases are believed to be derived from a common,ancient ancestral gene that diverged during early vertebrateevolution [12]. Whereas FAK is expressed at high levels invirtually all cells and tissues, PYK2 expression is much morerestricted and is preferentially expressed in cells of the endo-thelium, central nervous system, and hematopoietic lineages.

Activation of PYK2 occurs in response to integrin engage-ment and clustering in some cell types, but its cellular distri-bution is predominantly cytoplasmic. This is particularly truein cardiomyocytes, where PYK2 is found throughout thecytoplasm of both neonatal and adult cells [3] with only asmall portion localized to focal adhesions and costameres[28]. There are other important structural differences betweenPYK2 and FAK that reflect their distinct roles in

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cardiomyocyte signal transduction. Perhaps most important isthe fact that unlike FAK, PYK2 activation is sensitive tointracellular Ca2+ ([Ca2+]i) signals and undergoes dimeriza-tion in response to Ca2+-calmodulin binding to the FERM F2subdomain within the N-terminus of the molecule [36]. Thecalcium dependence of PYK2 activation may be related to thedissociation of the PYK2 FERM domain from the centralcatalytic domain as occurs during FAK activation [44],resulting in dimerization and intermolecular trans-autophosphorylation. Indeed, overexpression of the PYK2FERM domain (in the absence of calmodulin overexpression)inhibited the activation of full-length PYK2 by forming acomplex with the inactive enzyme [65]. These data indicatethat the PYK2 FERM domain is also involved in the regula-tion of PYK2 activity by regulating the formation of PYK2oligomers that are critical for its activity. Regardless of its rolein oligomerization, Ca2+-calmodulin binding to the PYK2FERM domain provided an answer to the question of howPYK2 activity was regulated by [Ca2+]i, and this may be animportant issue with regard to its regulation during cardiachypertrophy and HF.

As a Ca2+-calmodulin-dependent protein tyrosine kinase,PYK2 undergoes bimolecular transphosphorylation [59] inresponse to integrin engagement, increased [Ca2+]i, and acti-vation of PKCs in many cell types, including cardiomyocytes[3, 4, 22, 26, 28, 29, 35, 49, 50]. Like FAK, PYK2 serves as anactivatable scaffolding protein and transduces signals from G-protein-coupled receptors to the mitogen-activated proteinkinases (MAPK) and the phosphoinositol-3-kinase(PI-(3)K)-PDK1-Akt signaling pathway depending uponwhich adaptor proteins bind to the phosphorylated enzyme[6, 22, 56, 82]. Indeed, many of the same binding partners forFAK also bind PYK2,making it somewhat difficult to assign aspecific role for PYK2-dependent signaling in cells express-ing both PYK2 and FAK. Furthermore, PYK2 can function-ally compensate for the loss of FAK in some cell types [89].The adaptive capacity of cells to switch to PYK2-dependentsignaling after deletion or kinase inhibition of FAK is evi-dence for some degree of signaling redundancy between thetwo protein tyrosine kinases. In fact, global deletion of thePTK2b gene that encodes PYK2 produces viable offspringwithout obvious defects in cardiovascular development [54],indicating that FAK may also compensate for the loss ofPYK2 in most cell types.

Despite these caveats, PYK2 appears to serve a limitednumber of specific functions in cardiomyocytes, especiallywith respect to the recognition of, and response to a varietyof stressful stimuli. Unlike FAK, PYK2 undergoes tyrosinephosphorylation during Ca2+ overload, UV irradiation, andH2O2 and TNF-α treatment [84]. Hirotani et al. [29] demon-strated that PYK2 is an essential signaling component inendothelin- and phenylephrine-induced cardiomyocyte hyper-trophy, perhaps acting via the Ca2+- and/or PKC-dependent

activation of Rac1. Furthermore, recent studies have con-firmed that PYK2 is an important upstream regulator of thestress-activated protein kinases (p38MAPK and JNK1/2) incardiomyocytes [22, 28, 49]. Thus, PYK2 activation has beenimplicated in hypertrophic gene expression changes duringpathological cardiomyocyte hypertrophy [26, 28] and in theinduction of apoptosis [49].

Regulated PYK2 expression in cardiomyocytes Unlike FAK,which appears to be constitutively expressed in most celltypes, PYK2 expression also appears to vary in response tointracellular and extracellular stimuli. Although PYK2 ishighly expressed in the neonatal cardiomyocyte, PYK2 ex-pression in the adult heart is substantially reduced, but un-dergoes upregulation in response to cardiomyocyte stress. Themechanisms underlying the regulated expression of PYK2 incardiomyocytes and other cells are not known. As indicatedabove, embryonic fibroblasts isolated from FAK knockoutmice demonstrate a two- to threefold increase in PYK2 ex-pression levels, suggesting a compensatory role for PYK2 inmaintaining integrin-dependent signaling events in FAK-deficient cells [78]. A similar increase in PYK2 expressionand phosphorylation was observed in mice with conditionaldeletion of FAK in endothelial cells [89]. However, PYK2levels did not increase with cardiomyocyte-restricted FAKdeletion [14, 24, 61]. Nevertheless, our group was the first todemonstrate that PYK2 expression and phosphorylation wereboth significantly increased in cultured NRVM and adult ratventricular myocytes (ARVM) by increases in [Ca2+]I andcontractile activity [3] and in ARVM in vivo in response toacute LV pressure overload [5]. The increase in PYK2 levelswas at least partly mediated by an increase in PYK2 mRNA[64]. Similarly, Melendez et al. [50] showed that PYK2 ex-pression and PYK2 activity were substantially increased in amouse model of dilated cardiomyopathy due to tropomodulinoverexpression, and we have recently demonstrated thatPYK2 is substantially upregulated in the PKCε-overexpressing mouse during LV remodeling and HF [37].Nevertheless, very little is known about the factors that regu-late PYK2 expression in cardiomyocytes and other cell types.

PYK2-dependent signal transduction in pathological LVremodeling The upregulation and activation of PYK2 duringcardiac hypertrophy and its transition to HF suggests that thisfocal adhesion kinase plays an important role in the pathogen-esis of contractile dysfunction during cardiomyocyte stress. Ingeneral, PYK2 is considered a pro-apoptotic signaling mole-cule, in contradistinction to FAK, which has been shown to bepro-survival inmany cell types. Indeed, our group has recentlyshown that adenovirally mediated overexpression of CRNK,the C-terminal domain of PYK2, inhibits PYK2 activation incardiomyocytes, improves LV contractile function in normalhearts, and improves LV fractional shortening, pathological

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changes in gene expression, and survival during post-MIventricular remodeling [26]. These results are in stark contrastto those obtained by conditional deletion of FAK in adultcardiomyocytes, in which FAK depletion alone led to progres-sive LV dilatation and fibrosis [60] or the impairment ofcompensatory LVH in response to pressure overload [14].Thus, FAK and PYK2 appear to have very different, opposingfunctions in cardiomyocytes.

Termination of FAK- and PYK2-dependent signaling in theheart Activation of both FAK and PYK2 duringmechanotransduction signaling is dependent on phosphoryla-tion of specific tyrosine and serine residues, which eitheraugment kinase activity, or induce conformational alterationsthat promote adaptor protein binding and release. Kinaseactivation, however, is transient, which suggests a role forcellular phosphatases in attenuating or terminating focal ad-hesion signaling. Two phosphatases, Shp2 and PTEN, havebeen implicated in this process. PTEN is a dual specificity,lipid and protein phosphatase that is highly expressed incardiomyocytes and whose expression increases in responseto hypertrophic stimuli [72]. PTEN is known to inhibit car-diomyocyte growth, in part via the dephosphorylation of FAK(and PYK2), and inhibition of downstream signaling to thePI(3)K-AKT signaling pathway. PTEN was shown by Seqqatet al. [75] to be activated in response to β1-adrenergic stimu-lation following acute volume overload in vivo and bypro longed i sop ro t e r eno l t r ea tment in cu l tu redcardiomyocytes. PTEN activation was accompanied by FAKand PYK2 dephosphorylation and the induction of apoptosis.In mice, muscle-specific knockout of PTEN resulted in basalhypertrophy and mild reduction in LV systolic function [55].But in contrast to control mice, TAC in PTEN-knockout miceresulted in reduced pathological hypertrophy, less interstitialfibrosis, and reduced apoptosis with a marked preservation ofLV function, indicating that loss of PTEN prevents the devel-opment of maladaptive ventricular remodeling in response topathological biomechanical stress.

Shp2 is another FAK/PYK2 phosphatase, whose ac-tivity is reduced in response to biomechanical stress.Depletion of Shp2 by small interfering RNAs increasedFAK phosphorylation and downstream focal adhesionsignaling in cultured cardiomyocytes [48]. These find-ings demonstrate that basal Shp2 tyrosine phosphataseactivity controls the size of cardiomyocytes by down-regulating a pathway that involves FAK/Src and thePI(3)K-AKT-mTOR signaling pathways.

Speculation and future directions The application of induc-ible gene excision technology to the study of focal adhesionsignaling has already provided insight into the role of FAKand other components of the costamere in cardiomyocytesignal transduction. This approach, along with high-

resolution fluorescence microscopy, should continue to pro-vide important structure-function relationships between indi-vidual components of the cardiomyocyte costamere in healthand disease. In contrast, the roles of PYK2 and PYK2-dependent signaling in HF progression remain largely unex-plored, despite the fact that this kinase is upregulated duringpressure and volume overload prior to the development ofovert HF. Indeed, there is some controversy as to whether thekinase is cardioprotective in this setting, where it may protectthe ventricular myocardium against tachyarrhythmias duringvagal stimulation [40]. However, this cardioprotective, elec-trophysiological effect was examined in global PYK2 knock-out mice, where at least some of the deleterious effects ofPYK2 deletion may have resulted from PYK2 ablation innoncardiac cell types, including neuronal, inflammatory, andvascular cells which all express abundant amounts of PYK2.Inducible gene targeting in cardiomyocytes should clarify thisissue. FAK has already been considered a novel drug target forthe development of small molecule inhibitors in the treatmentof a variety of malignancies in which FAK overexpression is acommon finding [33]. However, it remains to be determinedwhether FAK inhibition will result in significantcardiotoxicity in patients treated with these agents, as has beenobserved in patients treated with other tyrosine kinase inhib-itors [9]. FAK inhibitors currently under evaluation also showsignificant inhibitory activity against PYK2, which will un-doubtedly complicate the interpretation of toxicity studies.Conversely, specific small molecule inhibitors of PYK2 arenot currently available, but might have utility in the treatmentof HF if indeed PYK2 has deleterious effects during HFprogression. Nevertheless, therapeutics aimed at focal adhe-sion signaling to prevent cardiac hypertrophy and HF mayultimately prove useful in reducing morbidity and mortalityduring LV remodeling.

Acknowledgments The author is supported by NIH 2PO1 HL062426.The author also gratefully acknowledges the support of the Dr. Ralph andMarian Falk Medical Research Trust.

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