Assembly and Signaling of Adhesion Complexes

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Assembly and Signaling of Adhesion Complexes Jorge L. Sepulveda,* Vasiliki Gkretsi,* and Chuanyue Wu Department of Pathology University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania 15261

I. Introduction II. Integrin-Mediated Cell–ECM Adhesion III. The PINCH–ILK–Parvin Complexes A. Molecular Activities of ILK, PINCH, and Parvins B. Assembly and Regulation of the PINCH–ILK–Parvin Complexes C. Signaling Through the PINCH–ILK–Parvin Complexes D. Proteins Interacting with the PINCH–ILK–Parvin Complexes IV. Adhesion Complexes in Cardiac Myocytes A. Structure of Adhesion Complexes in Cardiac Myocytes B. Models of Myofibrillogenesis C. Regulation of Myofibrillogenesis D. Role of Costameres and Intercalated Disks in Myofibrillogenesis E. Integrin Adhesion Complexes in Cardiac Development F. Integrin Pathways Regulate Hypertrophy G. Integrin Pathways Regulate Survival Acknowledgments References

Cell–extracellular matrix (ECM) adhesion is crucial for control of cell behavior. It connects the ECM to the intracellular cytoskeleton and transduces bidirectional signals between the extracellular and intracellular compartments. The subcellular machinery that mediates cell–ECM adhesion and signaling is complex. It consists of transmembrane proteins (e.g., integrins) and at least several dozens of membrane-proximal proteins that assemble into a network through multiple protein interactions. Furthermore, despite sharing certain common components, cell–ECM adhesions exhibit considerable heterogeneity in diVerent types of cells (e.g., the cell– ECM adhesions in cardiac myocytes are considerably diVerent from those in fibroblasts). Here, we will first briefly describe the general properties of the integrin-mediated cell–ECM adhesion and signal transduction. Next, we will focus on one of the recently discovered cell–ECM adhesion protein *Vasiliki Gkretsi and Jorge L. Sepulveda contributed equally to this work. Current Topics in Developmental Biology, Vol. 68 Copyright 2005, Elsevier Inc. All rights reserved.

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complexes consisting of PINCH, integrin-linked kinase (ILK), and Parvin and use it as an example to illustrate the molecular basis underlying the assembly and functions of cell–ECM adhesions. Finally, we will discuss in detail the structure and regulation of cell–ECM adhesion complexes in cardiac myocytes, which illustrate the importance and complexity of the cell–ECM adhesion structures in organogenesis and diseases. C 2005, Elsevier Inc.

I. Introduction One of the major prerequisites for the proper function of cells within a tissue is the formation of contacts with the surrounding extracellular matrix (ECM) and the neighboring cells. The interaction between cells and various components of the ECM is of vital importance for a number of cellular functions such as cell survival and death (apoptosis), proliferation, diVerentiation, cell shape modulation, actin organization, migration, and gene expression. Over the past two decades, exciting progress has been made in understanding the general molecular organization, assembly, and functions of the cell–ECM adhesions. Meanwhile, the complexity of cell–ECM adhesion structures in diVerent developmental stages, as well as various tissues and organs, has been well appreciated. Here, we will first briefly describe the general properties of the integrin-mediated cell–ECM adhesion and signaling (readers are referred to several excellent review articles: Burridge and Chrzanowska-Wodnicka, 1996; Calderwood et al., 2000; Dedhar, 2000; Geiger et al., 2001; Howe et al., 1998; Hynes, 2002; Martin, 2002; Schwartz et al., 1995; and Zamir and Geiger, 2001) for more detailed discussion on this topic). Next, we will focus on one of the recently discovered cell–ECM adhesion protein complexes, the integrin-linked kinase (ILK) complex, and use it as an example to illustrate the molecular basis underlying the assembly and functions of cell–ECM adhesions. Finally, we will discuss in detail adhesion complexes in cardiac myocytes to illustrate the complexity of the cell–ECM adhesion structures in organogenesis and diseases.

II. Integrin-Mediated Cell–ECM Adhesion Integrins are a family of heterodimeric, transmembrane glycoproteins that are primarily responsible for mediating cell–ECM interactions (Hynes, 2002). They interact with specific components of the ECM (collagen, fibronectin, vitronectin, laminin, etc.) by means of the extracellular domains and link them to the intracellular cytoskeletal and signaling complexes by means of the interactions of their cytoplasmic domains and multiple receptor-proximal cytoplasmic proteins. Apart from mediating the physical

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connection between ECM components and the actin cytoskeleton, integrins mediate bidirectional transduction of signals between the ECM and the intracellular compartment. Integrins can, therefore, be activated in two ways: either as a result of their detection of changes in the microenvironment of the cell (outside-in signaling) or in response to signals that originate from the inner compartment of the cell (inside-out signaling) (Geiger et al., 2001; Hynes, 2002). The ‘‘outside-in signaling’’ of integrins begins with the binding of the ECM ligands to integrins, and it is followed by clustering of integrins and the recruitment of actin filaments and signaling complexes to the cytoplasmic domain of integrins (Hynes, 2002). These initial complexes, which in cultured cells are often referred to as ‘‘focal complexes,’’ will, in turn, give rise to mature structures (often referred to as focal adhesions [Fas] in cultured cells) that consist of larger and more complicated protein assemblies. The connection of many of the integrin-associated proteins with the actin cytoskeleton is of great significance for the fate of the cell. It allows the cells to adhere properly to the ECM, modulate their shape by means of the connections to the cytoskeleton, and acquire certain morphology. In addition, this connection facilitates the transduction of extracellular signals to the interior of the cell, enabling it to respond appropriately by moving, changing morphology, diVerentiating, or coordinating several other functions. At the molecular level, most of the integrin-associated molecules in FAs are multifunctional (Brakebusch and Fassler, 2003; Burridge and Chrzanowska-Wodnicka, 1996; Calderwood et al., 2000; Geiger et al., 2001; Zamir and Geiger, 2001). They associate with integrins and actin cytoskeleton. In addition, they serve as scaVolds for the attachment of enzymes such as kinases and phosphatases that modify and regulate these complex interactions. Currently, more than 60 FA proteins including talin, -actinin, filamin, paxillin, focal adhesion kinase (FAK), ILK, PINCH, and Parvins have been identified (Niederreiter, 1994; Zamir and Geiger, 2001). The number of diVerent proteins that are involved in the assembly of FAs, along with the interactions that they mediate, indicates a high level of molecular complexity (Niederreiter, 1994; Zamir and Geiger, 2001), which in turn subtly underlines the importance of those proteins for cellular functions. The significance of such protein associations is particularly obvious in development. During normal embryonic development, cells are attached to or migrate on ECM, and in both cases, they interact with several components of the matrix by means of integrins and other associated FA proteins. These interactions are very dynamic and are continually modified until the developmental process has been successfully completed. In fact, lack of certain components of FAs, as well as proteins that make up the cell–cell junctions, has been shown to hinder normal development and lead to embryonic lethality or other developmental defects. Vinculin null mice, for

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example, are embryonic lethal with great defects in heart and brain development (Xu et al., 1998), E-cadherin knockout embryos are unable to form trophectoderm epithelium (Larue et al., 1994), whereas ILK knockout mice die at the preimplantation stage (Sakai et al., 2003). This demonstrates the absolute requirement of certain interactions between cells and the ECM for the normal development, morphogenesis, and life in general.

III. The PINCH–ILK–Parvin Complexes A. Molecular Activities of ILK, PINCH, and Parvins One protein that is emerging as a key component of the FAs is ILK. ILK was identified and cloned in 1996 based on its interaction with the 1-subunit of integrin (Hannigan et al., 1996), and it was shown to localize to cell–ECM adhesions (Li et al., 1999). It plays a crucial role in integrinmediated cell adhesion and signaling (Brakebusch and Fassler, 2003; Dedhar, 2000; GrashoV, 2004; Wu, 2001, 2004; Yoganathan et al., 2000). ILK consists of three structurally distinct regions: four ANK repeats in the N-terminal region, followed by a pleckstrin homology (PH)–like domain and a C-terminal domain, which exhibits significant homology to Ser/Thr kinase catalytic domains (GrashoV, 2004; Hannigan et al., 1996; Wu and Dedhar, 2001). ILK can phosphorylate several proteins, including protein kinase B PKB/Akt (Delcommenne et al., 1998), Gsk-3 (Delcommenne et al., 1998), and myosin light chain (Deng et al., 2001) and thereby regulates intracellular signaling (Hannigan et al., 2005). ILK also mediates multiple protein–protein interactions (Fig. 1) (Mackinnon et al., 2002; Wu, 2001; Wu et al., 2002). It binds to PINCH by way of its N-terminal ANK domain (Li et al., 1999a; Tu et al., 1999; Velyvis et al., 2001) and - or -Parvin, members of the CH-ILKBP/-Parvin/actopaxin/aYxin protein family (Fukuda et al., 2003a; Nikolopoulos and Turner, 2001; Tu et al., 2001; Yamaji, 2001; Zhang, 2004; Zhang et al., 2002a,b,c), by way of its C-terminal domain, resulting in the formation of a ternary complex within cells. PINCH consists of five LIM domains, each of which contains two Zn2þ fingers that mediate protein–protein interactions (Dawid et al., 1998; Jurata and Gill, 1998; Schmeichel and Beckerle, 1994). The N-terminal-most LIM1 domain mediates the interaction with ILK (Tu et al., 1999; Velyvis et al., 2001), whereas the C-terminal LIM domains mediate interactions with several other proteins including Nck-2 (Tu et al., 1998; Velyvis et al., 2003), thymosin 4 (Bock-Marquette et al., 2004), and RSU-1 (Kadrmas et al., 2004). Parvins contain one N-terminal region and two C-terminal Calponin-Homology (CH) domains (Nikolopoulos and Turner, 2000; Olski et al., 2001; Tu et al., 2001; Yamaji et al., 2001). In addition to ILK, other

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Figure 1 Schematic representation of the protein–protein interactions mediated by ILK and proteins associated with it. ILK binds to 1‐integrin, which in turn connects the components of the ECM with proteins in the intracellular compartment. ILK forms a stable ternary complex by binding to PINCH‐1 and ‐Parvin/actopaxin/CH‐ILKBP, which in turn binds to actin. ILK can also form a ternary complex with PINCH‐2 in the place of PINCH‐1 and/or ‐Parvin/aYxin, in the place of ‐Parvin. Thus, there are multiple possible combinations of proteins for the formation of the PINCH–ILK–Parvin complex (the binding of PINCH‐1 and PINCH‐2 to ILK is mutually exclusive, and the same happens with ‐Parvin and ‐Parvin). The third member of the Parvin family, ‐Parvin, has not been shown to bind to ILK, but judging by its structural similarity with the ‐ and ‐Parvins, there is a good likelihood that it may also bind to it. In the schematic diagram of this figure, the stable ternary PINCH–ILK–Parvin complex is shown in blue, and the alternative partners for ILK are enclosed in dashed boxes. PINCH‐1 can also bind to Nck‐2, which has been found to be associated with receptor tyrosine kinases (RTKs), thus connecting the focal adhesion signaling with growth factor molecular pathways. In addition to that, ILK can bind to Mig‐2, which was recently shown to form a complex with the protein migfilin and the actin–cross‐linking protein filamin. Interestingly, apart from its localization to FAs, migfilin has been shown to bind to the nuclear cardiac transcription factor CSX/Nkx‐2.5, and it has also been reported to localize at cell–cell adhesions, although it is not yet clear what recruits migfilin to these sites. All the reported bindings are shown with solid red arrows, whereas other potential protein–protein interactions are shown with blue dashed arrows.

proteins, including paxillin (Nikolopoulos and Turner, 2000), -actinin (Yamaji et al., 2004), and actin (Nikolopoulos and Turner, 2000; Olski et al., 2001), have been shown to interact with -Parvin and/or -Parvin. Thus, the PINCH–ILK–Parvin complex likely functions as a molecular

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platform at cell–ECM adhesions mediating multiple interactions and hence regulates cell–ECM adhesion and signaling. PINCH, ILK, and Parvins are widely expressed in human tissues and well conserved among diVerent species (Clark, 2003; Mackinnon et al., 2002; Wu and Dedhar, 2001; Zervas and Brown, 2002; Zervas et al., 2001). Genetic studies in Drosophila, C. elegans, and mouse have demonstrated their essential roles for embryonic development and normal cell function in vivo (Brakebusch and Fassler, 2003; Clark et al., 2003; Hobert et al., 1999; Lin et al., 2003; Mackinnon et al., 2002; Zervas et al., 2001). For example, loss of ILK leads to periimplantation lethality in the mouse (Brakebusch and Fassler, 2003), embryonic lethality in C. elegans (Mackinnon et al., 2002) and Drosophila (Zervas et al., 2001), characterized by muscle detachment and severe actin-related defects (Brakebusch and Fassler, 2003). Similarly, PINCH depletion seems to also lead to embryonic lethality during the phase of implantation (GrashoV et al., 2004). Interestingly enough, the interactions among PINCH, ILK, and Parvins are well conserved in C. elegans and Drosophila (Clark et al., 2003; Kadrmas et al., 2004; Lin et al., 2003; Mackinnon et al., 2002). They work together in a multiprotein complex in the invertebrate organisms as well. Thus, the formation of the PINCH–ILK–Parvin complex is likely crucial for the functions of these proteins. Indeed, the phenotypes resulting from loss of ILK or PINCH bear significant similarity to the ones observed when the complex formation is disrupted (Zhang et al., 2002c).

B. Assembly and Regulation of the PINCH–ILK–Parvin Complexes Recent work has shown that ILK, PINCH, and Parvin are recruited to cell– ECM sites as a preassembled complex (Zhang et al., 2002b), indicating that the formation of the complex precedes the integrin-mediated cell adhesion and spreading. The assembly of the PINCH–ILK–Parvin complex is known to be regulated by two main signaling pathways, although other levels of regulation may be also involved. First, the complex formation is regulated by the protein kinase C (PKC) signaling pathway (Zhang et al., 2002b). Inhibition of PKC leads to down-regulation of the assembled complex, suggesting that the PINCH–ILK–Parvin complex is an important downstream target of the PKC pathway through which many cellular processes such as cell migration, spreading, and proliferation are regulated. Second, the formation of the PINCH–ILK–Parvin complex is regulated by the phosphatidylinositol-3 (PI-3) kinase pathway, because inhibition of PI3K pathway by small compound inhibitors or by overexpressing PTEN also results in the inhibition of the complex assembly (Attwell et al., 2003).

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In mammalian cells, two structurally related PINCH proteins (termed PINCH-1 and PINCH-2), which are encoded by two diVerent genes, have been identified to date. PINCH-1 and PINCH-2 are co-expressed in a number of cell types and tissues (Braun et al., 2003; Wu, 2004; Zhang et al., 2002a). Both PINCH-1 and PINCH-2 can bind to ILK (Zhang et al., 2002a). Furthermore, the binding of PINCH-1 and PINCH-2 to ILK is mutually exclusive (Zhang et al., 2002a). As mentioned previously, both - and -Parvins are capable of binding to ILK (by way of their CH2 domain). Although little is known about -Parvin, it probably can also interact with ILK, based on sequence similarities with the other members of the Parvin family (Wu, 2004). Thus, in many types of cells, there exist multiple PINCH–ILK–Parvin complexes. It is worth noting, though, that the diVerent PINCH–ILK–Parvin complexes, although sharing certain common functions, are not redundant or interchangeable but rather each one plays its own distinct role within the cell. The PINCH-2–ILK–Parvin complex, for instance, cannot substitute for the PINCH-1–ILK–Parvin complex, because overexpression of PINCH-2 cannot rescue the defects in cell shape and survival induced by depletion of PINCH-1 (Fukuda et al., 2003a).

C. Signaling Through the PINCH–ILK–Parvin Complexes Activation of ILK, either by integrin clustering or by growth factors, aVects multiple cell signaling pathways that regulate cell survival, proliferation, and diVerentiation. ILK activation results in the phosphorylation and subsequent activation of PKB/Akt (Persad et al., 2000; Troussard et al., 2003; Wu and Dedhar, 2001), as well as extracellular signal–regulated kinase/mitogen-activated protein kinase (Erk/MAPK) (Wu and Dedhar, 2001). In addition, -Parvin, one of the partners of ILK in the PINCH– ILK–Parvin ternary complexes, was shown to regulate cell survival signaling by aVecting the membrane translocation of PKB/Akt (Fukuda et al., 2003b). Thus, the components of the ILK complexes seem to function in concert to mediate cellular signaling. The activation of PKB/Akt, for instance, results in a cascade of signaling events leading to protection of cells from apoptosis (Downward, 2004; Franke et al., 2003; Hemmings, 1997). ILK can also phosphorylate and subsequently inhibit glycogen synthase kinase 3-beta (GSK3- ) in a PI3K-dependent manner (Delcommenne et al., 1998; Mhashilkar et al., 2003; Tan et al., 2001; Wu and Dedhar, 2001). This leads to the induction of the transcriptional activities of AP-1 transcription factor, as well as the -catenin/lymphoid enhancer factor-1 (LEF1) transcription complex (Novak et al., 1998; Novak and Dedhar, 1999). This in turn up-regulates the expression of matrix-metalloproteinase-9 (MMP-9) (Troussard, 2000), c-myc (Novak and Dedhar, 1999), and cyclin D1,

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respectively (D’Amico et al., 2000), resulting in degradation of the ECM, invasion, and cell cycle progression. Activation of PKB/Akt also leads to induction of the expression of hypoxia-inducible factor 1
(HIF-1), which in turn stimulates the expression of vascular endothelial growth factor (VEGF), thus aVecting angiogenesis (Tan et al., 2004). Recent studies have identified several upstream regulators of ILK. For example, ILK activity can be negatively regulated by PTEN (Persad et al., 2000; Wu and Dedhar, 2001) and ILK-associated phosphatase (ILK-AP) (Kumar et al., 2004; Leung-Hagesteijn et al., 2001). Recently it was shown that -Parvin, one of its binding partners, is also capable of inhibiting it (Mongroo et al., 2004; Zhang et al., 2004). Loss of -Parvin was observed in some advanced breast tumors that express high levels of ILK (Mongroo et al., 2004). Moreover, overexpression of -Parvin in breast cancer cells, which express elevated levels of ILK and reduced levels of -Parvin, inhibited ILK kinase activity, as well as anchorage-independent growth and invasion (Mongroo et al., 2004). Thus, -Parvin likely functions as a negative regulator of ILK. Taking into account the fact that -Parvin promotes cell survival through ILK signaling (Fukuda et al., 2003b), it seems that - and -Parvin, two binding partners of ILK, exert antagonistic eVects on ILK signaling. In fact, the binding of - and -Parvin to ILK is mutually exclusive (Zhang et al., 2004). Thus, ILK signaling, apart from PI3K and growth factors is also regulated by its binding partners. Consistent with the fact that ILK regulates major signaling pathways, it has been implicated in many physiological or pathological processes such as cell diVerentiation, proliferation, migration, platelet aggregation, and angiogenesis (Friedrich et al., 2004; Huang et al., 2000; Kaneko et al., 2004; Tan et al., 2004; Terpstra, 2003; Wu et al., 1998; Yamaji et al., 2002). ILK was shown to be an important regulator of myogenic diVerentiation, aVecting the initial stages of this process by regulating MAPK activation (Huang et al., 2000). In fact, both the kinase activity of ILK and its ability to form a complex with PINCH are required for the regulation of myogenic diVerentiation, because overexpression of kinase-deficient ILK mutants or PINCHbinding–deficient mutants, unlike that of the wild type ILK, cannot inhibit the expression of myogenic proteins or the formation of myotubes (Huang et al., 2000). In addition to myogenic diVerentiation, ILK was shown to be involved in chondrocyte proliferation and diVerentiation (Terpstra et al., 2003) and platelet aggregation (Yamaji et al., 2002). Furthermore, it was shown to regulate tumor angiogenesis (Tan et al., 2004) by stimulating the expression of VEGF. In that case, inhibition of ILK leads to the inhibition of VEGF-mediated endothelial cell migration and angiogenesis (Tan et al., 2004). Finally, recent work by Friedrich et al., done in mice using the Cre-Lox system and in the developing zebra fish using the antisense technology, has shown that ILK plays a fundamental role in vascular development

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and endothelial cell survival in vertebrates (Friedrich et al., 2004). ILK deficiency had pleiotropic eVects in all embryos such as defects in branching morphogenesis of chorionic epithelium in mice and disordered patterning of vessels in zebra fish, whereas endothelial cells isolated from the mice showed defective adhesion to the ECM (Friedrich et al., 2004). There is compelling evidence suggesting that ILK plays a role in oncogenic transformation. First, overexpression of ILK promotes anchorage-independent growth and cell cycle progression (Hannigan et al., 1996; Radeva et al., 1997; White et al., 2001; Wu et al., 1998). Second, ILK overexpression in epithelial cells leads to down-regulation of E-cadherin and disruption of cell–cell adhesions (Hannigan et al., 1996; Wu et al., 1998), as well as inhibition of suspension-induced apoptosis (anoikis), all of which are important traits of cancer cells (Avizienyte, 2002; Cairns et al., 2003; Cavallaro and Christofori, 2001; Conacci-Sorrell, 2002; Hajra and Fearon, 2002; Hanahan and Weinberg, 2000; Popov et al., 2000). Third, modest overexpression of ILK in intestinal and mammary epithelial cells promotes invasion and is accompanied by translocation of -catenin to the nucleus, complex formation between -catenin and the LEF1 transcription factor, and subsequent transcriptional activation of prosurvival genes such as MMP-9, cyclins, and cMYC (Ben-Ze’ev et al., 1999; Novak and Dedhar, 1999; Novak et al., 1998). Finally, clinic studies have shown that ILK expression level increases in a number of malignancies, and there is often a correlation between ILK expression levels and the tumor grade. For instance, the ILK level is increased in the highest grade of prostate adenocarcinoma compared with lower grade or benign prostate hyperplasia (GraV et al., 2001; Yoganathan et al., 2002). It is also correlated with the ovarian tumor grade (Ahmed et al., 2003) and melanoma progression and poor prognosis (Dai et al., 2003). Both ILK activity and expression levels are also increased in polyps from patients with familial adenomatous polyposis (Marotta et al., 2001, 2003). This combined with all the aforementioned evidence indicates that ILK may be a useful biological marker for certain types of cancer (Ahmed et al., 2004) or even a potent target for anticancer therapy (Attwell et al., 2000; Hannigan et al., 2005; Mhashilkar et al., 2003; Yoganathan et al., 2000).

D. Proteins Interacting with the PINCH–ILK–Parvin Complexes The interactions of ILK with PINCH and -Parvin are necessary but not suYcient for ILK localization to cell–ECM adhesion sites (Zhang et al., 2002b). This suggests that there are other proteins that interact with the components of the PINCH–ILK–Parvin complex and mediate its localization to cell–ECM sites. Indeed, each of the proteins forming the ternary complex of PINCH–ILK–Parvin interacts with multiple proteins within the

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cell. For example, ILK can interact with 1 and 3 integrin cytoplasmic domains (Hannigan et al., 1996), paxillin (Nikolopoulos and Turner, 2001), ILK-AP (Kumar et al., 2004), and mitogen inducible gene-2 (Mig-2), a mammalian homologue of C. elegans UNC-112 (Mackinnon et al., 2002; Rogalski et al., 2000; Tu et al., 2003). Studies in C. elegans have shown that UNC-112 binds to PAT4/ILK and is essential for the recruitment of PAT4/ ILK to attachment sites (Mackinnon et al., 2002), thus making UNC-112 a possible candidate for recruiting ILK to cell–ECM sites. Mammalian Mig2 also interacts with ILK. In addition, it interacts with migfilin, a recently identified LIM domain-containing protein (Tu et al., 2003). Migfilin, in turn, binds to filamin A and C (hence the name migfilin) (Tu et al., 2003). These interactions are important for actin cytoskeleton organization and processes that require extensive shape modulation such as cell migration (Stossel and Hartwig, 2003; Tu et al., 2003). Migfilin consists of five distinct domains: an N-terminal domain, a proline-rich domain in the center, and three LIM domains in the C-terminal region. Three splice variants of migfilin have been identified to date: the long form migfilin with all the domains described earlier, a short form lacking the proline-rich domain (Tu et al., 2003), and FBLP-1, which lacks the last LIM domain (Takafuta, 2003). The C-terminal region of migfilin is responsible for the binding to Mig-2, the N-terminal accounts for the interaction with filamin (Tu et al., 2003), whereas the central proline-rich domain mediates the interaction with VASP (Y. Zhang and C. Wu, manuscript submitted), another actin-regulatory protein (Krause et al., 2002, 2003). Interestingly, Cal, the mouse homologue of migfilin, is capable of interacting with the cardiac homeobox transcription factor CSX/Nkx2-5 by way of the Cterminal LIM domains (Akazawa et al., 2004). Taking into consideration the fact that the LIM domain motif with the tandem zinc fingers is known to mediate multiple protein–protein interactions (Bach, 2000; Dawid et al., 1998; Jurata and Gill, 1998; Khurana et al., 2002; Schmeichel and Beckerle, 1994), it is anticipated that other binding partners of migfilin exist as well. In a wide variety of fibroblasts, epithelial, and endothelial cells, migfilin is present at the cell–ECM adhesion sites where it is being recruited by Mig-2, and it is also associated with actin filaments as a result of its interaction with filamin (Tu et al., 2003). Consistent with its association with the CSX/Nkx25 transcription factor, the mouse homologue of migfilin was also detected in the nucleus (Akazawa et al., 2004). The nuclear traYcking of migfilin has not been studied extensively, but existing evidence indicates that migfilin enters the nucleus in response to high intracellular calcium levels, because treatment of the cells with a calcium ionophore promotes the nuclear accumulation of the protein (Akazawa et al., 2004). As for the nuclear export of migfilin, the nuclear export signal (NES) within the proline-rich domain was shown to be responsible (Akazawa et al., 2004). Consistent with the latter,

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expression of the short-form migfilin that lacks the proline-rich domain and thus the NES leads to nuclear accumulation of the protein, whereas expression of the long-form migfilin does not have the same result (Gkretsi et al., 2005). Studies in C. elegans reveal that UNC-112, the invertebrate homologue of Mig2, is essential for the localization of integrins in the muscle cell membrane, indicating that Mig-2 itself is important for integrin-related cell functions (Rogalski et al., 2000). Depletion of Mig2 from mammalian cells impairs cell spreading, showing that Mig-2 is important for cell-shape modulation (Tu et al., 2003). Migfilin, the binding partner of Mig-2, is also critical for cell-shape modulation. Depletion of migfilin impairs cell spreading and reduces the level of filamentous actin (F-actin) compared with the level of free G-actin, thus playing an important role in actin remodeling (Tu et al., 2003). Intriguingly, a recent study demonstrated that Mig-2 was transcriptionally elevated in uterine leiomyomas compared with normal myometrium (Kato et al., 2004). This suggests a putative role for Mig-2 in the hormone-mediated growth of benign smooth muscle tumor cells of uterine leiomyomas. Moreover, the significantly lower level of Mig-2 in leiomyosarcomas compared with leiomyomas and normal myometrium indicates that there must exist a mechanism responsible for this diVerential expression of the protein in diVerent types of tumors. In epithelial and endothelial cells, migfilin localizes not only to cell–ECM but also to cell–cell adhesions. It seems to be a component of the adherens junctions (Gkretsi et al., 2004). Moreover, the domains of migfilin that mediate its localization to cell–cell adhesions partially overlap with the ones mediating its localization to cell–ECM adhesions, although it is not yet clear how migfilin is recruited to these sites. The role of migfilin in cell–cell adhesions is incompletely understood. Recent work shows that depletion of migfilin by silencing RNA in HT-1080 fibrosarcoma cells leads to a phenotype characterized by tremendously disorganized adherens junctions, which are far weaker than the ones in the control-treated cells (Gkretsi et al., 2005). This is indicative of the importance of migfilin in the proper organization of adherens junctions. New insight into the functional role of migfilin in cells is gained from a recent study in which Cal, the mouse homologue of migfilin, is shown to be important for cardiac diVerentiation (Akazawa et al., 2004). Interestingly, this study shows that the mouse homologue of migfilin possesses itself a transcription-promoting activity, and by association with the CSX/Nkx2-5 transcription factor, it is capable of transactivating certain genes such as the atrial natriuretic peptide (ANP) promoter and the transcription factor GATA-4 (Akazawa et al., 2004). Thus, migfilin seems to play a very significant part in the regulation of transcription of cardiac genes and cardiac development in general.

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IV. Adhesion Complexes in Cardiac Myocytes A. Structure of Adhesion Complexes in Cardiac Myocytes Cardiac myocytes are subject to repeated contraction cycles lasting for the prolonged lifetime of the cells, which place heavy physical demands on the myofibrils. Moreover, cardiac myocytes need to respond to various types of mechanical strain as the heart adapts to variations in pressure or volume. Adhesion complexes play critical roles in the mechanical and physiological properties of myocytes and their response to mechanical and chemical signals by: (1) providing stable structural linkages between adjacent myocytes and between the contractile apparatus (sarcomere), the cytoskeleton, the plasma membrane (sarcolemma), and the ECM; (2) transmitting mechanical signals from the ECM and adjacent cells to transduction pathways leading to adaptive or pathological phenotypic changes in gene expression and cytoskeletal organization (outside-in signaling); and (3) integrating cellular signaling pathways by providing a scaVold for organization of signaling components and by responding to those signals by undergoing conformational changes that rearrange the cytoskeletal–ECM interactions (inside-out signaling). In addition to the classic FA similar to those in other cell types (see earlier), myocyte adhesion complexes are organized in sarcolemmal domains related to cell–cell junctions at the termini of adjacent myocytes (adherens junctions and intercalated disks) or in subsarcolemmal structures in close contact with peripheral myofibrils, known as costameres. Costameres align circumferentially with the Z-disks of sarcomeres, which serve to anchor sarcomeric actin thin filaments and contain a multitude of proteins involved in myofibrillar assembly, cytoskeletal organization, and signal transduction (Fig. 2). Three molecular types of adhesion complexes are present in cardiac myocyte costameres, all of which are interconnected to cytoskeletal networks and participate in signaling pathways (Fig. 2): 1. Integrin/FA complexes composed of - and -integrins, vinculin, talin, paxillin, and other molecules 2. Dystroglycan complexes composed of transmembrane laminin receptors (dystroglycan) and sarcoglycans in connection with dystrophin and associated proteins

Figure 2 Protein components of the sarcomere Z‐disk, adhesion complexes and cytoskeletons. The protein interactions in costameres and intercalated disks are depicted (recently reviewed [Borg et al., 2000; Calaghan et al., 2004; Clark et al., 1998; Ervasti, 2003; Lapidos et al., 2004; Nagafuchi, 2001; Ross, 2004]). Protein–protein interactions are represented by lines ending in circles.

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3. Cadherin/catenin complexes composed of transmembrane N-cadherins linked to - and -catenin and plakoglobin. Three types of junctional structures are present in the intercalated disks at the myocyte termini: 1. Fascia adherens connecting the actin cytoskeleton and the myofibril to the sarcolemma where cadherin/catenin–based adhesion complexes promote homotypic intercellular adhesion 2. Desmosome, connecting the intermediate filament network to the intercalated disk and firmly connecting adjacent myocytes 3. Gap junctions composed of connexins 40, 43, and 45, which electrically couple adjacent myocytes. The various types of adhesion complexes and structures connect to the four types of cellular cytoskeleton components: 1. Microfilaments: In addition to the sarcomeres, composed of thin filaments (F-actin), thick filaments (myosin), and numerous structural and signaling proteins, nonsarcomeric microfilaments of - and -actin are dispersed throughout the myocyte. 2. Intermediate filaments composed mainly of desmin, but also synemin and syncoilin, cross-link and organize the sarcomeric structures and provide bridges between various adhesion complexes. 3. The microtubule network provides rigidity to the cell and aVects protein synthesis, intracellular traYcking, and intracellular signaling. 4. The ankyrin-spectrin membrane skeleton provides stability and organization to the sarcolemma and connections to the other cytoskeleton components. In the following, we will focus on the assembly of integrin–FA complexes and the role they play in cardiomyocyte development, myofibrillogenesis, and transduction of mechanical and chemical signals that lead to cardiac hypertrophy. When appropriate, we will mention the role of other types of adhesion complexes and their interactions with integrin-dependent pathways. B. Models of Myofibrillogenesis Integrin-dependent adhesion complexes play an important role in organizing the structured assembly of myofibrils to form sarcomeres in the myocyte, a process known as myofibrillogenesis. This process has been observed in various experimental systems, leading to two major models on how myofibrillogenesis proceeds.

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In the mini-sarcomere model, mainly derived from observation in cultured primary cardiac myocytes, sarcomeric -actinin, together with nonmuscle and muscle isoforms of -actin and tropomyosin, begins to appear in periodic dots called Z-bodies assembled at subsarcolemmal regions of the spreading edges of the myocytes (Dabiri et al., 1997; Handel et al., 1991). At this stage, nonsarcomeric myosin IIB fibrils are interspersed between the Z-bodies, forming minisarcomeres. Later, the growing Z-bodies organize in parallel arrangements. With the incorporation of the giant protein titin, the Z-bodies start to be evenly spaced along the longitudinal dimension, and thick filaments appear when sarcomeric myosin is incorporated in place of nonsarcomeric myosin. Mature myofibrils are formed when Z-bodies from closely apposed neighboring myofibrils fuse laterally to form the Z-disk (Dabiri et al., 1997). The I-Z-I model, originating from observations in chick embryos (Ehler et al., 1999), explanted precardiac mesoderm (Rudy et al., 2001), and embryoid bodies derived from embryonic stem cells (Guan et al., 1999), suggests that -actinin and the N-terminus of titin are first organized in membrane-attached Z-bodies. Subsequently, actin filaments attach to the Z-bodies to form I-Z-I stresslike fibers (Rudy et al., 2001). In this model, myosin IIB is not present, consistent with the fact that myosin IIB knockout mice show normal myofibrillogenesis during early embryonic development (Tullio et al., 1997). Later, sarcomeric myosin filaments attach to form mature myofibrils, a process facilitated by the binding of myomesin to the M-line region of titin (Ehler et al., 1999). In this model, the actin-capping protein tropomodulin (Tmod1) localizes to the sarcolemma in close association with spectrin, even before myofibrillogenesis is initiated, and has been suggested to play a role in early Z-body formation (Ehler et al., 2004). This is in contrast with the cultured myocyte model, in which Tmod1 is the last protein incorporated at the pointed ends of the actin filaments, when thin filament length becomes fixed (Rudy et al., 2001). However, a critical role for Tmod1 in the initial steps of myofibrillogenesis has not been well established, because I-Z-I structures can form in the absence of Tmod1, even though myofibril maturation is severely perturbed (Fritz-Six et al., 2003). Additional proteins found in the early Z-bodies include NRAP (Lu et al., 2003), actininassociated LIM protein (ALP, LMPDZ1) (Henderson et al., 2003), and palladin (Bang et al., 2001b). NRAP has been suggested to initiate the formation of Z-bodies by virtue of its interaction with -actinin (Carroll et al., 2004). After Z-body nucleation and formation of I-Z-I fibrils, Z-bodies fuse laterally and release NRAP, which becomes restricted to the intercalated disks and absent from mature sarcomeres (Carroll and Horowits, 2000; Carroll et al., 2004). ALP co-localizes with -actinin in early premyofibrils (Henderson et al., 2003), possibly through its two -actinin binding domains (Klaavuniemi et al., 2004), and may play a role in stabilizing actin filament

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anchorage at the Z-bodies (Pashmforoush et al., 2001). Palladin is critical for assembly of FA complexes in other cell types, suggesting that its presence in I-Z-I bodies in nascent myofibrils may play a role in organization of mature costameres (Bang et al., 2001b).

C. Regulation of Myofibrillogenesis Myofibrillogenesis is aVected by costameres and depends on intact signaling by Rho, because C3 exo-enzyme application to developing cardiac myocytes resulted in disassembly of costameres, with loss of periodic staining for integrins, talin, and vinculin and disappearance of striated myofibrils (Wang et al., 1997). Signaling involving small G proteins of the Rho family (RhoA, Rac1, Cdc42) is critical for actin polymerization in other cell types, and high RhoA, Rac1, or Cdc42 activity induces strong sarcomeric assembly in cultured cardiac myocytes (Aikawa et al., 1999; Aoki et al., 1998; Clerk et al., 2001; Nagai et al., 2003; Wei et al., 2001). Increased actin polymerization leads to induction of the serum response factor (SRF), an important transcription factor regulating expression of various sarcomeric and cytoskeletal genes, including sarcomeric actin and myosin subunits, FAK, 1-integrin, talin, zyxin, and vinculin (Gineitis and Treisman, 2001; Schratt et al., 2002; Sepulveda et al., 2002; Sotiropoulos et al., 1999; Wei et al., 2001). It is conceivable that initial myofibrillogenesis induced by high Rho activity leads to increased actin polymerization and decreased availability of G-actin, a negative regulator of SRF activity (Sotiropoulos et al., 1999). Stimulation of SRF activity then results in increased availability of sarcomeric and FA proteins, leading to assembly of mature myofibrils and costameres. In addition to myofibrillogenesis in embryonic development, hypertrophic signaling converging on SRF, SRF transcriptional partners such as GATA4 and TEF1, and other transcription factors, such as NFATc and MEF2C, is involved in hypertrophic myofibrillogenesis and costamere assembly (Akazawa and Komuro, 2003). Clearly, the giant molecule titin plays a critical role in sarcomeric assembly, either at the initial stages (I-Z-I model) or after formation of the initial Z-bodies (Granzier and Labeit, 2004). Titin binds myosin thick filaments at the M-bands and connects them to -actinin in the Z-disks (Sorimachi et al., 1997). The number of alternatively spliced Z-repeats, which are -actinin binding sites in the titin molecule, is diVerent in the various types of striated muscle and determines the degree of -actinin–dependent cross-linking of actin thin filaments in the Z-disk. Similarly, various diVerentially spliced ‘‘spring’’ domains of titin provide elasticity to the sarcomeres (Granzier and Labeit, 2004). One interesting aspect of the titin–-actinin interaction is that

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it is enhanced by high levels of phosphatidylinositol 4,5 bisphosphate (PI4,5P2) and may, therefore, be regulated by the activity of phosphatidylinositol-4-phosphate-5-OH-kinase (PIP5K1C), which also co-localizes to the costameres (Young and Gautel, 2000), perhaps through its ability to bind talin (Ling et al., 2002). Because PI4,5P2 also promotes formation of FAs (Gilmore and Burridge, 1996; Ling et al., 2002), this may be another mechanism linking sarcomere assembly and costamere formation. By interacting with various proteins along the sarcomere, titin provides a platform for further organization of the myofibrils and links to signal transduction and metabolic pathways. For example, titin binds the LIMdomain protein FHL2, which anchors adenylate kinase, phosphofructokinase, and creatine kinase to the M-bands and I-bands of the sarcomeres (Lange et al., 2002). Interestingly, FHL2 binds to 1-integrin in cardiac muscle at the periphery of Z-disks (Samson et al., 2004) and can also localize to the nucleus, where it can function as a co-activator (Johannessen et al., 2003; Morlon and Sassone-Corsi, 2003; Muller et al., 2000) or a repressor (Hill and Riley, 2004) in a manner regulated by RhoA and Rho-dependent kinase (Muller et al., 2002). These findings suggest that FHL2 may integrate integrin and Rho signaling with mechanical tension readouts provided by titin, resulting in changes in metabolic and transcriptional activity. Another titin-binding protein is telethonin (TCAP), which binds the Nterminus of titin and anchors it to the Z-disk (Mues et al., 1998). In the Z-disk, TCAP interacts with muscle LIM protein (MLP, CSRP3), an actinin–, and NRAP-binding protein (Gehmlich et al., 2004). Interestingly, absence of MLP results in disruption of myofibril and Z-disk alignment (Arber et al., 1997), and mutation of MLP in humans is associated with dilated cardiomyopathy (Knoll et al., 2002). MLP-deficient cardiac myocytes do not mount a hypertrophic response to mechanical strain (Knoll et al., 2002), suggesting that -actinin, MLP, TCAP, titin, and NRAP may be components of protein complexes that function as mechanical sensors and/or are required for sarcomeric reorganization in response to stretch. Like FHL2, MLP is a cytoskeleton-linked LIM domain protein that acts as a scaVold for assembly of multiprotein complexes, both at the Z-disk and in the nucleus, where MLP acts as a co-activator for MyoD (Kong et al., 1997), and MLP-related proteins CSRP1 and CSRP2 act as SRF and GATA4 co-activators (Chang et al., 2003). Additional proteins interacting with titin are nebulin (Ma and Wang, 2002) and obscurin (Bang et al., 2001a). Nebulin is a large protein expressed in skeletal muscle sarcomeres, where it is postulated to serve as a molecular ruler to define the length of the thin filaments, possibly because it interacts with Tmod1, which caps the pointed ends of F-actin at the end of the I-band and maintains thin filament size (McElhinny et al., 2001), and

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with myopalladin, which anchors nebulin to -actinin and the Z-disk (Bang et al., 2001b). Nebulin was proposed to regulate thin filament length in skeletal but not cardiac muscle, because the cardiac homologue, called nebulette, extends only approximately 25% along the length of the thin filament. However, recent data indicate that nebulin is also expressed in the heart and its N-termini and C-termini co-localize with the barbed (Z-disk) and pointed ends (I-band) of F-actin, respectively (Kazmierski et al., 2003), suggesting that it may also regulate thin filament size in cardiac sarcomeres. Because of its large size, spanning the sarcomere, and its interactions with titin, it was proposed that the giant protein obscurin plays an important role in integrating and aligning the neighboring nascent myofibrils (Borisov et al., 2004), a role played in more mature myofibrils by the intermediate filament desmin. In addition, because obscurin binds small ankyrin proteins present in the sarcoplasmic reticulum and the T-tubules system, obscurin might play a role in aligning the sarcoplasmic reticulum and T system to the Z and M lines of the sarcomeres (Bagnato et al., 2003; KontrogianniKonstantopoulos et al., 2003). Interestingly, obscurin is upregulated during cardiac hypertrophy, where it may play a role in assembly of new sarcomeric units (Borisov et al., 2003). Because obscurin possesses a Rho–GEF domain (Young et al., 2001), it is tempting to speculate that it may play a role in sarcomere-linked signaling pathways involving small G proteins.

D. Role of Costameres and Intercalated Disks in Myofibrillogenesis In either model of myofibrillogenesis, it is apparent that spatial information is conveyed from the ECM and the sarcolemma to the formation and orientation of the nascent myofibrils, possibly through costameres and intercalated disks (Tokuyasu, 1989). However, mature integrin-based costameres are unlikely to be involved in the initial appearance of Z-bodies, because FA proteins such as talin, vinculin, and paxillin are absent from the Z-bodies and only appear in costameres associated with mature myofibrils (Sanger et al., 2000). Nevertheless, 1-integrin is critical for early myofibrillogenesis because 1-integrin–deficient cardiac myocytes have a severe delay in myofibrillogenesis (Fassler et al., 1996). Addition of antibodies against integrins 1A, 1D, 3A, and 3B resulted in disruption of myofibrillar organization (Kim et al., 1999). Similarly, expression of mutated 1 or 5 intracellular domains caused defects in cardiogenesis and myocyte survival (Keller et al., 2001; Valencik and McDonald, 2001; Valencik et al., 2002). It is possible that localization of -actinin to the membrane is mediated by its direct interaction with the cytoplasmic domain of -integrins (Cattelino

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et al., 1999). These primitive costameres containing integrins and -actinin might serve as ECM-oriented anchorage sites for organization of the Z-bodies and subsequently the Z-disk of the mature myofibrils. -actinin and -integrin represent in the primitive costamere the two ends of a network of interacting proteins connecting the Z-disk of the mature sarcomeres with the mature costameres (Borg et al., 2000). Costameric proteins such as talin (PfaV et al., 1998), paxillin (Tanaka et al., 1996), and ILK (Chen, Wu, and Sepulveda, manuscript submitted) assemble in mature costameres possibly by their ability to bind the cytoplasmic tail of 1-integrin. With maturation of the myofibrils, -actinin in the Z-disk becomes a terminal docking station for a network of multiple Z-disk proteins (Clark et al., 2002), which provide tensile strength to the anchoring of the thin filaments of the sarcomeres and, in addition, seem to supply a mechanism for transducing mechanical strain in the cell. By virtue of their connections to costameres, Z-disk proteins can also transmit signals from and to the sarcolemma and ECM. Later, we will review the role of costameric and Z-disk proteins in signal transduction. After organization of sarcomeres in mature myofibrils, the myofibrils detach from the membrane and begin contracting. However, the Z-disks remain co-localized and connected to the adhesion complexes (integrin, dystroglycan, and spectrin based) mainly through -actin filaments (Calaghan et al., 2004). -Actin is connected to the Z-disk through -actinin (Calderwood et al., 2000), with the costameric integrin complexes possibly by binding talin (Lee et al., 2004), vinculin (Steimle et al., 1999), and -Parvin (Nikolopoulos and Turner, 2001), with the dystroglycan complexes by binding dystrophin (Rybakova et al., 2000), and with the spectrin membrane skeleton by means of the cytolinker protein plectin (Sevcik et al., 2004) (Fig. 2). Two other proteins that can directly bridge mature costameres and Z-disks are vinculin, which binds both -actinin (Wachsstock et al., 1987) and talin (Papagrigoriou et al., 2004), and filamin C, which binds actin filaments and -integrin (Goldmann and Isenberg, 1993; Loo et al., 1998). In addition to the formation of Z-bodies near the sarcolemma at primitive costamere sites, nascent premyofibrils are anchored by their ends to the intercalated disks containing vinculin, NRAP, and the cadherin–catenin complexes (Carroll and Horowits, 2000; Imanaka-Yoshida et al., 1998; Luo et al., 1999). Cadherin–catenin based adhesion complexes in the fascia adherens are also essential for myofibrillogenesis, because extracellular antibodies to N-cadherin and intracellular microinjection of antibodies against N-cadherin, - or -catenin inhibited the formation and organization of myofibrils (Imanaka-Yoshida et al., 1998; Wu et al., 1999). Studies with N-cadherin null cardiac myocytes show that whereas initial myofibrillogenesis is unaVected, the alignment of myofibrils is severely perturbed, resulting in their random orientation (Luo and Radice, 2003). In addition, disruption

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of vinculin expression with antisense oligonucleotides (Shiraishi et al., 1997) or in knockout mice (Xu et al., 1998) inhibits myofibrillogenesis. Deletion of plakoglobin also results in heart defects, even though myofibrillogenesis is not aVected (Isac et al., 1999). These results are consistent with the model that the fascia adherens, composed of N-cadherin, - and -catenins, plakoglobin, and vinculin (Fig. 2), serves to anchor nascent myofibrils and coordinate myofibril orientation across adjacent myocytes. In addition to the fascia adherens in intercalated disks, N-cadherin–catenin complexes are also present in a costameric fashion in embryonic rat cardiac myocytes and on the dorsal side of cultured chicken cardiac myocytes (Wu et al., 1999, 2002). Interestingly, cadherin/catenin costameres and integrin/talin costameres were observed in mutually exclusive locations, and only the latter persisted in the adult heart (Wu et al., 2002). In addition to their role in organizing myofibrillogenesis, cadherin–catenin complexes play an important role in signaling transduction during early cardiac development, either by patterning cell–cell interactions in precardiac mesoderm (Linask et al., 1997) or by participating in canonical Wnt signaling pathways (Nakamura et al., 2003).

E. Integrin Adhesion Complexes in Cardiac Development The cardiac myocytes express integrins 1, 3B, 5, 6A, 6B 7B, 7C, 7 (X2), 10, and 11 together with 1, 3, and 5, which are diVerentially expressed throughout development and in response to hypertrophic stimuli (Maitra et al., 2000; Ross, 2004). Figure 3 summarizes the relative abundance of integrin isoforms in cardiac myocytes. During embryonic development, integrins 1, 5, 3B, and 6 dimerize with integrin 1A. After birth, there is an induction of the expression of integrin 1D, which becomes the predominant -integrin isoform in the adult, whereas 1A is downregulated (Maitra et al., 2000). Similarly, integrins 1 and 5 are downregulated after birth, whereas 3B and 6 undergo transitory induction during the neonatal period. In the adult, the predominant -integrins are 3B, 7 variants, and 11. Hypertrophy is accompanied by further induction of integrin 1D, as well as 1, 5, and 7, whereas 6 integrins are downregulated (Babbitt et al., 2002). These changes eVectively switch the receptor density for various ECM components during development and in response to hypertrophy. For example, the fibronectin receptor 5 1 is maximally expressed in the fetal period, repressed in the adult, and re-induced during hypertrophy, whereas the 6 1 laminin receptor has maximal expression during the neonatal period and is downregulated in hypertrophy. These results are in agreement with the notion that fibronectin and 5 1A are associated

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Figure 3 Summary of the relative abundance of integrin isoforms in cardiac myocytes. The relative abundance of - and ‐integrin isoforms throughout development and after induction of hypertrophy in cardiac myocytes is represented by the height of the respective line. - and ‐integrins are graphed separately. Broken lines represent uncertainty in the abundance of the respective isoform.

with proliferation, whereas laminin and 6 1D or 7 1D is associated with diVerentiation, at least in skeletal myocytes (Shin et al., 1995). Interestingly, fibronectin plating of postnatal cardiac myocytes is associated with 1integrin–dependent hypertrophy and transcriptional induction of fetal genes (Chen et al., 2004; Ogawa et al., 2000). Another consequence of isoform switch from 1A to 1D may relate to the fact that 1D binds talin more tightly, therefore providing increased resilience to mechanical stress at the costameres (Ross, 2004). What is the role of integrin-based costameres in cardiac development? Studies in embryonic stem cells showed that deletion of the 1-integrin gene results in severe deficiency in myofibrillogenesis and diVerentiation of stem cells into atrial, ventricular, and pacemaker myocytes (Fassler et al., 1996). In chimeric embryos, cells lacking 1-integrin were able to diVerentiate into myocytes but had severely compromised survival, disappearing from the hearts at 6 months after birth (Fassler et al., 1996). A similar approach using cardiac restricted expression of a dominant-negative 1D-integrin cytoplasmic tail construct in transgenic mice also resulted in severe myocyte loss, leading to a fatal dilated cardiomyopathy within 6 months after birth

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(Valencik et al., 2002). A less severe phenotype was seen with cardiac expression of a similar 1A dominant–negative construct (Keller et al., 2001). Surprisingly, cardiac myocyte–specific deletion of the 1-integrin gene in mice did not disrupt cardiogenesis, and animals were able to survive into adulthood (Shai et al., 2002). However, myocyte membrane integrity and glucose metabolism were impaired, resulting in decreased cardiac function and development of dilated cardiomyopathy by 6 months of age. The 1integrin–deficient hearts were particularly susceptible to hemodynamic stress, as evidenced by increased mortality after aortic banding. In focal areas, loss of myofibrils and intercalated disks, together with mitochondrial abnormalities, was observed. The less severe phenotype in these mice may have resulted from incomplete expression of Cre recombinase under the MLC2v promoter, resulting in significant residual 1-integrin expression (Shai et al., 2002). In contrast to 1-integrins, there is more redundancy in -integrins expressed in the heart. Complete knockout of integrin 1 (Gardner et al., 1996) did not result in any cardiac phenotype. Deletion of 5 (Yang et al., 1999) resulted in early embryonic lethality because of mesodermal defects similar to those observed in mice lacking fibronectin (George et al., 1997), paxillin (Hagel et al., 2002), or vinculin (Xu et al., 1998), indicating that intact fibronectin-integrin-FA signaling is important in adhesion, migration, proliferation, and/or diVerentiation of mesoderm precursors. Cardiac-restricted deletion of these genes will be required to assess their specific role in cardiac myocyte development.

F. Integrin Pathways Regulate Hypertrophy In many cell types, FAs are active sites of signal transduction. Outside-in signaling often modulates cytoskeletal dynamics, locomotion, and proliferation in response to adhesion to diVerent ECM substrates. Similar roles are played by integrin costameres in cardiac myocytes, where outside-in signaling aVects sarcomeric assembly, hypertrophic gene expression, and survival. One mechanism for induction of outside-in signaling occurs in response to increased deposition of fibronectin (FN) in the ECM in animal models of cardiac hypertrophy and in patients with cardiac failure (Boluyt et al., 1995; Grimm et al., 1998; Heling et al., 2000; Laser et al., 2000; Pardo Mindan and Panizo, 1993; Rothermund et al., 2002). The increased synthesis of FN is, in part, mediated by stimulation of cardiac fibroblasts by angiotensin II produced in response to mechanical overload (Craig et al., 2001; Grimm et al., 1998; Kawano et al., 2000; Tsutsumi et al., 1998). The ECM remodeling associated with interstitial fibrosis can lead to contractile and diastolic dysfunction and consequent heart failure. Moreover, outside-in signaling

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induced by FN leads to hypertrophy characterized by increased cellular area, sarcomere assembly, and changes in gene expression (Chen et al., 2004; Ogawa et al., 2000, 2002). That integrin-mediated pathways are critical for the response to hypertrophic stimuli is suggested by the observations that inhibition of outside-in integrin signaling blocks induction of the hypertrophic phenotype caused by phenylephrine (PE) (Pham et al., 2000a; Taylor et al., 2000; Wei et al., 2001) or by mechanical stretch (Aikawa et al., 2002; Liang et al., 2000). Hypertrophic agonists such as endothelin-1 (Kovacic-Milivojevic et al., 2001) and PE (Pham et al., 2000a) induce increased costameric localization of 1D-integrin, as well as of FAK, p130Cas, and paxillin. In another study, PE stimulation for 48 hours resulted in increased costameric localization of integrins 1A, 1, 3A, and 5 (Kim et al., 2003). Moreover, PE induced increased tyrosine phosphorylation of FAK, p130Cas, and paxillin (Taylor et al., 2000). Mechanical stretch induces expression of 1-integrin (Sharp et al., 1997) and costameric localization of activated FAK (Torsoni et al., 2003). In a feline model, 48 hours of pressure overload resulted in increased association of FN and vitronectin with the insoluble fraction of the myocardium (Nagai et al., 1999). The increase in sarcolemmal-associated FN and vitronectin was accompanied by similar increases in insoluble FAK, c-Src, Nck, Shc, p130Cas, 3 integrin, and by tyrosine phosphorylation of FAK and c-Src (Nagai et al., 1999; Willey et al., 2003). In our own work, we observed that ILK forms a complex with -Parvin and PINCH-1 in cardiac myocytes (PIP complex). Moreover, the PIP complex localizes in a diVuse, random pattern in unstimulated myocytes plated on gelatin or poly-L-lysine, whereas FN, PE, or a combination of FN and PE induces costameric localization of endogenous ILK and -Parvin, or transfected PINCH-1 and PINCH-2 (Chen, Wu, and Sepulveda, manuscript submitted). The tyrosine kinase FAK, which can bind directly to the cytoplasmic tail of 1-integrin (Pham et al., 2000a; Schaller et al., 1995), has been proposed to be a critical player in integrin signaling in cardiomyocytes. For example, inhibition of FAK with a dominant negative FAK mutant blocked the hypertrophic response to endothelin-1 (Heidkamp et al., 2002) and phenylephrine (Taylor et al., 2000), ultimately causing apoptosis of cardiomyocytes with detachment of the cardiac myocytes from the extracellular matrix (anoikis) (Heidkamp et al., 2002). The pathway involving integrin, FAK, p130Cas, c-Src, Grb2, and Ras is critical for activation of p38 MAPK by mechanical stretch (Aikawa et al., 2002; Willey et al., 2003). Similarly, the integrin/p130Cas/Crk/Pyk2/c-Src pathway is required for activation of JNK in cardiac myocytes in response to endothelin-1 (Kodama et al., 2003). Furthermore, mechanical stretch leads to tyrosine phosphorylation of FAK and stabilization of FAs and costameres (Sharp et al., 1997).

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What are the downstream mediators of the hypertrophic signaling induced by integrins? We have described how small G proteins of the Rho family play a role in myofibrillogenesis in coordination with integrin pathways. RhoA, Cdc42, and Rac1 have all been implicated in cardiac myocyte hypertrophy (Aikawa et al., 1999; Clerk et al., 2001; Nagai et al., 2003). For example, expression of activated RhoA induced marked myofibrillogenesis, sarcomere organization, and expression of ANP in cardiac myocytes (Hoshijima et al., 1998). The exact molecules connecting integrins and activation of Rho proteins remain speculative in cardiac myocytes but may involve the following pathways: 1. FAK activation by integrins leads to binding of the adaptor p130Cas (Polte and Hanks, 1995). In fibroblasts, tyrosine phosphorylation and activation of p130Cas promotes its interaction with Crk and the adaptor DOCK1 (Kiyokawa et al., 1998b), which in association with ELMO1 (Lu et al., 2004) functions as a two-part Rac1 guanidine nucleotide exchange factor (GEF) (Kiyokawa et al., 1998a). 2. Another possible mechanism involves ILK. We have some evidence that in certain conditions ILK dominant-negative constructs can inhibit cardiac hypertrophy (Chen, Wu and Sepulveda, manuscript submitted). Because ILK can connect the cytoplasmic tail of 1-integrin with paxillin, PINCH, and Parvins, it provides at least three possible mechanisms for activation of Rac1. The first mechanism involves PIX (ARHGEF6), which can bind -Parvin (Rosenberger et al., 2003) and act as a Rac1 and Cdc42 GEF (Mishima et al., 2004). The second possible mechanism involves binding of paxillin to both ILK and Parvin (Nikolopoulos and Turner, 2001; Nikolopoulos and Turner, 2002), recruitment of p95Pkl (GIT2) and PIX (ARHGEF7) (Turner et al., 1999). PIX can heterodimerize with PIX and activate Rac1 and Cdc42 (Rosenberger et al., 2003). Finally, PINCH-1 can bind to Nck-2 (Tu et al., 1998), which in turn can recruit DOCK1 (Kiyokawa et al., 1998b) and induce Rac1. In addition to Rho proteins, MAPK pathways, in particular the ERK branch, have been implicated in hypertrophy. For example, ERK1/2 can phosphorylate GATA4 and activate its transcriptional activity in the context of PE, endothelin, or stretch-induced hypertrophy (Charron et al., 2001; Kitta et al., 2001; Liang et al., 2001). There are many possible pathways linking integrin activation and ERK stimulation, involving molecules such as PKC, FAK, c-Src, p130/Crk, Grb2, Pyk2, and Nck2 (Aikawa et al., 2002; Bayer et al., 2002; Heidkamp et al., 2003; Laser et al., 2000; Melendez et al., 2004; Pham et al., 2000a; Taylor et al., 2000). The issue is further complicated by evidence that tyrosine kinase receptors, such as the epidermal growth receptor, are involved in hypertrophic stimulation and cross-talk

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with G-protein–coupled receptors (GPCR) and integrin-dependent pathways (Anderson et al., 2004; Asakura et al., 2002; Kodama et al., 2003; Ross, 2004). Most of these pathways converge to activation of the Ras-Raf1MEK1/2-ERK1/2 pathway (Chiloeches et al., 1999), although alternative pathways involving Rap1 and B-Raf may be involved, at least in diVerent cell types (Barberis et al., 2000). Further intersection is provided by the fact that -integrin cytoplasmic domains can recruit PKC via RACK1 binding (Liliental and Chang, 1998). Rack1/2 bind PKC, a known mediator of cardiac myocyte hypertrophy and cardioprotection (Pass et al., 2001). PKC activates hypertrophy mainly through the Ras/ERK pathway (Chiloeches et al., 1999). An involvement of ILK in stimulation of ERK has also been suggested by studies in skeletal myocytes (Huang et al., 2000) and non-myocytes (Fukuda et al., 2003b). It is also clear that induction of MAPK pathways and activation of Rho proteins by hypertrophic stimuli are interconnected. For example, PKC activation by mechanical stretch is required for RhoA and Rac1 induction, and these G-proteins regulate MAPK phosphorylation in cardiac myocytes (Pan et al., 2004). Rac1 and RhoA activation in response to endothelin-1 and PE was required for activation of ERK and JNK (Clerk et al., 2001). One eVect of integrin and Rho activation relates to increased actin polymerization and myofibrillogenesis, as described earlier. In turn, increased actin polymerization, or rather the decrease of G-actin, results in stimulation of SRF target genes, such as skeletal -actin, a marker of hypertrophy (Gineitis and Treisman, 2001; Wei et al., 2001). In addition, increased actin polymerization is required for endothelin-1 activation of GATA4 by the Rhodependent kinase and ERK (Yanazume et al., 2002), possibly because the actin cytoskeleton is involved in translocating ERK to the nucleus of cardiac myocytes (Kawamura et al., 2003), where it can activate GATA4 by phosphorylation. Inactivation of GSK3 provides another link between integrin stimulation and hypertrophy, because GSK3 has a potent antihypertrophic role (e.g., by phosphorylating and increasing the nuclear export of GATA4 and NFATc) (Haq et al., 2000; Morisco et al., 2000, 2001). GSK3 can be directly phosphorylated by ILK or by Akt (Delcommenne et al., 1998). Because in non-myocytes ILK, PINCH1, and -Parvin play a role in recruiting Akt to the membrane (Fukuda et al., 2003a, 2003b) and ILK can activate Akt by direct phosphorylation of serine 473 (Troussard et al., 2003), it is likely that ILK-dependent Akt phosphorylation is involved in GSK3 inactivation during hypertrophic stimulation. Confirmation of this possibility is given by the observation that in cardiac myocytes, expression of a dominantnegative ILK mutant (211A) resulted in inhibition of Akt S473 phosphorylation in response to Ras-GAP, which is a hypertrophy-inducing molecule (Yue et al., 2004). It is also possible that in cardiac myocyte phosphorylation

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of GSK3 in response to endothelin or PE stimulation is directly mediated by ILK rather than through Akt, because these two GPCR agonists are potent inducers of GSK3 phosphorylation but poor inducers of Akt (Haq et al., 2000; Pham et al., 2000b). Another protein binding to 1-integrin cytoplasmic tail is melusin, which is expressed in a costameric pattern and is induced by pressure overload in cardiac myocytes (Brancaccio et al., 1999, 2003). Lack of melusin prevented the hypertrophic response and blunted GSK3 inactivation in response to pressure overload but not PE or angiotensin II, suggesting that melusin participates in integrin-dependent pathways specific to transducing mechanical strain (Brancaccio et al., 2003). Given their mutual eVects in GSK3 inactivation, it would be interesting to determine whether there is any interaction between ILK and melusin. Integrins also aVect general assembly pathways associated with hypertrophic growth. For example, RGD peptides can induce the ribosomal protein S6-kinase, an eVect mediated by 3 rather than 1 integrin (Balasubramanian and Kuppuswamy, 2003). In our global analysis of gene expression changes after fibronectin-induced hypertrophy, many of the pathways induced were involved in mRNA, protein, cholesterol, and fatty acid synthesis, consistent with the increase growth and sarcomere assembly observed in hypertrophic cardiac myocytes (Chen et al., 2004). In summary, it seems that integrin activation is critical for induction of hypertrophy by mechanical strain or GPCR agonists and that increased abundance of 1-integrin agonists in the ECM, such as FN, is suYcient to induce a mild hypertrophic phenotype. These eVects are mediated by a variety of cross-talking pathways, including MAPK and Rho family G proteins. The two integrin-binding protein kinases, ILK and FAK, together with their binding partners, seem to play a critical role in the initiation of integrin outside-in signaling, and their ability to act as self-regulating platforms for assembly of multiprotein signaling complexes is probably more important than their enzymatic activity.

G. Integrin Pathways Regulate Survival Increased assembly of FAs and costameres allows hypertrophic cardiac myocytes to respond to higher degrees of mechanical strain by increasing their attachment to the ECM, while providing enhanced responsiveness to extracellular and intracellular signaling events that fine tune the hypertrophic response. In addition, increased turnover of FAs and costameres is necessary for the myocytes to grow, while providing the survival signal usually associated with cell attachment. Failure of these mechanisms can have severe consequences. For example, mice subjected to pressure overload for more than 4 weeks transition from an appropriate hypertrophic response to cardiac failure, a process accompanied by increased cardiac myocyte

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apoptosis, possibly related to decreased attachment to the ECM (a process called anoikis) (Ding et al., 2000). For this reason, it is significant that hypertrophic stimuli such as PE and stretch provide antiapoptotic signals, in part mediated by the PI3K–Akt system (Franchini et al., 2000). Integrin signaling is a necessary component of this survival pathway, because interference with FAK signaling by overexpression of dominant FAK-related nonkinase (FRNK) induces anoikis (Heidkamp et al., 2002; Taylor et al., 2000). Apoptosis is also induced in cardiac myocytes by overexpression of the FAK-related kinase Pyk2 in the presence of the calcium ionophore ionomycin, apparently by causing excessive c-Src activity, which could be prevented by overexpression of paxillin (Melendez et al., 2004). Because paxillin can anchor c-Src (as well as its inhibitor Csk) to costameres (Turner, 2000), these data suggest that survival signals require proper localization of c-Src–dependent signaling complexes to costameres. The ILK pathway also seems to be involved in cardiac myocyte survival, because overexpression of dominant-negative ILK lacking N-terminal, PINCH-binding sequences, resulted in loss of the protective eVect of FN and PE against H2O2- or serum deprivation–induced cardiac myocyte apoptosis (Chen, Wu, and Sepulveda, manuscript submitted). The links between integrin signaling and prosurvival pathways are not fully understood in cardiac myocytes. Activation of 1-integrin by laminin or antibodies is suYcient to reduce apoptosis induced by -adrenergic agonists, an eVect mediated by ERK (Communal et al., 2003). Stretch induction of integrin/ FAK/c-Src/Ras/p38 MAPK pathway has been demonstrated in cardiac myocytes (Aikawa et al., 2002). Survival in myocytes is enhanced by p38 but decreased by p38 (Wang et al., 1998), although this concept has been challenged by in vivo data (Zhang et al., 2003). However, it is not clear which p38 isoform is induced by integrin signaling. Another possible antiapoptotic mechanism involves activation of NFB (nuclear factor-B) and phosphorylation of STAT-3 (signal transducer and activators of transcription-3) (Taylor et al., 2000), a pathway clearly invoked by cardiotrophin-1 signaling through the gp130 receptor (Craig et al., 2001). The critical player in integrin-dependent survival seems to be the PI3K– Akt pathway. Akt promotes cardiac myocyte survival by several potential mechanisms, including induction of NFB (Franchini et al., 2000) and inactivation by phosphorylation of the pro-apoptotic proteins BAD, caspase 9, and forkhead transcription factors (Kuwahara et al., 2000; van Empel and De Windt, 2004). One mechanism leading to Akt activation involves assembly of multiprotein complexes containing Fak, c-Src, Grb2, and the p85 subunit of phosphatidylinositol 3-kinase after pressure overload (Franchini et al., 2000). In fact, direct binding of FAK to PI3K-p85 has been demonstrated (Chen and Guan, 1994). However, at least in fibroblasts, FAK or Src family kinases are not required for Akt S473 phosphorylation in response to

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integrin activation (Velling et al., 2004). Our observations that ILK plays a role in cardiac myocyte survival suggest that ILK provides another connection between integrin signaling and Akt stimulation. This hypothesis has been strengthened by the recent observation that the G-actin sequestering peptide thymosin 4 binds to PINCH1 and induces ILK activity in cardiac myocytes, resulting in AKT stimulation and increased protection against myocardial infarction (Bock-Marquette et al., 2004). We are currently investigating the mechanisms by which ILK-dependent pathways participate in the antiapoptotic response associated with cardiac hypertrophy.

Acknowledgments This work was supported by NIH grants GM65188 and DK54639 to C. W. and HL068714 to J. S.

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