**4. Structural connections of integrins to the cytoskeleton**

Given the crucial functions played by integrins during skeletal muscle development, as determined by the studies in vitro and on animal models mentioned above, it is surprising that few defects in integrin function have been associated with muscle disease in patients. In fact, with the exception of 7-integrin and filamin C (Mayer et al., 1997), mutations in integrin effectors have been linked to defects in the heart but not in skeletal muscle. This is in contrast with the mutations in DPC, which have been identified as being the most common causes of muscular dystrophy (mutations the dystrophin gene alone affect approximately 1:3500 male births)(Goyenvalle et al., 2011). This could be due to mutations in integrins or integrin effectors being very rare, or to the pathology not being clearly identified, which would complicate the selection of patients for genetic screening. Although integrins and the DPC provide a similar link between laminin and the actin cytoskeleton, ablation of the two protein complexes leads to a different spectrum of muscle defects, and despite extensive analysis, the specific functions of each protein complex remain unclear.

#### **4.1 71-integrin**

71-integrin is a receptor for laminin in the basement membrane, localizes to costameres, NMJs and MTJs, and is the sole integrin known to be expressed in adult skeletal muscle (Bao et al., 1993; Martin et al., 1996). The intracellular domain of 7-integrin is spliced to produce two main isoforms, termed 7a and 7b. Their expression is tightly regulated during

Downregulation of filamin C in C2C12 myoblasts via siRNA causes an impairment of cellcell fusion, defective elongation of myotubes, and impaired gene expression during myoblast differentiation, including myogenin, caveolin 3 and 7-integrin (Dalkilic et al., 2006). An important function for filamin C in muscle differentiation was confirmed by analyzing mice in which its expression was genetically ablated. Filamin C-knockout mice died at birth likely because of respiratory failure, and presented with severe defects in myogenesis abnormal morphology of myofibres and a loss of muscle mass. While these defects partly overlap with those of 1-integrin and tln1/2-dKO mice, the phenotypes differ in that fusion and sarcomere defects are less pronounced, indicating that filamin C is not essential for 1-integrin function in muscle and that some of its effects are likely due to the

Mutations in filamin C have been identified in patients with late-onset myopathies, characterized by progressive muscle weakness. Mutations in the C-terminal dimerization domain lead to myofibrillar myopathy, characterized by the accumulation of intracellular aggregates constituted of filamin C and various Z-disk associated proteins (Vorgerd et al., 2005; Lowe et al., 2007). The mutations are localized to the dimerization domain of filamin C, and cause the formation of a truncated protein that cannot form dimers, implying that dimerization is important for its function. Recently, mutations in filamin C have also been identified in patients with distal myopathies. These mutations are localized in the N-terminal actin-binding domain of filamin C, and induce increased actin binding. However, unlike the situation in patients where mutations are in the C-terminus, no protein aggregates accumulate in myofibres, suggesting that the pathological mechanisms differs from those observed in

Given the crucial functions played by integrins during skeletal muscle development, as determined by the studies in vitro and on animal models mentioned above, it is surprising that few defects in integrin function have been associated with muscle disease in patients. In fact, with the exception of 7-integrin and filamin C (Mayer et al., 1997), mutations in integrin effectors have been linked to defects in the heart but not in skeletal muscle. This is in contrast with the mutations in DPC, which have been identified as being the most common causes of muscular dystrophy (mutations the dystrophin gene alone affect approximately 1:3500 male births)(Goyenvalle et al., 2011). This could be due to mutations in integrins or integrin effectors being very rare, or to the pathology not being clearly identified, which would complicate the selection of patients for genetic screening. Although integrins and the DPC provide a similar link between laminin and the actin cytoskeleton, ablation of the two protein complexes leads to a different spectrum of muscle defects, and despite extensive analysis, the specific functions of each protein complex remain unclear.

71-integrin is a receptor for laminin in the basement membrane, localizes to costameres, NMJs and MTJs, and is the sole integrin known to be expressed in adult skeletal muscle (Bao et al., 1993; Martin et al., 1996). The intracellular domain of 7-integrin is spliced to produce two main isoforms, termed 7a and 7b. Their expression is tightly regulated during

interaction with other binding partners (Dalkilic et al., 2006).

patients with mutations in the dimerization domain (Duff et al., 2011).

**4. Structural connections of integrins to the cytoskeleton** 

**4.1 71-integrin** 

myoblast differentiation and muscle regeneration, and this regulation is conserved across mammals, suggesting that the specific roles played by these isoforms are important (Collo et al., 1993; Ziober et al., 1993; Cohn et al., 1999). The 7b isoform is expressed at higher levels in proliferating myoblasts and adult fibres, while the 7a isoform is expressed upon terminal differentiation. These 7-integrin splice variants bind with equal affinity to laminin, thus differences probably reside in binding to intracellular integrin effectors. It has been suggested that the splice variants may differ in the regulation of myoblast differentiation (Samson et al., 2007), as 7a interacts with Def-6, a guanine nucleotide exchange factor (GEF) for the Rho GTPase Rac-1 that has been implicated in the regulation of myoblast fusion. However, mice in which 7-integrin is ablated (7-KO) are viable and present with normal muscle development, indicating that 7-integrin is not essential for myogenesis *in vivo* (Mayer et al., 1997). Instead, 71-integrin plays an important structural role in skeletal muscle by mediating a connection of actin to the sarcolemma at the MTJ. In 7-KO mice this connection fails, leaving a space filled with vesicular and amorphous material, and the mice developed a progressive myopathy, characterised by muscle weakness and a mild accumulation of centrally nucleated fibres (Mayer et al., 1997; Miosge et al., 1999). 7b-integrin has been shown have a protective effect against mechanical damage. Following exercise, expression of 7-integrin is upregulated in muscle, and exercise-induced damage is increased in 7-KO mice (Boppart et al., 2006). A protective function for 7-integrin is supported by studies in which the 7bX2 splice variant was overexpressed in mice. The transgenic mice showed a reduced activation of the MAPK pathway, associated with injury, and of AKT, mTOR and p70s6k, associated with hypertrophy, and presented with reduced muscle damage in response to exercise (Boppart et al., 2008). It is interesting to note that 71-integrin is increased in the muscle of patients with DMD and of *mdx* mice (Hodges et al., 1997). Thus, upregulation of 71-integrin might be a natural mechanisms to increase the resistance of muscle to injury in the absence of dystrophin and indeed, enhanced 7-integrin expression alleviates muscular dystrophy in transgenic mice lacking dystrophin and utrophin (Burkin et al., 2001; Burkin et al., 2005).

Mutations in 7-integrin have been associated with muscle disease in humans: three Japanese patients were identified with a deficiency in 7-integrin, caused by deletion or frame-shift mutations in the *itga7* gene (Hayashi et al., 1998). Similar to the phenotype of 7- KO mice, the muscle in patients presented with no signs of necrosis and creatine kinase values that were only slightly elevated, indicating no major damage to the sarcolemma. However, the clinical phenotype was severe: patients presented with delayed motor milestones from early childhood, and in one case mental retardation. Follow up of one of the patients showed a severe progression of the disease, comparable to that of DMD, which led the patient to be wheelchair bound by the age of 12 (Nakashima et al., 2009). Thus, while the initial classification was that of a congenital myopathy, patients with a clinical presentation of congenital muscular dystrophy should also be considered for screening for integrin 7 deficiency. As no new patients have been diagnosed with a deficiency of 7-integrin since the initial identification, mutations appear to be rare.

#### **4.2 Talin**

Of the proteins that bind to the cytoplasmic domain of integrins, studies have revealed important functions for talin in mediating the connection to myofilaments at the MTJ. In

Integrins in the Development and Pathology of Skeletal Muscle 9

stimuli and in reinforcing the connection of integrins to the actin cytoskeleton (Giannone et al., 2003; del Rio et al., 2009; Margadant et al., 2011). Cardiac myocyte-specific excision of the vinculin gene reveals an essential function for the structural integrity of the heart, where ICDs become disorganized and present with an altered distribution of the ICD proteins cadherins and connexin 43. Likely because of these defects, sudden death was found in about half of the transgenic mice, caused by ventricular tachycardia. The mice that survived developed dilated cardiomyopathy and died by 6 months of age (Zemljic-Harpf et al., 2007). Mutations in the splice variant metavinculin, which includes an additional exon, have been identified in patients with dilated or hypertrophic cardiomyopathy, including deletions and missense mutations (Maeda et al., 1997; Olson et al., 2002; Vasile et al., 2006). A missense mutation in a vinculin-specific exon (L277M) was identified in a patient with hypertrophic cardiomyopathy, which led to a reduction in vinculin levels in ICDs (Vasile et al., 2006). Skeletal muscle problems were not reported for this patient, but the rarity of mutations in vinculin-specific exons, and the fact that the identified mutations are clustered in the metavinculin-specific exon, may be attributed to the fact that mutations in the ubiquitously expressed vinculin splice variant may lead to early lethality, as it occurs in mice (Xu et al.,

Integrins are important sensors of mechanical forces applied to cells (Geiger et al., 2009; Moore et al., 2010). For instance, the size of focal adhesions can be modulated by altering the stiffness of the ECM, actomyosin contractility, and by applying forces to specific integrin subunits (Giannone et al., 2003; Jiang et al., 2003; Moore et al., 2010). It is therefore significant that, in skeletal muscle, integrins are expressed specifically at costameres and MTJs, where mechanical stress generated by muscle contraction is transmitted through the plasma membrane to the ECM (Mayer, 2003). As we will see, integrins signaling is important for modultating hypertrophy in the heart in response to mechanical and soluble stimuli. Perhaps surprisingly, it is still unclear whether integrins are also important for

No mutations in 1-integrin have been identified in patients, likely because compromised function would result in early lethality, as it occurs in the knockout mouse model (Fassler and Meyer, 1995; Schwander et al., 2003). However, mice with a heart-restricted ablation of 1-integrin present impaired contractility and develop ventricular fibrosis and cardiac hypertrophy in response to transverse aortic constriction (TAC, a procedure in which the lumen of the aorta is artificially restricted)(Shai et al., 2002). These data indicate that 1 integrins are essential for a normal response of cardiomyocytes to mechanical stress, and subsequent analysis identified several proteins associated with 1-integrin that mediate

ILK is closely associated to 1 and 3-integrins (Hannigan et al., 1996; Zervas et al., 2001; Wickstrom et al., 2010), and binds to several proteins that relay biochemical signals and

**5. Integrin signaling: Responses to mechanical stimuli** 

regulating hypertrophy in skeletal muscle.

1998).

**5.1 1-integrin** 

these effects.

**5.2 Integrin-linked kinase (ILK)** 

*Drosophila*, ablation of the talin gene (*mys*), induces detachment of actin filaments from the integrin cytoplasmic domain at muscle termini (Brown et al., 2002). Two talin isoforms are expressed in vertebrates, with talin 2 being most expressed in skeletal and cardiac muscle, while talin 1 is ubiquitous (Monkley et al., 2001). Muscle-specific ablation of talin 1 was achieved using conditional gene inactivation in muscle, as knockout of the talin 1 gene causes early embryonic lethality. In contrast, mice in which talin 2 was ablated were viable (Monkley et al., 2000; Conti et al., 2009). Both talin1-KO and talin2-KO mice presented with defects in skeletal muscle similar to those obtained following ablation of 7-integrin, consisting in structural failure at the MTJ, and a limited accumulation of centrally nucleated fibres, with no obvious damage to the sarcolemma. Consistent with the expression data, the phenotype was more severe in talin2-KO mice (Conti et al., 2008; Conti et al., 2009). Interestingly, adult muscle expresses a splice variant of integrin 1-integrin, termed 1D, which binds to F-actin with greater affinity than the ubiquitous 1A isoform (Belkin et al., 1997; van der Flier et al., 1997). The data suggest a model whereby a strong connection between the ECM and actin is established at the MTJ by complexes of 71D-integrin and talin 2, and, to a lesser extent, talin 1. In the absence of 7-integrin or of talin 2, stress induced by muscle contraction leads to mechanical failure at the MTJ.

#### **4.3 Centrally nucleated fibres in myopathies**

The reason for the accumulation of centrally nucleated fibres in muscles lacking integrins or talin is unclear. In muscular dystrophies, central nuclei are associated with regenerating fibres that are thought to form because the absence of DPC proteins causes fragility to the plasma membrane and necrosis of myofibres (Davies and Nowak, 2006). This does not occur when integrins are affected: patients with null mutations in the *itga7* gene have only mildly elevated plasma creatine kinase, and there is no evidence of damage to the sarcolemma in mice deficient in 7-integrin and talin 1 or 2 (Hayashi et al., 1998; Conti et al., 2008; Conti et al., 2009). Thus integrins appear dispensable for maintaining the structural integrity of the sarcolemma and other mechanisms must account for the presence of centrally nucleated fibres. One possibility is that cytoskeletal alterations might affect nuclear positioning. For example, internal nuclei were observed in mouse models lacking proteins that regulated actin organization and membrane trafficking, such as myotubularin 1, dynamin 2 and -actin, without evidence of damage to the sarcolemma (Buj-Bello et al., 2002; Bitoun et al., 2005; Sonnemann et al., 2006), but whether integrins regulate nuclear anchorage, it is at present unknown. Alternatively, integrins might be essential to provide survival signals to myofibres. In particular, signaling from FAK, which associates with talin, is important to suppress apoptosis in cultured cells (Lim et al., 2008). These signals might be perturbed in 7-KO and talin2-KO mice, leading to loss of myofibres and regeneration.

#### **4.4 Vinculin**

Vinculin is a ubiquitous component of focal adhesions that establishes a connection between integrins and an array of cytoskeletal proteins, including paxillin, talin, actin and the Arp2/3 complex, among others (Ziegler et al., 2006). In skeletal muscle, vinculin localizes to costameres, MTJ and NMJ (Bao et al., 1993), and in cardiac muscle, to costameres and intercalated disks (ICDs). Its expression levels are regulated by mechanical stress, and studies on cells in culture have revealed a function for vinculin in sensing mechanical stimuli and in reinforcing the connection of integrins to the actin cytoskeleton (Giannone et al., 2003; del Rio et al., 2009; Margadant et al., 2011). Cardiac myocyte-specific excision of the vinculin gene reveals an essential function for the structural integrity of the heart, where ICDs become disorganized and present with an altered distribution of the ICD proteins cadherins and connexin 43. Likely because of these defects, sudden death was found in about half of the transgenic mice, caused by ventricular tachycardia. The mice that survived developed dilated cardiomyopathy and died by 6 months of age (Zemljic-Harpf et al., 2007).

Mutations in the splice variant metavinculin, which includes an additional exon, have been identified in patients with dilated or hypertrophic cardiomyopathy, including deletions and missense mutations (Maeda et al., 1997; Olson et al., 2002; Vasile et al., 2006). A missense mutation in a vinculin-specific exon (L277M) was identified in a patient with hypertrophic cardiomyopathy, which led to a reduction in vinculin levels in ICDs (Vasile et al., 2006). Skeletal muscle problems were not reported for this patient, but the rarity of mutations in vinculin-specific exons, and the fact that the identified mutations are clustered in the metavinculin-specific exon, may be attributed to the fact that mutations in the ubiquitously expressed vinculin splice variant may lead to early lethality, as it occurs in mice (Xu et al., 1998).

#### **5. Integrin signaling: Responses to mechanical stimuli**

Integrins are important sensors of mechanical forces applied to cells (Geiger et al., 2009; Moore et al., 2010). For instance, the size of focal adhesions can be modulated by altering the stiffness of the ECM, actomyosin contractility, and by applying forces to specific integrin subunits (Giannone et al., 2003; Jiang et al., 2003; Moore et al., 2010). It is therefore significant that, in skeletal muscle, integrins are expressed specifically at costameres and MTJs, where mechanical stress generated by muscle contraction is transmitted through the plasma membrane to the ECM (Mayer, 2003). As we will see, integrins signaling is important for modultating hypertrophy in the heart in response to mechanical and soluble stimuli. Perhaps surprisingly, it is still unclear whether integrins are also important for regulating hypertrophy in skeletal muscle.

#### **5.1 1-integrin**

8 Neuromuscular Disorders

*Drosophila*, ablation of the talin gene (*mys*), induces detachment of actin filaments from the integrin cytoplasmic domain at muscle termini (Brown et al., 2002). Two talin isoforms are expressed in vertebrates, with talin 2 being most expressed in skeletal and cardiac muscle, while talin 1 is ubiquitous (Monkley et al., 2001). Muscle-specific ablation of talin 1 was achieved using conditional gene inactivation in muscle, as knockout of the talin 1 gene causes early embryonic lethality. In contrast, mice in which talin 2 was ablated were viable (Monkley et al., 2000; Conti et al., 2009). Both talin1-KO and talin2-KO mice presented with defects in skeletal muscle similar to those obtained following ablation of 7-integrin, consisting in structural failure at the MTJ, and a limited accumulation of centrally nucleated fibres, with no obvious damage to the sarcolemma. Consistent with the expression data, the phenotype was more severe in talin2-KO mice (Conti et al., 2008; Conti et al., 2009). Interestingly, adult muscle expresses a splice variant of integrin 1-integrin, termed 1D, which binds to F-actin with greater affinity than the ubiquitous 1A isoform (Belkin et al., 1997; van der Flier et al., 1997). The data suggest a model whereby a strong connection between the ECM and actin is established at the MTJ by complexes of 71D-integrin and talin 2, and, to a lesser extent, talin 1. In the absence of 7-integrin or of talin 2, stress

The reason for the accumulation of centrally nucleated fibres in muscles lacking integrins or talin is unclear. In muscular dystrophies, central nuclei are associated with regenerating fibres that are thought to form because the absence of DPC proteins causes fragility to the plasma membrane and necrosis of myofibres (Davies and Nowak, 2006). This does not occur when integrins are affected: patients with null mutations in the *itga7* gene have only mildly elevated plasma creatine kinase, and there is no evidence of damage to the sarcolemma in mice deficient in 7-integrin and talin 1 or 2 (Hayashi et al., 1998; Conti et al., 2008; Conti et al., 2009). Thus integrins appear dispensable for maintaining the structural integrity of the sarcolemma and other mechanisms must account for the presence of centrally nucleated fibres. One possibility is that cytoskeletal alterations might affect nuclear positioning. For example, internal nuclei were observed in mouse models lacking proteins that regulated actin organization and membrane trafficking, such as myotubularin 1, dynamin 2 and -actin, without evidence of damage to the sarcolemma (Buj-Bello et al., 2002; Bitoun et al., 2005; Sonnemann et al., 2006), but whether integrins regulate nuclear anchorage, it is at present unknown. Alternatively, integrins might be essential to provide survival signals to myofibres. In particular, signaling from FAK, which associates with talin, is important to suppress apoptosis in cultured cells (Lim et al., 2008). These signals might be perturbed in 7-KO and

Vinculin is a ubiquitous component of focal adhesions that establishes a connection between integrins and an array of cytoskeletal proteins, including paxillin, talin, actin and the Arp2/3 complex, among others (Ziegler et al., 2006). In skeletal muscle, vinculin localizes to costameres, MTJ and NMJ (Bao et al., 1993), and in cardiac muscle, to costameres and intercalated disks (ICDs). Its expression levels are regulated by mechanical stress, and studies on cells in culture have revealed a function for vinculin in sensing mechanical

induced by muscle contraction leads to mechanical failure at the MTJ.

talin2-KO mice, leading to loss of myofibres and regeneration.

**4.4 Vinculin** 

**4.3 Centrally nucleated fibres in myopathies** 

No mutations in 1-integrin have been identified in patients, likely because compromised function would result in early lethality, as it occurs in the knockout mouse model (Fassler and Meyer, 1995; Schwander et al., 2003). However, mice with a heart-restricted ablation of 1-integrin present impaired contractility and develop ventricular fibrosis and cardiac hypertrophy in response to transverse aortic constriction (TAC, a procedure in which the lumen of the aorta is artificially restricted)(Shai et al., 2002). These data indicate that 1 integrins are essential for a normal response of cardiomyocytes to mechanical stress, and subsequent analysis identified several proteins associated with 1-integrin that mediate these effects.

#### **5.2 Integrin-linked kinase (ILK)**

ILK is closely associated to 1 and 3-integrins (Hannigan et al., 1996; Zervas et al., 2001; Wickstrom et al., 2010), and binds to several proteins that relay biochemical signals and

Integrins in the Development and Pathology of Skeletal Muscle 11

ILK is important for the sensing of mechanical stress in the heart. In the Zebrafish main squeeze (*msq*) mutant, isolated through a genetic screen, a missense mutation (L308P) was identified in the ILK gene (Bendig et al., 2006). Fish develop normally, but their hearts loose contractility, resulting in pericardial edema. The *msq* mutation disrupts the interaction with -parvin, and morpholino-mediated knockdown of -parvin phenocopies the ILK phenotype. These data suggests that the integrin-ILK--parvin complex is essential for transducing mechanical stimuli into signaling pathways important for cardiac contractility. In mice, conditional ablation of ILK in the heart causes dilated cardiomyopathy and sudden death in response to aortic pressure overload, with altered signaling from proteins involved in hypertrophy. A missense mutation in the ILK gene (A262V) has been identified in a patient affected by dilated cardiomyopathy (Knoll et al., 2007), and expression of ILK was elevated in patients affected by pathological cardiac hypertrophy, with a concomitant activation of signaling effectors associated with hypertrophic responses, including Rac, Cdc42, the ERK1/2 pathway and the kinase p70 S6 (Lu et al., 2006). It is at present unclear

Kindlin binds directly to 1-integrins and ILK. Three isoforms are expressed in vertebrates, named kindlin 1, 2 and 3. The main isoform expressed in skeletal and cardiac muscle is kindlin 2 (Ussar et al., 2006), which is localized at costameres and ICDs, again suggesting that it may play a structural role in areas of elevated mechanical stress (Dowling et al., 2008a). Mutations in kindlin 1 and 3 have been identified in patients affected by skin and immune disorders, respectively (Jobard et al., 2003; Siegel et al., 2003; Malinin et al., 2009; Svensson et al., 2009), but no mutations in kindlin 2 have been found in humans. *In vitro*  studies have shown that kindlin 2 is important for differentiation of myoblasts (Dowling et al., 2008b), and knockdown of kindlin 2 in *Zebrafish* caused defective development of several organs, including skeletal and cardiac muscle, with disruption of ICDs and failure in the attachment of myofibrils to the membrane (Dowling et al., 2008a). Thus, kindlin 2 may be a good candidate gene for screening in patients affected by dilated cardiomyopathy or

Focal adhesion kinase (FAK) is closely associated with integrins, and following integrin engagement with ECM ligands, it becomes phosphorylated at tyrosine 397 (Y397). This creates a binding site for the SH2 domain of Src family kinases, and leads to the activation of several signaling effectors, including Rho and Rac, PI3K, Akt and the ERK1/2 signaling

The tyrosine phosphorylation of FAK is rapidly increased following pressure overload in the rat heart (Franchini et al., 2000), and FAK activates hypertrophic signaling through PKB/AKT, the ERK1/2 and the JNK/c-JUN pathways. Additionally, FAK signaling regulates expression of the MEF2 transcription factors, which regulate the expression of several sarcomeric proteins (Nadruz et al., 2005). Insights on the function of FAK in striated muscle were obtained by generating mice with a conditional FAK ablation in cardiomyocytes. These mice developed defects that included thinner ventricular walls, ventricular septal defects and reduced cell numbers (DiMichele et al., 2006; Hakim et al.,

whether ILK plays any role in regulating hypertrophy in skeletal muscle.

**5.3 Kindlin** 

congenital myopathies.

pathway (Franchini et al., 2009).

**5.4 FAK** 

regulate actin dynamics, including paxillin, -and -parvins and PKB. Ablation of ILK in invertebrates leads to detachment of myofibres at the MTJ, a phenotype similar to that obtained following ablation of talin (Zervas et al., 2001; Brown et al., 2002). Thus, in invertebrates, talin and ILK share a common function in the connection of actin to integrins at the MTJ. In vertebrates, however, MTJ defects following ablation of ILK differ from those observed in talin 1- or talin 2-KO mice. MTJ defects in ILK-deficient muscle consisted in discontinuities in the basal lamina and a detachment of actin filaments at the MTJ was not reported (Wang et al., 2008). ILK was important to stabilize MTJs in response to exercise, a process that might involve the relay of biochemical signals in association with the insulin growth factor receptor 1 (IGF-R1), which forms a complex with 1-integrins and plays a role during muscle repair (Musaro et al., 2001). IGF-R1 signaling was impaired in ILK-deficient muscle (Wang et al., 2008). Normally, in response to exercise, the insulin growth factor receptor 1R (IGF-1R) activates PKB/Akt, which in turn activates the kinase mTOR that is involved in the generation of new myofibrils. This activation was impaired in ILK-deficient muscle. Interestingly, 1-integrin was associated with IGF-R1, and this association increased in response to IGF-1. The data suggest a model whereby 1-integrin forms a complex with IGF-R1 that controls activation of ILK, the PKB/Akt and mTOR pathways to regulate skeletal muscle regeneration in response to exercise (Wang et al., 2008).

Fig. 2. **Integrin function in skeletal and cardiac muscle.** In skeletal muscle (right), integrins establish a connection between the ECM and actin filaments at the myotendinous junction (MTJ). In cardiac muscle (left), integrins activate hypetrophic signaling pathways, including PKB/AKT and mTOR, JNK/c-jun and ERK1/2, in response to mechanical and soluble stimuli. In addition, vinculin ablation leads to destabilization of intercalated disks (ICD). It is unclear at present whether integrins mediate hypertrophic responses in skeletal muscle. Abbreviations are: ECM = extracellular matrix; ILK = integrin-linked kinase; FAK = focal adhesion kinase; TLN = talin 1 or talin 2; VCL = vinculin; CTNA1 = -catenin.

ILK is important for the sensing of mechanical stress in the heart. In the Zebrafish main squeeze (*msq*) mutant, isolated through a genetic screen, a missense mutation (L308P) was identified in the ILK gene (Bendig et al., 2006). Fish develop normally, but their hearts loose contractility, resulting in pericardial edema. The *msq* mutation disrupts the interaction with -parvin, and morpholino-mediated knockdown of -parvin phenocopies the ILK phenotype. These data suggests that the integrin-ILK--parvin complex is essential for transducing mechanical stimuli into signaling pathways important for cardiac contractility. In mice, conditional ablation of ILK in the heart causes dilated cardiomyopathy and sudden death in response to aortic pressure overload, with altered signaling from proteins involved in hypertrophy. A missense mutation in the ILK gene (A262V) has been identified in a patient affected by dilated cardiomyopathy (Knoll et al., 2007), and expression of ILK was elevated in patients affected by pathological cardiac hypertrophy, with a concomitant activation of signaling effectors associated with hypertrophic responses, including Rac, Cdc42, the ERK1/2 pathway and the kinase p70 S6 (Lu et al., 2006). It is at present unclear whether ILK plays any role in regulating hypertrophy in skeletal muscle.

#### **5.3 Kindlin**

10 Neuromuscular Disorders

regulate actin dynamics, including paxillin, -and -parvins and PKB. Ablation of ILK in invertebrates leads to detachment of myofibres at the MTJ, a phenotype similar to that obtained following ablation of talin (Zervas et al., 2001; Brown et al., 2002). Thus, in invertebrates, talin and ILK share a common function in the connection of actin to integrins at the MTJ. In vertebrates, however, MTJ defects following ablation of ILK differ from those observed in talin 1- or talin 2-KO mice. MTJ defects in ILK-deficient muscle consisted in discontinuities in the basal lamina and a detachment of actin filaments at the MTJ was not reported (Wang et al., 2008). ILK was important to stabilize MTJs in response to exercise, a process that might involve the relay of biochemical signals in association with the insulin growth factor receptor 1 (IGF-R1), which forms a complex with 1-integrins and plays a role during muscle repair (Musaro et al., 2001). IGF-R1 signaling was impaired in ILK-deficient muscle (Wang et al., 2008). Normally, in response to exercise, the insulin growth factor receptor 1R (IGF-1R) activates PKB/Akt, which in turn activates the kinase mTOR that is involved in the generation of new myofibrils. This activation was impaired in ILK-deficient muscle. Interestingly, 1-integrin was associated with IGF-R1, and this association increased in response to IGF-1. The data suggest a model whereby 1-integrin forms a complex with IGF-R1 that controls activation of ILK, the PKB/Akt and mTOR pathways to regulate

skeletal muscle regeneration in response to exercise (Wang et al., 2008).

Fig. 2. **Integrin function in skeletal and cardiac muscle.** In skeletal muscle (right), integrins establish a connection between the ECM and actin filaments at the myotendinous junction (MTJ). In cardiac muscle (left), integrins activate hypetrophic signaling pathways, including PKB/AKT and mTOR, JNK/c-jun and ERK1/2, in response to mechanical and soluble stimuli. In addition, vinculin ablation leads to destabilization of intercalated disks (ICD). It is unclear at present whether integrins mediate hypertrophic responses in skeletal muscle. Abbreviations are: ECM = extracellular matrix; ILK = integrin-linked kinase; FAK = focal

adhesion kinase; TLN = talin 1 or talin 2; VCL = vinculin; CTNA1 = -catenin.

Kindlin binds directly to 1-integrins and ILK. Three isoforms are expressed in vertebrates, named kindlin 1, 2 and 3. The main isoform expressed in skeletal and cardiac muscle is kindlin 2 (Ussar et al., 2006), which is localized at costameres and ICDs, again suggesting that it may play a structural role in areas of elevated mechanical stress (Dowling et al., 2008a). Mutations in kindlin 1 and 3 have been identified in patients affected by skin and immune disorders, respectively (Jobard et al., 2003; Siegel et al., 2003; Malinin et al., 2009; Svensson et al., 2009), but no mutations in kindlin 2 have been found in humans. *In vitro*  studies have shown that kindlin 2 is important for differentiation of myoblasts (Dowling et al., 2008b), and knockdown of kindlin 2 in *Zebrafish* caused defective development of several organs, including skeletal and cardiac muscle, with disruption of ICDs and failure in the attachment of myofibrils to the membrane (Dowling et al., 2008a). Thus, kindlin 2 may be a good candidate gene for screening in patients affected by dilated cardiomyopathy or congenital myopathies.

#### **5.4 FAK**

Focal adhesion kinase (FAK) is closely associated with integrins, and following integrin engagement with ECM ligands, it becomes phosphorylated at tyrosine 397 (Y397). This creates a binding site for the SH2 domain of Src family kinases, and leads to the activation of several signaling effectors, including Rho and Rac, PI3K, Akt and the ERK1/2 signaling pathway (Franchini et al., 2009).

The tyrosine phosphorylation of FAK is rapidly increased following pressure overload in the rat heart (Franchini et al., 2000), and FAK activates hypertrophic signaling through PKB/AKT, the ERK1/2 and the JNK/c-JUN pathways. Additionally, FAK signaling regulates expression of the MEF2 transcription factors, which regulate the expression of several sarcomeric proteins (Nadruz et al., 2005). Insights on the function of FAK in striated muscle were obtained by generating mice with a conditional FAK ablation in cardiomyocytes. These mice developed defects that included thinner ventricular walls, ventricular septal defects and reduced cell numbers (DiMichele et al., 2006; Hakim et al.,

Integrins in the Development and Pathology of Skeletal Muscle 13

the ECM and the actin cytoskeleton, the assortment of proteins that are associated with each complex differs, and it is likely that specific signaling pathways elicit different biochemical responses. Studies in the heart indicate that integrins translate mechanical stimuli into hypertrophic responses. Are they important for regulating hypertrophy in skeletal muscle? It is also unclear how integrins regulate the process of myoblast fusion, for instance whether defects in the function of integrins lead to altered actin organization at sites of cell-cell fusion. Are signaling cascades activated by integrins important for the activation or recruitment of other effectors that mediate the breakdown of plasma membrane occurring during cell-cell fusion? Few patients affected by congenital myopathy have been identified with mutations in an integrin (7). It is unclear still how defects in integrin function lead to the observed muscle defects, as, unlike for the DPC, breakdown of the sarcolemma is not usually apparent. This may be elucidated with the identification of additional patients, and by an in-depth analysis of 7-KO mice. Also, do mutations in other integrins or associated proteins underlie genetically undiagnosed cases of muscular dystrophy or congenital myopathy? Talin, kindlin and vinculin are good candidate genes, as genetic studies in animal models showed essential roles for these proteins in conferring structural integrity to

We would like to thank Dr Yalda Jamshidi (St. George's University of London) and Dr Sarah Farmer (Institute of Child Health, UCL, London) for comments on the manuscript. This work was supported by funding from the Institute of Child Health and Great Ormond Street Hospitals Biomedical Research Centre (BRC 09DN09), Association Francaise contres les Myopathies (AFM 14572) (F.J.C.) and from the Muscular Dystrophy Association (S.C.B.).

Bader, B. L., Rayburn, H., Crowley, D. and Hynes, R. O. (1998) 'Extensive vasculogenesis,

Baker, L. P., Daggett, D. F. and Peng, H. B. (1994) 'Concentration of pp125 focal adhesion

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Barresi, R. and Campbell, K. P. (2006) 'Dystroglycan: from biosynthesis to pathogenesis of

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human disease', *Journal of cell science* 119(Pt 2): 199-207.

splicing', *The Journal of cell biology* 139(6): 1583-95.

angiogenesis, and organogenesis precede lethality in mice lacking all alpha v

kinase (FAK) at the myotendinous junction', *Journal of cell science* 107 ( Pt 6): 1485-

a component of the myotendinous junction on skeletal muscle', *Journal of cell science*

V. E., Burridge, K. and Tarone, G. (1997) 'Muscle beta1D integrin reinforces the cytoskeleton-matrix link: modulation of integrin adhesive function by alternative

H. A., Fishman, M. C. and Rottbauer, W. (2006) 'Integrin-linked kinase, a novel

skeletal or cardiac muscle.

**7. Acknowledgements** 

integrins', *Cell* 95(4): 507-19.

106 ( Pt 2): 579-89.

**8. References** 

97.

2007; Peng et al., 2008). However, the function of FAK in the postnatal heart is still unclear, as studies provide contrasting data on its function in cardiac hypertrophy, reporting either an increase in hypertrophy following mechanical or chemical stimuli (Peng et al., 2008), or an impaired hypertrophic response, with reduced expression of ANF and ERK1/2 (Hakim et al., 2007). The reason for the discrepancy is unclear, but it might be due to differences in the timing of FAK deletion, in the extent of aortic constriction, or in the genetic background of the mice.

The conditional inactivation of FAK in skeletal muscle has not been reported. In myoblasts, the application of mechanical forces to integrins results in FAK phosphorylation, and induction of hypertrophy in skeletal muscle leads to increased FAK expression and activation. Conversely, unloading of skeletal muscle leads to a sharp decrease in FAK activation (Fluck et al., 1999; Carson and Wei, 2000; Laser et al., 2000; Taylor et al., 2000; Gordon et al., 2001; Kovacic-Milivojevic et al., 2001). The inactivation of FAK in skeletal muscle would address its function and clarify whether its activity enhances or inhibits muscle hypertrophy.

#### **5.5 Melusin**

Melusin binds directly to 1-integrins and is expressed in skeletal and cardiac muscle, where it colocalises at costameres with integrins and vinculin (Brancaccio et al., 2003). Its domain structure includes in the N-terminus repeats of CHORD domain, which bind Zn2+, and in the C-terminus the integrin binding site and an acidic region resembling domains in calreticulin and calsequestrin that bind to calcium. In addition, while melusin is not endowed with catalytic activity, it includes binding sites for SH2- and SH3-domain proteins. The *itgb1 bp2* gene, encoding melusin, was inactivated in mice (Brancaccio et al., 2003). The mutant mice developed normally and were fertile. The basal structure and function of the heart were normal. However, when subjected to pressure overload via TAC, melusin-null mice presented with an impaired hypertrophic response, characterized by a reduction in myocyte cross-sectional area, ventricular wall thickness and induction of hypertrophic markers such as atrial neuretic factor and -MHC. These changes led to an enlarged left ventricular chamber, a decrease in contractile function and eventually cardiac arrest, and may involve signaling through GSK3 and Akt, as phosphorylation in these proteins was reduced. Interestingly, unlike what is observed in FAK-deficient mice, infusion with angiotensin II or phenylephrine did not cause an aberrant hypertrophic response in melusin-null mice, indicating that melusin is required to specifically sense mechanical but not biohemical stimuli (Brancaccio et al., 2006; 2003). No overt defects in skeletal muscle were observed in melusing-knockout mice.

#### **6. Conclusions and future perspectives**

In recent years integrins have emerged as key players in skeletal muscle, both during development, where they are essential for somitogenesis, myoblast fusion and assembly of the sarcomere, and in the adult, where they play important structural roles, in particular in conferring mechanical integrity to the MTJ of skeletal muscle, and to ICDs in cardiac muscle in the heart. Key questions remain to be addressed. For instance, how do the functions of integrins differ from those of the DPC? While both protein complexes create a link between the ECM and the actin cytoskeleton, the assortment of proteins that are associated with each complex differs, and it is likely that specific signaling pathways elicit different biochemical responses. Studies in the heart indicate that integrins translate mechanical stimuli into hypertrophic responses. Are they important for regulating hypertrophy in skeletal muscle? It is also unclear how integrins regulate the process of myoblast fusion, for instance whether defects in the function of integrins lead to altered actin organization at sites of cell-cell fusion. Are signaling cascades activated by integrins important for the activation or recruitment of other effectors that mediate the breakdown of plasma membrane occurring during cell-cell fusion? Few patients affected by congenital myopathy have been identified with mutations in an integrin (7). It is unclear still how defects in integrin function lead to the observed muscle defects, as, unlike for the DPC, breakdown of the sarcolemma is not usually apparent. This may be elucidated with the identification of additional patients, and by an in-depth analysis of 7-KO mice. Also, do mutations in other integrins or associated proteins underlie genetically undiagnosed cases of muscular dystrophy or congenital myopathy? Talin, kindlin and vinculin are good candidate genes, as genetic studies in animal models showed essential roles for these proteins in conferring structural integrity to skeletal or cardiac muscle.

#### **7. Acknowledgements**

We would like to thank Dr Yalda Jamshidi (St. George's University of London) and Dr Sarah Farmer (Institute of Child Health, UCL, London) for comments on the manuscript. This work was supported by funding from the Institute of Child Health and Great Ormond Street Hospitals Biomedical Research Centre (BRC 09DN09), Association Francaise contres les Myopathies (AFM 14572) (F.J.C.) and from the Muscular Dystrophy Association (S.C.B.).

#### **8. References**

12 Neuromuscular Disorders

2007; Peng et al., 2008). However, the function of FAK in the postnatal heart is still unclear, as studies provide contrasting data on its function in cardiac hypertrophy, reporting either an increase in hypertrophy following mechanical or chemical stimuli (Peng et al., 2008), or an impaired hypertrophic response, with reduced expression of ANF and ERK1/2 (Hakim et al., 2007). The reason for the discrepancy is unclear, but it might be due to differences in the timing of FAK deletion, in the extent of aortic constriction, or in the genetic background

The conditional inactivation of FAK in skeletal muscle has not been reported. In myoblasts, the application of mechanical forces to integrins results in FAK phosphorylation, and induction of hypertrophy in skeletal muscle leads to increased FAK expression and activation. Conversely, unloading of skeletal muscle leads to a sharp decrease in FAK activation (Fluck et al., 1999; Carson and Wei, 2000; Laser et al., 2000; Taylor et al., 2000; Gordon et al., 2001; Kovacic-Milivojevic et al., 2001). The inactivation of FAK in skeletal muscle would address its function and clarify whether its activity enhances or inhibits

Melusin binds directly to 1-integrins and is expressed in skeletal and cardiac muscle, where it colocalises at costameres with integrins and vinculin (Brancaccio et al., 2003). Its domain structure includes in the N-terminus repeats of CHORD domain, which bind Zn2+, and in the C-terminus the integrin binding site and an acidic region resembling domains in calreticulin and calsequestrin that bind to calcium. In addition, while melusin is not endowed with catalytic activity, it includes binding sites for SH2- and SH3-domain proteins. The *itgb1 bp2* gene, encoding melusin, was inactivated in mice (Brancaccio et al., 2003). The mutant mice developed normally and were fertile. The basal structure and function of the heart were normal. However, when subjected to pressure overload via TAC, melusin-null mice presented with an impaired hypertrophic response, characterized by a reduction in myocyte cross-sectional area, ventricular wall thickness and induction of hypertrophic markers such as atrial neuretic factor and -MHC. These changes led to an enlarged left ventricular chamber, a decrease in contractile function and eventually cardiac arrest, and may involve signaling through GSK3 and Akt, as phosphorylation in these proteins was reduced. Interestingly, unlike what is observed in FAK-deficient mice, infusion with angiotensin II or phenylephrine did not cause an aberrant hypertrophic response in melusin-null mice, indicating that melusin is required to specifically sense mechanical but not biohemical stimuli (Brancaccio et al., 2006; 2003). No overt defects in skeletal muscle

In recent years integrins have emerged as key players in skeletal muscle, both during development, where they are essential for somitogenesis, myoblast fusion and assembly of the sarcomere, and in the adult, where they play important structural roles, in particular in conferring mechanical integrity to the MTJ of skeletal muscle, and to ICDs in cardiac muscle in the heart. Key questions remain to be addressed. For instance, how do the functions of integrins differ from those of the DPC? While both protein complexes create a link between

of the mice.

muscle hypertrophy.

were observed in melusing-knockout mice.

**6. Conclusions and future perspectives** 

**5.5 Melusin** 


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**2** 

*1Italy 2USA* 

**Facioscapulohumeral** 

**Muscular Dystrophy: From Clinical** 

*1Universita' degli Studi di Modena e Reggio Emilia, 2University of Massachussets Medical School,* 

**Data to Molecular Genetics and Return** 

Monica Salani2, Elisabetta Morini1, Isabella Scionti1 and Rossella Tupler1,2

Facioscapulohumeral muscular dystrophy (FSHD or Dejerine–Landouzy muscular dystrophy, OMIM #158900) is the third most common hereditary myopathy, with prevalence of 1 in 20,000 (Padberg, 1982; Mostacciuolo et al., 2009). This disease is characterized by the progressive wasting of a highly selective set of muscle groups (Padberg, 1982) and it has been traditionally classified as an autosomal dominant trait (Lunt, 1998; Padberg, 1992). FSHD genetic locus has been mapped on chromosome 4q35 by genetic linkage analysis (Wijmenga et al., 1990). Interestingly, this muscular dystrophy has not yet been related to a classical mutation within a protein-coding gene, but rather the disease has been associated with DNA rearrangements in a polymorphic genomic region consisting of an array of tandemly repeated 3.3 kb segments, named D4Z4 (Wijmenga et al., 1992b). D4Z4 contains an ORF encoding a putative homeobox protein called "DUX4." The existence of native transcripts of DUX4 from D4Z4 single repeats is still controversial (Gabriels et al., 1999; Hewitt et al., 1994; Lyle et al., 1995), although recent data show evidences of the presence of the DUX4 transcript from the last D4Z4 unit in FSHD myoblasts (Lemmers et al., 2010a). The number of D4Z4 repeats varies from 11 to 100 in the general population, whereas less than 11 repeats are usually present in sporadic and familial FSHD patients. A very low copy number of 4q35 D4Z4 repeats (1–3) often correlates with an earlier onset and more severe disease. However no FSHD-linked array has been found to have zero copies of the repeat unit (Tupler et al., 1996; van der Maarel et al., 2007) suggesting that the repeat itself plays a critical role in the disease. Alleles with 4-7 repeats are the most frequent in the FSHD population and are associated with the more common form of FSHD that usually presents in adulthood, whereas alleles with 8-10 repeats typically display milder disease phenotypes with reduced penetrance. Nearly identical and equally polymorphic D4Z4 sequences reside on the subtelomere of chromosome 10q (Bakker et al., 1995; Deidda et al., 1995). The proportion of individuals in the general population carrying 4q or 10q chromosome ends with repeat arrays entirely or partially transferred between both chromosomes, is considerable (van Deutekom et al., 1996b; Lemmers et al., 1998; van Overveld et al., 2000). Rearrangements between 4q and 10q subtelomeres occur in 20% of

**1. Introduction** 

both hypertrophic and dilated cardiomyopathy', *Molecular genetics and metabolism* 87(2): 169-74.

