**2. Integrins**

Integrins are transmembrane receptors that connect via the extracellular domain to extracellular matrix (ECM) ligands such as collagen, laminin and fibronectin, and via the intracellular domain to the actin cytoskeleton and to a variety of signaling and adaptor proteins. Each integrin is a heterodimer composed of an - and a -subunit. In mammalian cells 18 and 8 subunits have been characterized, and are known to assemble to form 24 distinct integrin heterodimers, with the combination of - and - subunits determining ligand specificity. These play essential functions during development and in adult tissues. Accordingly, genetic ablation of individual subunits in mice leads to defects in tissues including brain, skin vasculature, lung, kidneys, inner ear, placenta, skeletal and cardiac muscle (Hynes, 2002).

The cytoplasmic domain of both the - and -integrin subunits is devoid of catalytic activity, but it binds to an array of proteins that mediate integrin effects on cell function. It is currently estimated that over 150 proteins are associated with integrin adhesion sites (Zaidel-Bar et al., 2007). Of these, we will discuss those that to date have been shown to be important in skeletal muscle. Some play structural roles, conferring mechanical integrity to myofibres by connecting integrins to the actin cytoskeleton, and others play signaling roles, by eliciting a biochemical response to mechanical stimuli caused by muscle contraction.

#### **2.1 Developmental expression of integrins in skeletal muscle**

Several integrins are expressed in myoblasts and muscle fibres, including v3-integrin, 4 integrin (associated with 1 or 2), and 1-integrin with the 1, 2, 3, 5, 6, 7 or 9 subunits (Gullberg et al., 1998; Mayer, 2003; Thorsteinsdottir et al., 2011). Expression of these subunits is regulated, with regards both to the stage of muscle development and to the localization within muscle fibres (Thorsteinsdottir et al., 2011). Some integrin subunits (1A, 4, 5, 6, 7b and v) are detected in the somites and in myoblasts. During myotube formation, expression of most subunits is downregulated, and adult muscle fibres express the 1D subunit, paired with 7a, 7b, and 9. The subcellular distribution of these integrin

proteins that bind to the ECM, and of cytoplasmic proteins that connect to the cytoskeleton and transmit biochemical signals. The DPC is composed of several proteins, which include - and -dystroglycan that bind to laminin, dystrophin that connects to the cytoskeleton, and associated proteins such as sarcoglycans and neuronal nitric oxide synthase (nNOS). These proteins have the important function to confer mechanical integrity to the plasma membrane, which otherwise would break following muscle contraction. Indeed, this occurs in patients with mutations in DPC components, and present with several types of severe muscle disease, including Duchenne Muscular Dystrophy (DMD) and various forms of

While integrins also establish a connection between the ECM and cytoskeletal and signalling proteins, the two complexes are biochemically distinct. As we will see below, integrins appear dispensable for the mechanical integrity of the sarcolemma, but have important

Integrins are transmembrane receptors that connect via the extracellular domain to extracellular matrix (ECM) ligands such as collagen, laminin and fibronectin, and via the intracellular domain to the actin cytoskeleton and to a variety of signaling and adaptor proteins. Each integrin is a heterodimer composed of an - and a -subunit. In mammalian cells 18 and 8 subunits have been characterized, and are known to assemble to form 24 distinct integrin heterodimers, with the combination of - and - subunits determining ligand specificity. These play essential functions during development and in adult tissues. Accordingly, genetic ablation of individual subunits in mice leads to defects in tissues including brain, skin vasculature, lung, kidneys, inner ear, placenta, skeletal and cardiac

The cytoplasmic domain of both the - and -integrin subunits is devoid of catalytic activity, but it binds to an array of proteins that mediate integrin effects on cell function. It is currently estimated that over 150 proteins are associated with integrin adhesion sites (Zaidel-Bar et al., 2007). Of these, we will discuss those that to date have been shown to be important in skeletal muscle. Some play structural roles, conferring mechanical integrity to myofibres by connecting integrins to the actin cytoskeleton, and others play signaling roles, by eliciting a biochemical response to mechanical stimuli caused by muscle contraction.

Several integrins are expressed in myoblasts and muscle fibres, including v3-integrin, 4 integrin (associated with 1 or 2), and 1-integrin with the 1, 2, 3, 5, 6, 7 or 9 subunits (Gullberg et al., 1998; Mayer, 2003; Thorsteinsdottir et al., 2011). Expression of these subunits is regulated, with regards both to the stage of muscle development and to the localization within muscle fibres (Thorsteinsdottir et al., 2011). Some integrin subunits (1A, 4, 5, 6, 7b and v) are detected in the somites and in myoblasts. During myotube formation, expression of most subunits is downregulated, and adult muscle fibres express the 1D subunit, paired with 7a, 7b, and 9. The subcellular distribution of these integrin

**2.1 Developmental expression of integrins in skeletal muscle** 

Limb Girdle Muscular Dystrophy (Bushby, 1999; Barresi and Campbell, 2006).

functions during all stages of muscle development.

**2. Integrins** 

muscle (Hynes, 2002).

subunits is also regulated: 7a is found at the MTJ, 7b at the sarcolemma, MTJ and neuromuscular junction (NMJ), 3- and v-integrins are localized to the NMJ, and 9 integrin appears to be uniformly distributed along the sarcolemma (Wang et al., 1995; Martin et al., 1996). We will discuss here the functions identified for integrins in muscle, and refer the reader to recent reviews for details on the regulation of somitogenesis and NMJ formation by integrin-ECM interactions (Singhal and Martin, 2011; Thorsteinsdottir et al., 2011).

While the expression pattern of the different subunits is well characterized, the precise function of many remains to be addressed. Genetic ablation of integrins in mice has not always been informative in this regard. For instance, mice with an ablation of 1-, 9- and v-integrins present no defects in skeletal muscle (Gardner et al., 1996; Bader et al., 1998; Huang et al., 2000). Mice with a genetic ablation of 3- and 6-integrins, die too early to study the long-term functions of integrins in skeletal muscle maintenance (Georges-Labouesse et al., 1996; Kreidberg et al., 1996). The distribution of laminin 5, which is the main ligand for 3-integrin, suggests a possible function in maturation of the muscle fibre and of the NMJ, since its initial expression throughout the basal lamina of developing myotubes becomes restricted to the NMJ in the first 3 weeks following birth (Nishimune et al., 2008). This is also consistent with 3-integrin expression being concentrated at the presynaptic NMJ (Martin et al., 1996).

Muscle defects have also been identified in mice with ablation of 5- and 7-integrins (Taverna et al., 1998; Mayer et al., 1997). 5-integrin is a receptor for fibronectin, and is expressed transiently during myotube differentiation. Ablation of 5-integrin in mice leads to early embryonic lethality with defects in mesoderm, vascular development and neural crest (Yang et al., 1993; Goh et al., 1997), but mice chimeric for this subunit survive postnatally and develop a form of muscular dystrophy (Taverna et al., 1998). No patients have been identified with mutations in 5-integrin possibly because, extrapolating from the data obtained in the mutant mouse models, null mutations are likely to be non viable. 7 integrin has been shown to play important functions in muscle in animal models and human patients, where mutations lead to a form of congenital muscular dystrophy (Mayer et al., 1997; Hayashi et al., 1998). Whilst it is possible that an in-depth analysis of the integrin subunit knockout mice would reveal muscle defects, for example in response to stressors such as exercise or mechanical damage, the apparent absence of a reported phenotype for some of these mice might be explained by redundancy. This possibility is supported by the generation of mice with a muscle specific ablation of the 1-subunit, which leads to the concomitant ablation of all 1-integrins (Schwander et al., 2003). These mice die shortly after birth, probably because of respiratory failure, with severe developmental defects in the muscle caused by impaired myoblast fusion and altered assembly of the sarcomere.

#### **3. Integrins in skeletal muscle development**

Fusion of myoblasts is essential for the formation of a syncytial myofibre, and it occurs in distinct steps: (i) migration of myoblasts to achieve cell proximity; (ii) contact between

Integrins in the Development and Pathology of Skeletal Muscle 5

Fig. 1. **Integrins are essential for myoblast fusion.** Prior to fusion, migrating myoblasts elongate and make contact between their plasma membranes. Integrins localise at the cell interface, and are important for the formation of fusion pores, i.e. the breakdown of plasma membrane that precedes mixing of cytoplasmic content. In vitro experiments suggest that integrins interact heterophylically with an as yet unidentified counterreceptor (X in upper image). The mechanisms by which this occurs are unclear, but fusion defects are also observed following ablation of filamin C, talin 1 or talin 2, which are important actin regulators, suggesting that changes in cytoskeletal dynamics are important. Abbreviations:

FLNC = filamin C; TLN = talin 1 or talin 2; FAK = focal adhesion kinase; = as yet

muscular dystrophies, including calpain-3 and sarcoglycans (Zhou et al., 2010).

for 1-integrin.

**3.2 Filamin C** 

unidentified 1-integrin associated -subunit. X = putative (unidentified) counter receptor

Filamins are actin binding proteins that cross-link actin filaments into orthogonal networks. They bind to over 30 proteins, including integrins and actin, through which they perform many functions, including modulation of cell adhesion to the ECM, cell migration, mechanical strengthening of the plasma membrane, and the activation of signaling networks. Mammalians express three filamin isoforms, termed filamin A, B and C. Filamins A and B are widely expressed, and play essential functions in the development of a variety of tissues. Expression of filamin C is mostly restricted to skeletal and cardiac muscle, where it localizes to the sarcolemma and to the Z-disk, and interacts with several proteins associated with

myoblasts and alignment of the plasma membranes; (iii) breakdown of the plasma membrane at the site of fusion, leading to the formation of fusion pores (iv) merging of the cytoplasmic contents (Chen et al., 2007). While the identity of the proteins leading to plasma membrane breakdown is unknown, studies in recent years have led to the identification of several components of the fusion machinery, most notably elucidating the importance of actin remodeling (Rochlin et al., 2010).

#### **3.1 1-integrin and talin**

A direct involvement of integrins in the regulation of myoblast fusion in vertebrates has been obtained using genetically modified mice. Ablation of 1-integrin in developing muscle has revealed important functions in cell-cell fusion and assembly of the sarcomere (Schwander et al., 2003). 1-deficient mice died at birth, with histological analysis showing that many myoblasts failed to fuse. *In vitro* analysis showed that fusion defects could be rescued when wild-type and 1-deficient myoblasts were mixed, suggesting that heterophilic interactions of 1-integrin with an unidentified receptor may be important. Analysis of cultured myoblasts by electron microscopy showed that plasma membranes aligned properly, but fusion pores failed to open, indicating that integrins are not essential for the alignment of myoblasts, but affect a subsequent step in fusion. The analysis of mice lacking the integrin effectors talin 1 and talin 2 suggests that signaling to the cytoskeleton may be important in this respect.

Talin 1 and 2 are expressed by two distinct genes (*tln1* and *tln2*) and present a high degree of homology (74% identity in the amino acid sequence)(Senetar and McCann, 2005). They bind to cytoskeletal proteins such as actin and vinculin, and signaling effectors that include focal adhesion kinase (FAK) and PIPK1, which regulate the assembly of focal adhesions (Critchley, 2004). The two isoforms are essential to mediate 1-interin functions in myoblasts ablation of talin 1 and talin 2 in muscle (tln1/2-dKO) resulted in defects similar to those observed following ablation of 1-integrin: mice died shortly after birth, with abnormal development of the musculature, including defects in myoblast fusion, sarcomere assembly and in the clustering of 7-integrin, vinculin and integrin-linked kinase (ILK) at the MTJ (Conti et al., 2009). The tetraspanin CD9, which has been implicated in sperm-egg fusion (Kaji et al., 2000; Hemler, 2001), was mislocalised in 1-deficient muscle, but localized normally at the interface of tln1/2-dKO myoblasts, and integrin activation was also normal, suggesting that outside-in signaling mechanisms may be responsible for the fusion defects (Conti et al., 2009). In this respect, it is interesting to note that several of the proteins implicated in myoblast fusion are controlled by integrins, specifically, the Rho GTPases Rac1 and Cdc42, and associated proteins such as Dock180 (Laurin et al., 2008; Pajcini et al., 2008; Vasyutina et al., 2009). In mouse, vinculin, an actin- and talin-binding protein (see below) accumulates at the interface of fusing myoblasts, and genetic ablation of Rac and Cdc42 causes a reduction in this accumulation (Vasyutina et al., 2009). Furthermore, ablation of two other integrin effectors, FAK and filamin C, leads to compromised myoblast fusion (Dalkilic et al., 2006; Quach and Rando, 2006). These data are indicative of a possible involvement of integrins in regulating actin dynamics at the sites of fusion, although this still needs to be demonstrated directly.

myoblasts and alignment of the plasma membranes; (iii) breakdown of the plasma membrane at the site of fusion, leading to the formation of fusion pores (iv) merging of the cytoplasmic contents (Chen et al., 2007). While the identity of the proteins leading to plasma membrane breakdown is unknown, studies in recent years have led to the identification of several components of the fusion machinery, most notably elucidating the importance of

A direct involvement of integrins in the regulation of myoblast fusion in vertebrates has been obtained using genetically modified mice. Ablation of 1-integrin in developing muscle has revealed important functions in cell-cell fusion and assembly of the sarcomere (Schwander et al., 2003). 1-deficient mice died at birth, with histological analysis showing that many myoblasts failed to fuse. *In vitro* analysis showed that fusion defects could be rescued when wild-type and 1-deficient myoblasts were mixed, suggesting that heterophilic interactions of 1-integrin with an unidentified receptor may be important. Analysis of cultured myoblasts by electron microscopy showed that plasma membranes aligned properly, but fusion pores failed to open, indicating that integrins are not essential for the alignment of myoblasts, but affect a subsequent step in fusion. The analysis of mice lacking the integrin effectors talin 1 and talin 2 suggests that signaling to the cytoskeleton

Talin 1 and 2 are expressed by two distinct genes (*tln1* and *tln2*) and present a high degree of homology (74% identity in the amino acid sequence)(Senetar and McCann, 2005). They bind to cytoskeletal proteins such as actin and vinculin, and signaling effectors that include focal adhesion kinase (FAK) and PIPK1, which regulate the assembly of focal adhesions (Critchley, 2004). The two isoforms are essential to mediate 1-interin functions in myoblasts ablation of talin 1 and talin 2 in muscle (tln1/2-dKO) resulted in defects similar to those observed following ablation of 1-integrin: mice died shortly after birth, with abnormal development of the musculature, including defects in myoblast fusion, sarcomere assembly and in the clustering of 7-integrin, vinculin and integrin-linked kinase (ILK) at the MTJ (Conti et al., 2009). The tetraspanin CD9, which has been implicated in sperm-egg fusion (Kaji et al., 2000; Hemler, 2001), was mislocalised in 1-deficient muscle, but localized normally at the interface of tln1/2-dKO myoblasts, and integrin activation was also normal, suggesting that outside-in signaling mechanisms may be responsible for the fusion defects (Conti et al., 2009). In this respect, it is interesting to note that several of the proteins implicated in myoblast fusion are controlled by integrins, specifically, the Rho GTPases Rac1 and Cdc42, and associated proteins such as Dock180 (Laurin et al., 2008; Pajcini et al., 2008; Vasyutina et al., 2009). In mouse, vinculin, an actin- and talin-binding protein (see below) accumulates at the interface of fusing myoblasts, and genetic ablation of Rac and Cdc42 causes a reduction in this accumulation (Vasyutina et al., 2009). Furthermore, ablation of two other integrin effectors, FAK and filamin C, leads to compromised myoblast fusion (Dalkilic et al., 2006; Quach and Rando, 2006). These data are indicative of a possible involvement of integrins in regulating actin dynamics at the sites of fusion, although this

actin remodeling (Rochlin et al., 2010).

**3.1 1-integrin and talin** 

may be important in this respect.

still needs to be demonstrated directly.

Fig. 1. **Integrins are essential for myoblast fusion.** Prior to fusion, migrating myoblasts elongate and make contact between their plasma membranes. Integrins localise at the cell interface, and are important for the formation of fusion pores, i.e. the breakdown of plasma membrane that precedes mixing of cytoplasmic content. In vitro experiments suggest that integrins interact heterophylically with an as yet unidentified counterreceptor (X in upper image). The mechanisms by which this occurs are unclear, but fusion defects are also observed following ablation of filamin C, talin 1 or talin 2, which are important actin regulators, suggesting that changes in cytoskeletal dynamics are important. Abbreviations: FLNC = filamin C; TLN = talin 1 or talin 2; FAK = focal adhesion kinase; = as yet unidentified 1-integrin associated -subunit. X = putative (unidentified) counter receptor for 1-integrin.

#### **3.2 Filamin C**

Filamins are actin binding proteins that cross-link actin filaments into orthogonal networks. They bind to over 30 proteins, including integrins and actin, through which they perform many functions, including modulation of cell adhesion to the ECM, cell migration, mechanical strengthening of the plasma membrane, and the activation of signaling networks. Mammalians express three filamin isoforms, termed filamin A, B and C. Filamins A and B are widely expressed, and play essential functions in the development of a variety of tissues. Expression of filamin C is mostly restricted to skeletal and cardiac muscle, where it localizes to the sarcolemma and to the Z-disk, and interacts with several proteins associated with muscular dystrophies, including calpain-3 and sarcoglycans (Zhou et al., 2010).

Integrins in the Development and Pathology of Skeletal Muscle 7

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

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

the initial identification, mutations appear to be rare.

**4.2 Talin** 

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

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