**3. Changes in ECM assembly and function during myogenesis**

Skeletal muscle growth and development is a complex process controlled by interactions between muscle cells and surrounding microenvironment. Several cellular events take place during skeletal myogenesis, that is, migration of muscle precursor cells, proliferation of myoblasts, cell cycle arrest, and myoblast terminal differentiation, followed by transcription of muscle-specific genes and myoblast fusion. Muscle cell differentiation is governed by an ordered sequence of the expression of muscle regulatory factors (MRFs) such as MyoD (Myoblast determination protein), Myf-5 (Myogenic factor-5), myogenin, and MRF-4 [24]. The commitment of muscle precursor cells requires MyoD expression, whereas the proliferation arrest and terminal myoblast differentiation are driven by myogenin, a key transcription factor, which activates skeletal muscle-specific genes encoding creatine kinase, myosin heavy chain, and acetylcholine receptor. The formation of myotubes expressing muscle-specific genes is essential for the specialization of myofiber function.

The importance of extracellular matrix molecules as a part of myogenesis signaling mechanism has also been demonstrated. An inhibition of cell-surface transmembrane proteoglycan sulfation results in delayed proliferation and altered MyoD expression, indicating that heparan sulfate is required for proper progression of the early myogenic program [25]. Neither the expression of myogenin nor its localization to myoblast nuclei was sufficient to drive skeletal muscle differentiation, if the cell–ECM interactions were inhibited [26]. Inhibition of proteo‐ glycan sulfation in myoblast cultures strongly affects ECM synthesis and deposition, and induces the expression of the osteogenic markers (alkaline phosphatase and osteocalcin), without alterations in expression of specific muscle transcription factors, such as MyoD and Myf-5 [27]. The above observations support the idea that extracellular matrix provides stimuli for muscle cell development, which are independent of muscle-specific factor expression.

Myogenesis is accompanied by remodeling of ECM proteins as well as by changes in integrin receptor expression pattern [28]. Fibronectin and laminins display an opposite pattern of changes in time during myogenesis, that is, myoblasts secrete a large amount of fibronectin, which is replaced by laminins in myotubes. As a consequence, the location of these proteins in muscle is different, that is, fibronectin is absent in regions manifesting active myogenesis, whereas laminin adjoins myotubes. In myoblasts subjected to differentiation in vitro, fibro‐ nectin is detected primarily in the extracellular environment as a thick mesh. At the same time, laminin appears ultimately in the cytosolic fraction, which confirms delayed synthesis of this

protein during myogenesis, in comparison to fibronectin [29]. During myogenic differentia‐ tion, the laminin synthesis increases, and laminin begins to accumulate in the medium in soluble form, followed by the formation of insoluble cell-associated fraction [30]. Both fibronectin and laminin per se can affect myogenesis. Fibronectin promotes myoblast adhesion and proliferation; however, it inhibits differentiation and participates in collagen fibrillogen‐ esis, thus providing the ECM assembly [1]. Fibronectin also stimulates adhesion of fibroblasts and may facilitate dedifferentiation of myoblasts. This protein is required for somitogenesis, and it may function to regulate fiber organization and limit fast-twitch muscle fiber length [31]. Laminin is crucial for several processes involved in myogenesis, as it enhances myoblast proliferation, migration, and alignment preceding the fusion. Myotube formation is markedly impaired in the absence of laminin [2]. Changes in integrin receptor expression pattern reflect the ECM remodeling during myogenesis. Proliferating and migrating myoblasts express high amounts of the fibronectin-binding alpha5beta1 integrin, while during myotube formation they switch to the laminin-binding alpha7beta1 integrin, which is the major integrin receptor in adult muscles [32]. Moreover, there is a negative cooperativity between alpha7 and alpha5 integrin subunits. Transfection with integrin alpha7 resulted in the marked reduction of alpha5beta1 surface complex expression and its decreased affinity to fibronectin in myoblasts. Such a relationship may play an important role in determining functional regulation of integrins during myogenesis. A critical phase of myogenesis is the fusion of mononucleated myoblasts and the formation of long multinucleated myotubes. Myoblast fusion and myotube formation are associated with increased expression of integrin alpha3, particularly abundant in myotube membrane [29]. Overexpression of the full-length integrin alpha3 subunit induces myoblast fusion, whereas the inhibition of integrin alpha3 extracellular domain impairs this process [33]. Myogenesis is largely normal in the absence of alpha4, alpha5, alpha6, and alpha7 integrin subunits, indicating the redundancy in integrin functions. In contrast, disruption of the integrin beta1 in vivo and in vitro profoundly influences myogenesis. Lack of integrin beta1 had no apparent effect on the migration and proliferation of myoblasts; however, clear alterations occur at the later stages of myogenesis and are manifested by impaired fusion [34]. According to an early study, muscle-specific integrin beta1, appearing in a doublet form, was used as a marker of differentiation [35]. Integrin beta1 subunit is also involved in muscle cell survival. In response to the activation of integrin beta1, focal adhesion kinase phosphorylates tyrosine at residue 397, leading to the activation of cell survival signal transduction and inhibition of apoptosis [36]. Moreover, FAK appears as a mediator by which integrins may regulate myoblast fusion. Specific disruption of gene encoding FAK suppresses the transcrip‐ tion of caveolin 3 and integrin subunit beta1D isoform, both considered as essential for morphological muscle differentiation. As a consequence, the cell fusion and myotube forma‐ tion are defective, while the expression of muscle terminal differentiation genes, such as sarcomeric alpha-actin, alpha-actinin, and vinculin, remain unaltered [37]. It suggests a specific role of FAK in the regulation of cell fusion, as a part of the myogenic differentiation program.

proteins) to mimic the muscle extracellular environment. Usually, the primary muscle stem cells show distinct proliferation and differentiation pattern, as well as different muscle-specific and ECM-related gene expressions, dependent on the coating type used [9]. These experiments reveal that the loss of mitogenic and/or myogenic potential of muscle stem cells, due to their transfer from the specific niche to an ex vivo situation, could be reduced by using some ECM components/mixture coating. For example, fibronectin and laminin could be used for sorting myoblasts from fibroblasts. Such observations are of great interest and importance in tissue

Skeletal muscle growth and development is a complex process controlled by interactions between muscle cells and surrounding microenvironment. Several cellular events take place during skeletal myogenesis, that is, migration of muscle precursor cells, proliferation of myoblasts, cell cycle arrest, and myoblast terminal differentiation, followed by transcription of muscle-specific genes and myoblast fusion. Muscle cell differentiation is governed by an ordered sequence of the expression of muscle regulatory factors (MRFs) such as MyoD (Myoblast determination protein), Myf-5 (Myogenic factor-5), myogenin, and MRF-4 [24]. The commitment of muscle precursor cells requires MyoD expression, whereas the proliferation arrest and terminal myoblast differentiation are driven by myogenin, a key transcription factor, which activates skeletal muscle-specific genes encoding creatine kinase, myosin heavy chain, and acetylcholine receptor. The formation of myotubes expressing muscle-specific genes is

The importance of extracellular matrix molecules as a part of myogenesis signaling mechanism has also been demonstrated. An inhibition of cell-surface transmembrane proteoglycan sulfation results in delayed proliferation and altered MyoD expression, indicating that heparan sulfate is required for proper progression of the early myogenic program [25]. Neither the expression of myogenin nor its localization to myoblast nuclei was sufficient to drive skeletal muscle differentiation, if the cell–ECM interactions were inhibited [26]. Inhibition of proteo‐ glycan sulfation in myoblast cultures strongly affects ECM synthesis and deposition, and induces the expression of the osteogenic markers (alkaline phosphatase and osteocalcin), without alterations in expression of specific muscle transcription factors, such as MyoD and Myf-5 [27]. The above observations support the idea that extracellular matrix provides stimuli for muscle cell development, which are independent of muscle-specific factor expression.

Myogenesis is accompanied by remodeling of ECM proteins as well as by changes in integrin receptor expression pattern [28]. Fibronectin and laminins display an opposite pattern of changes in time during myogenesis, that is, myoblasts secrete a large amount of fibronectin, which is replaced by laminins in myotubes. As a consequence, the location of these proteins in muscle is different, that is, fibronectin is absent in regions manifesting active myogenesis, whereas laminin adjoins myotubes. In myoblasts subjected to differentiation in vitro, fibro‐ nectin is detected primarily in the extracellular environment as a thick mesh. At the same time, laminin appears ultimately in the cytosolic fraction, which confirms delayed synthesis of this

**3. Changes in ECM assembly and function during myogenesis**

engineering and stem cell therapies.

8 Composition and Function of the Extracellular Matrix in the Human Body

essential for the specialization of myofiber function.

A characteristic feature of proliferating and quiescent undifferentiated myoblasts is the high expression of a disintegrin and metalloprotease, ADAM12, which combines features of adhesion molecules and proteinases [38]. ADAM12 cleaves insulin-like growth factor binding proteins IGFBP3 and IGFBP5, and heparin binding-EGF. The cysteine-rich domain of ADAM12 supports cell adhesion by binding to syndecan-4, whereas the cytoplasmic domain interacts with signaling proteins, that is, tyrosine kinase Src phosphatidylinositol-3-kinase, and cytoskeletal alpha-actinin 1 and 2 [38]. ADAM12 in transiently upregulated at the onset of differentiation, whereas other ADAMs, such as ADAM9, 10, 15, 17, and 19 are expressed at all stages of myogenesis [35]. Inhibition of ADAM12 by siRNA approach in myogenic cell cultures was accompanied by lower expression of both quiescent markers (p130 and p27 proteins) and differentiation markers (cell cycle inhibitor p21 and myogenin). Overexpression of ADAM12 induces a quiescent-like phenotype and does not stimulate differentiation. Possible role of ADAM12 in myogenesis is associated with the preservation of "reserve pool" of myoblasts, which do not trigger the myogenic differentiation program and maintain regeneration potential. A 100 kDa long isoform of ADAM12 is increased in myoblasts differentiating for 3 days in the presence of IL-1beta [39] and IGF-I [29], suggesting similar effects of proinflam‐ matory cytokines and anabolic growth factors on ECM regulation at early stages of myogen‐ esis. On the other hand, there are studies that implicate the involvement of ADAM12 in the fusion of muscle cells. The expression of ADAM12 and integrin alpha9 subunit parallels and culminates at the time of myoblast fusion, and inhibition of ADAM12/alpha9beta1 integrin interaction dramatically impairs this process [40]. ADAM12 is linked to the cytoskeleton via alpha-actinin [35], and thus the cytoskeleton may regulate the distribution of ADAM12 on the cell surface, where localized proteolysis and/or cell–cell contacts occur [41]. The most impor‐ tant modifications of the ECM structure and function associated with skeletal myogenesis are depicted in **Figure 2**.

**Figure 2.** Schematic illustration of ECM remodeling and ECM-related proteins level/activity during skeletal myogene‐ sis. The most important events during myogenic development are presented in the upper panel.
