**3. Skeletal muscle development and the extracellular matrix**

collagens in this extracellular region are COL XI, XII, XIV, XV and XVIII and are mostly expressed during muscle development [3]. The main structural constituent in ECM microfibrils is collagen isoform COL VI. Microfibrils provide structural support during the physical strains of continuing contraction–relaxation cycles. Directly overlaying the sarcolemma membrane is the basal lamina consisting of the main non-fibrillar collagen isoform COL IV and a few minor constituents, including COL VI, XV and XVIII. The collagen network of the basement mem‐ brane interacts with two crucial plasmalemma adhesion complexes, the integrin complex and the dystrophin–glycoprotein complex [10]. These sarcolemma-bridging protein assemblies form the laminin–dystroglycan axis [13] and the fibronectin-integrin (α7β1) axis [14], whereby

small leucine-rich repeat proteoglycans; TIMP, tissue inhibitors of matrix metalloproteinases.

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

**Figure 2** Diagrammatic presentation of the various layers of the extracellular matrix (ECM) from skeletal muscle and an overview of the molecular components that form the interstitial matrix, microfibrils and the basal lamina. Abbrevia‐ tions: COL, collagen; DG, dystroglycan; FN, fibronectin; LAM, laminin; INT, integrin; MMP, matrix metalloproteinase; NMJ, neuromuscular junction; PG, proteoglycan; PRELP, proline-arginine-rich end leucine-rich repeat protein; SLRP,

> Skeletal muscle fibres derive from the mesoderm [31] and represent one of the most abundant cell types in the body. Myofibres play a key physiological role in the provision and regulation of locomotion, breathing, postural control, heat homeostasis and metabolic integration [32–

34]. The development of contractile fibres is a highly complex process and changes in the ECM play an essential role during myogenesis. Adaptations occur at the level of the basal lamina, the interstitial ECM and the collagen-rich tendon and encompass a variety of fundamental processes of development, such as the determination of cell fate, as well as proliferation, cell division, patterning and tissue transitions [35]. The most critical developmental processes occur during the initial activation of precursor cells, various differentiation steps and the final maturation of innervated myofibres. These major developmental mechanisms are regulated by a large number of genetic and signalling factors [36]. Many specialized extracellular components are involved in muscle development, which is reflected by the transient expres‐ sion patterns of certain ECM molecules, such as the minor collagen isoforms COL XI, XII, XIV, XV and XVIII [3].

The molecular and cellular events that occur during the establishment of the myogenic cell lineage and resulting formation of multi-nucleated contractile fibres entail initially a fibrillar and fibronectin-rich matrix. In developmental terms, almost all myofibres of the skeletal musculature derive from mesodermal structures named somites that develop during early embryonic segmentation on both sides of the neural tube [37–39]. The developing ECM of somites is represented by a fibronectin core, a basement membrane and an outer fibronectincontaining matrix [35]. **Figure 3** outlines the initiation and control of embryonic and adult myogenesis by myogenic factors and through a complex series of spatio-temporal dependent signalling cascades. Fibronectin and its interactions with the integrin complex play a central role in polarizing and guiding somitic cells [40–42] and the ECM is crucial for somite formation and as a guiding cue during morphogenesis [43–45]. The small SLRP-type proteoglycan decorin was shown to be majorly involved in skeletal muscle development by promoting proliferation and differentiation of muscle cells through suppressing myostatin activity [46– 48].

The key regulator of early myogenesis that initiates the developmental commitment into the myogenic cell lineage is the paired-type homeobox gene *PAX3* [49–51]. Interestingly, another member of the PAX transcription factor family, the *PAX3* orthologue *PAX7*, acts as a regulator of mature skeletal muscle regeneration and postnatal growth mechanisms [52]. Following the induction of mesodermal precursors, the subsequent segmentation into somites and the formation of the primary myotome involves a variety of signalling molecules and transcription factors, such as the secreted and lipid-modified family of Wnt-glycoproteins and the large group of myogenic basic helix-loop-helix muscle regulatory factors, such as MyoD, Myf5, MRF4 and myogenin [53–55]. Myogenic factors act at multiple regulatory points during embryonic myogenesis, whereby the overall genetic pathway that is responsible for the transcriptional activation of skeletal muscle-specific genes is highly complex and partially redundant [56]. A key step during muscle development is the fusion of myogenic cells that result in the formation of innervated and multi-nucleated myofibres (**Figure 3**). Although specific regulatory processes differ between embryonic, foetal, postnatal and mature regen‐ erative myogenesis [57], the basic biological mechanisms of skeletal muscle development and injury-related adult muscle regeneration are extraordinarily similar [58,59]. A large pool of satellite cells provides a high level of regenerative capacity of the matured post-mitotic fibres and is located between the sarcolemma and the basal lamina [60]. These mono-nucleated myogenic stem cells are activated during cycles of fibre regeneration and cellular maintenance. To provide maximum skeletal muscle performance, activated stem cells undergo a complex pattern of proliferation, differentiation and cellular fusion to form multi-nucleated and functional contractile myofibres [61]. Following maturation, the tissue mass of skeletal muscles is then regulated by catabolic and anabolic mechanisms that include signalling factors such as NF-kB and FoxO, as well as the mTOR pathway [62].

34]. The development of contractile fibres is a highly complex process and changes in the ECM play an essential role during myogenesis. Adaptations occur at the level of the basal lamina, the interstitial ECM and the collagen-rich tendon and encompass a variety of fundamental processes of development, such as the determination of cell fate, as well as proliferation, cell division, patterning and tissue transitions [35]. The most critical developmental processes occur during the initial activation of precursor cells, various differentiation steps and the final maturation of innervated myofibres. These major developmental mechanisms are regulated by a large number of genetic and signalling factors [36]. Many specialized extracellular components are involved in muscle development, which is reflected by the transient expres‐ sion patterns of certain ECM molecules, such as the minor collagen isoforms COL XI, XII, XIV,

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

The molecular and cellular events that occur during the establishment of the myogenic cell lineage and resulting formation of multi-nucleated contractile fibres entail initially a fibrillar and fibronectin-rich matrix. In developmental terms, almost all myofibres of the skeletal musculature derive from mesodermal structures named somites that develop during early embryonic segmentation on both sides of the neural tube [37–39]. The developing ECM of somites is represented by a fibronectin core, a basement membrane and an outer fibronectincontaining matrix [35]. **Figure 3** outlines the initiation and control of embryonic and adult myogenesis by myogenic factors and through a complex series of spatio-temporal dependent signalling cascades. Fibronectin and its interactions with the integrin complex play a central role in polarizing and guiding somitic cells [40–42] and the ECM is crucial for somite formation and as a guiding cue during morphogenesis [43–45]. The small SLRP-type proteoglycan decorin was shown to be majorly involved in skeletal muscle development by promoting proliferation and differentiation of muscle cells through suppressing myostatin activity [46–

The key regulator of early myogenesis that initiates the developmental commitment into the myogenic cell lineage is the paired-type homeobox gene *PAX3* [49–51]. Interestingly, another member of the PAX transcription factor family, the *PAX3* orthologue *PAX7*, acts as a regulator of mature skeletal muscle regeneration and postnatal growth mechanisms [52]. Following the induction of mesodermal precursors, the subsequent segmentation into somites and the formation of the primary myotome involves a variety of signalling molecules and transcription factors, such as the secreted and lipid-modified family of Wnt-glycoproteins and the large group of myogenic basic helix-loop-helix muscle regulatory factors, such as MyoD, Myf5, MRF4 and myogenin [53–55]. Myogenic factors act at multiple regulatory points during embryonic myogenesis, whereby the overall genetic pathway that is responsible for the transcriptional activation of skeletal muscle-specific genes is highly complex and partially redundant [56]. A key step during muscle development is the fusion of myogenic cells that result in the formation of innervated and multi-nucleated myofibres (**Figure 3**). Although specific regulatory processes differ between embryonic, foetal, postnatal and mature regen‐ erative myogenesis [57], the basic biological mechanisms of skeletal muscle development and injury-related adult muscle regeneration are extraordinarily similar [58,59]. A large pool of satellite cells provides a high level of regenerative capacity of the matured post-mitotic fibres

XV and XVIII [3].

48].

The systematic profiling of myogenesis using mass spectrometry-based proteomics has covered postnatal growth and development [63,64], but focused mostly on studying cell culture models [65–67] and specifically the skeletal muscle secretome during myoblast differentiation and myotube formation [68–72]. The concept that skeletal muscle cells act as secretory tissues has recently been reviewed by Pedersen [73]. Secreted myokines probably exert autocrine, paracrine or endocrine effects within the neuromuscular system and also in relation to other organ systems [74]. The skeletal muscle secretome is estimated to consist of several hundred muscle-derived peptides and proteins [75]. During muscle development,

**Figure 3.** Outline of the initiation and control of embryonic and adult myogenesis. Shown is the transition from meso‐ dermal progenitor cells to myoblasts, followed by cellular fusion to produce multi-nucleated myotubes and finally in‐ nervated and functional myofibres. The critical role of stem cells during both embryonic development and regeneration of injured adult muscle fibres is highlighted, as well as changes in the ECM. Fibronectin (FN) and its in‐ teractions with the integrin complex are of central importance for polarizing and guiding somitic cells and the proteo‐ glycan decorin is majorly involved in myogenesis by promoting proliferation and differentiation through supressing myostatin activity. Changes in the ECM and the formation of a collagen (COL) matrix during muscle development are marked.

changes in ECM proteins have recently been studied in relation to human myogenesis and established that altered protein expression underlies the dramatic phenotypic conversion of primary mono-nucleated muscle cells during differentiation to form multi-nucleated myo‐ tubes [76]. The temporal profiling of the human myoblast proteome during *in vitro* differen‐ tiation highlighted the importance of ECM rearrangement during early myogenesis and showed a drastic increase in key ECM components, including several α-isoforms of collagen COL VI and COL XVIII, as well as the heparan sulfate proteoglycan HSPG2 of the basement membrane, the elastin–microfibril interface–located ECM glycoprotein EMILIN2 and nidogen isoform NID2 [76]. These findings confirm the developmental concept that enhanced synthesis of ECM proteins occurs during the transition from myoblasts to syncytial myotubes [72] and that complex interactions at the cell–ECM interface facilitate the fusion of myoblasts [35].
