**4. ECM and the motor function of skeletal muscle**

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

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

**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.

depicted in **Figure 2**.

Skeletal muscle provides structural support, enables the body to maintain posture, and controls motor movements. Muscle tissue is strong, flexible, stress-resistant, and in view of its mechanical properties, it consists of contractile elements (i.e. sarcomeres) and elastic compo‐ nents, supported by extracellular matrix. Majority of ECM elements, which account for muscle strength and elasticity, reside in the basement membrane, especially in basal lamina. The basic structure of basal lamina consists of different networks of triple-helical collagen IV, composed of alpha chains, and the major noncollagenous protein, laminin, which is a heterodimer of alpha, beta and gamma chains. The collagen network contains covalent cross-links; moreover, distinct networks are linked by another noncollagenous protein, nidogen (entactin). These major elements display several further functions: (i) they possess multiple sites binding other protein of basal lamina, (ii) they anchor components of reticular lamina to basal lamina, and (iii) they serve as ligands for membrane-associated receptors (i.e., integrins, dystroglycans, etc.), which interact with cytoskeleton [2]. Taken together, in the context of the mechanical function of skeletal muscle, the extracellular matrix may be considered as a series of networks that connect reticular lamina, basal lamina, sarcolemma, and cytoskeletal structure.

Overload of healthy skeletal muscle leads to myofiber hypertrophy and ECM remodeling, the processes that are thought to contribute to muscle growth. Several ECM components are controlled by the level of mechanical loading, and multiple intracellular proteins involved in mechanotransduction signaling are suggested, including focal adhesion kinase (FAK), paxillin, integrin-linked kinase (ILK), and mitogen-activated protein kinase (MAPK) [1]. The latter is crucial for the conversion of mechanical load to tissue adaptation, transmitting signaling from the cytosol to the nucleus. Laminin, integrin alpha7, and integrin-linked kinase (ILK) are all critical for mechanical stability of skeletal muscle [42]. ILK is recruited to the myotendinous junction, which requires the presence of laminin in the ECM and integrin alpha7 in sarcolemma. Moreover, ILK is essential for strengthening the adhesion of the muscle fibers with the ECM and acts with the dystrophin/dystroglycan adhesion complex in maintaining mechanical stability of skeletal muscles.

Endurance and resistance exercises accelerate the turnover of ECM components in skeletal muscle. Several studies reveal an increase in collagen synthesis and accumulation induced by exercise (summarized in [43]). Transcription of genes encoding types I, III, and IV collagen increases after endurance training. In another study, endurance exercise augments concentra‐ tion of type IV collagen in slow (soleus), but not in fast (rectus femoris) muscle. Matrix metalloproteinases are activated in human skeletal muscle in response to voluntary exercise, and the expression and time pattern indicate differences between the MMPs in regards of production sites as well as in the regulating mechanism. TIMPs are often activated together with MMPs in response to physical activity, indicating the simultaneous stimulation and inhibition of the ECM degradation. Probably, MMPs' activation precede TIMPs' activation, and the latter serve as "guardians" of degradation termination, providing limits in the ECM breakdown [1]. Levels of MMP-2, 14, and TIMP-1 mRNA in muscle tissue increase after 10 days of training. MMP-2 and 9 proteins were both present in the ECM, around myofibers and capillaries, but MMP-2 was also visible within the skeletal muscle fibers [44].

Mechanical loading induces the secretion of TGF-beta, PDGF, and bFGF in tendon fibroblasts; moreover, it increases the expression of collagen and other ECM components, such as proteo‐ glycans. TGF-beta stimulates collagen formation and reduces its degradation, also via activation of the TIMPs, together with a suppression of MMPs, leading to the ECM accumu‐ lation. TGF-beta is known to function as a modulator of ECM proteins and to induce both collagen gene activation and protein formation. In a human model of microdialysis of the Achilles tendon, both local and systemic levels of TGF-beta increase in response to 1 h of running, proving a release of this cytokine from tissues that are mechanically activated during exercise and suggesting a role in the response to mechanical loading in vivo [1]. Mechanical loading induces FGF release from skeletal muscle cells in vitro. Several isoforms of FGFs exist; of these, basic FGF (or FGF2) and, to a lesser extent, the acidic FGF (FGF1) stimulate fibroblast proliferation and collagen synthesis. Interleukin-6 (IL-6) is considered as a physical activityassociated myokine released from working muscles [45]. It can stimulate fibroblasts to increase the synthesis of collagens, glycosaminoglycans, hyaluronic acid, and chondroitin sulfates. Increased expression of IL-6 is necessary for the regulation of ECM remodeling during the hypertrophic response of skeletal muscle to overload [46]. Mechanical activity increases expression of IL-1beta in human and rabbit tendon cells, leading to increased MMPs activity, diminished collagen synthesis, and initiating tissue degradation and remodeling in response to loading. IGF-I is directly involved in skeletal muscle ECM synthesis after mechanical loading. This growth factor increases the expression of types I and III collagen in intramuscular fibroblasts. Bioavailability of IGF-I is controlled by IGF-binding proteins, and increased proteolysis of IGFBPs occurs in response to prolonged training in humans. Interestingly, MMPs can degrade IGFBPs, which provides a possible mechanism of regulation of the free IGF-I in skeletal muscle tissue and circulation.

The blood flow in skeletal muscle is tightly coupled with the metabolic demands of contracting myofibers. During exercise, local mechanisms cause rapid dilation of muscle arterioles to increase the flow of blood to the working muscle. It appears that fibronectin fibrils in the extracellular matrix transduce signals from actively shortening skeletal muscle fibers to local blood vessels to increase blood flow. Skeletal muscle contraction alters the conformation of ECM fibronectin, which results in transient exposure of specific matricryptic sites. These sequences are not exposed in the soluble form of ECM molecules, but may be expressed due to structural or conformational changes, providing "a reserve" of signaling sites activated during ECM remodeling. Matricryptic fibronectin sites (FNIII-1) interact with FNIII-1H receptors on smooth muscle cells and/or skeletal muscle fibers. This activates the neuronal nitrogen oxide (NO) synthase to release NO, which leads to smooth muscle relaxation, vasodilation, and increased blood flow. Thus, FNIII-1 sites in ECM fibronectin serve as important mechanical coupling between skeletal muscle contraction and arteriolar dilation [47]. **Figure 3** summarizes the cellular mechanisms activated during exercise leading to skeletal muscle ECM remodeling.

**Figure 3.** Proposed schema illustrating the mechanisms of alterations in the ECM in skeletal muscle induced by me‐ chanical loading. → means activation/stimulation, ┤means inhibition. Gray block arrows indicate total stimulation of particular processes resulting from the regulation of upstream pathways.
