**2. Molecular and cellular structure of the extracellular matrix**

In skeletal muscles, the ECM is involved in a variety of processes during development, contractile maturation, fibre regeneration following injury, physiological adaptations to changed functional demands and the natural aging process [8]. As outlined in **Figure 1**, the ECM is a highly dynamic non-cellular system that undergoes frequent cycles of modifications, degradation and reassembly. The muscle ECM functions on the one hand as an embedding and stabilizing structural support and on the other hand as a cellular interaction and signalling medium. The molecular lattice of collagens and proteoglycans with its associated matricellular proteins, enzyme systems and adhesion receptors mediates various physiological and biochemical mechanisms, including (i) the overall maintenance of muscle tissue stability and elasticity, (ii) the mechanical transduction of force from the contractile fibres to their anchoring tissues, (iii) cytoskeletal coupling to enable the efficient execution of frequent excitation– contraction–relaxation cycles, (iv) the provision of signalling pathways at the fibre periphery, (v) the preservation of neuromuscular homeostasis, and (vi) the physical scaffold and embed‐ ding medium of cellular constituents, such as contractile fibres, capillaries, motor neurons and satellite cell pools. Cell–matrix interactions are of central importance during cell adhesion and cell migration and thus essential during both embryonic and adult myogenesis.

issues related to high energy demand, elevated levels of cellular stress and enormous physi‐ cal strains during excitation–contraction–relaxation cycles [1]. The survival of skeletal muscle fibres therefore depends heavily on (i) a high degree of physiological adaptability, (ii) a unique level of tissue plasticity, (iii) efficient molecular chaperoning to prevent proteotoxic insults and (iv) a sophisticated repair machinery that can counter-act frequent cellular injuries [2]. A crucial stabilizing element that is intrinsically involved in this continuous maintenance of contractile tissues is the extracellular matrix (ECM) [3]. On the one hand, the complex layers of the muscle ECM provide the physical structure for force transmission between contracting fibres and their surrounding tissue environment [4], and on the other hand the ECM functions as an embed‐ ding medium for essential supportive components of muscles such as capillaries and motor

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

The composition and organization of the ECM adapts considerably in response to changed functional or structural demands during myogenesis, fibre maturation and exercise-induced changes [6]. During the natural aging process and in association with a variety of muscular disorders, a hyperactive connective tissue may trigger myofibrosis with a detrimental impact on muscle elasticity and fibre contractility [7]. Physiological or pathological changes in the muscle ECM frequently mirror the different phases of altered muscle structure and function [8]. The main component of the ECM is represented by collagen, which exits in a large number of isoforms that connect with proteoglycans, matricellular proteins and adhesion receptors to

This chapter provides an overview of the molecular components of the ECM from skeletal muscle and describes the proteomic concept of the ECM complexome. The formation, matu‐ ration and flexibility within the various layers of the ECM in developing, maturing and adapting skeletal muscles is outlined, as well as the crucial role of myofibrosis in neuromus‐

In skeletal muscles, the ECM is involved in a variety of processes during development, contractile maturation, fibre regeneration following injury, physiological adaptations to changed functional demands and the natural aging process [8]. As outlined in **Figure 1**, the ECM is a highly dynamic non-cellular system that undergoes frequent cycles of modifications, degradation and reassembly. The muscle ECM functions on the one hand as an embedding and stabilizing structural support and on the other hand as a cellular interaction and signalling medium. The molecular lattice of collagens and proteoglycans with its associated matricellular proteins, enzyme systems and adhesion receptors mediates various physiological and biochemical mechanisms, including (i) the overall maintenance of muscle tissue stability and elasticity, (ii) the mechanical transduction of force from the contractile fibres to their anchoring tissues, (iii) cytoskeletal coupling to enable the efficient execution of frequent excitation– contraction–relaxation cycles, (iv) the provision of signalling pathways at the fibre periphery, (v) the preservation of neuromuscular homeostasis, and (vi) the physical scaffold and embed‐

form an elaborate extracellular network and tight cell–matrix interactions [9].

**2. Molecular and cellular structure of the extracellular matrix**

neurons [5].

cular pathology.

**Figure 1.** Overview of the complex functions and interactions of the extracellular matrix from skeletal muscles.

The distinct layers of the ECM that surround muscle fibres, muscle fascicles and the entire skeletal muscle are formed by the endomysium, perimysium and epimysium, respectively [3]. **Figure 2** shows diagrammatically the arrangements of the ECM from skeletal muscles and lists the main molecular constituents, including various isoforms of collagen, proteoglycans, matricellular proteins, crosslinking proteins and matrix metalloproteinases. Crucial adhesion systems that maintain sarcolemma–matrix interactions are marked. They include the collagenlaminin-α/β-dystroglycan-dystrophin/utrophin axis and the collagen–fibronectin–integrin axis that link the basal lamina via the plasmalemma to the underlying membrane cytoskeleton [10].

The stabilizing linkage between the outside and inside of muscle cells, provided by the ECM– sarcolemma–cytoskeleton axis, is of critical importance for maintaining normal contractile functions [8]. Primary or secondary abnormalities in individual binding partners of these surface complexes may result in severe neuromuscular disorders, as discussed in below section on the role of the ECM in skeletal muscle pathology and myofibrosis.

Collagens form tight helical structures and function as the main structural protein species in the extracellular space. Muscle-associated collagens are highly abundant in the interstitial matrix, ECM microfibrils and the basal lamina [11]. Collagen isoform COL I, the most abundant protein in the mammalian body [12], is the primary collagen in the perimysium and tendon. The interstitial matrix contains mostly collagen COL I, COL III and COL V. Minor types of

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

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 the ECM glycoprotein fibronectin mediates the connection between laminin-211 (α2β1γ1 merosin) and collagen COL IV [15].

A variety of regulatory ECM proteins are involved in matrix assembly and the modulation of cell–matrix interactions, such as dermatopontin, nidogen/entactin, periostin (osteoblastspecific factor OSF-2) and osteopontin [16–18]. Matricellular proteins represent non-architec‐ tural ECM components and are crucial factors during muscle development and fibre repair. Matrix metalloproteinases are an important class of ECM-associated enzymes that regulate the degradation of ECM proteins and support tissue integrity during phases of collagen deposition and muscle regeneration [19]. The main isoforms present in skeletal muscles are matrix metalloproteinases MMP-1, MMP-2, MMP-9, MMP-10 and MMP-13 [20,21]. The tissue inhibitors of matrix metalloproteinases, named TIMP, are endogenous regulatory factors involved in the formation, adaptation and controlled degradation of the ECM [22]. TIMP molecules play a crucial role in the migration and differentiation of muscle stem cells during regeneration following cellular injury [19].

A large variety of proteoglycans fill the gaps between collagen molecules and thereby form an integral part of the highly complex ECM structure. Proteoglycan molecules are highly glycosylated with glycosaminoglycans at multiple sites along the peptide backbone [23]. Skeletal muscles contain small leucine-rich repeat proteoglycans (SLRP), heparan sulfate proteoglycans and chondroitin sulphate proteoglycans. Muscle-associated proteoglycans of the type SLRP are asporin, biglycan, decorin, mimecan (osteoglycin), fibromodulin and lumican. Asporin is mostly found in the cartilage matrix. Biglycan is a small SLRP-type proteoglycan that interacts with α-sarcoglycan and γ-sarcoglycan of the dystrophin-glyco‐ protein complex [24]. Decorin is the primary proteoglycan molecule of the perimysium and tendon structures [25]. Fibromodulin is involved in collagen fibril formation, which is illustrated by the biomedical fact that fibromodulin-deficient tendons exhibit abnormal collagen fibrils [26]. The heparan sulfate proteoglycan syndecan is transiently up-regulated during tissue differentiation and is involved in stem cell maintenance and muscle regeneration [27]. Perlecan is located to the basement membrane and its expression is also transiently increased during muscle differentiation [28]. The chondroitin sulphate proteoglycan named aggrecan forms large aggregates in cartilage [29]. A proline-arginine-rich end leucine-rich repeat protein (PRELP) is presented by prolargin of the basal lamina. At the highly differen‐ tiated neuromuscular junction region, the large proteoglycan molecule agrin is present and forms via α-dystroglycan a tight linkage to the utrophin-glycoprotein complex. Agrin is essential for the normal development of the neuromuscular junction and agrin-induced clustering processes are crucial for the anchoring of the acetylcholine receptor complex in the junctional folds [30].
