**5. Neuromuscular disorders and the extracellular matrix**

A general myopathological parameter of a variety of acquired and inherited muscle diseases [115], as well as the gradual loss of contractile strength during the natural aging process [116], is the progressive accumulation of ECM components, especially collagens [7]. Inflammatory processes and tissue infiltration often accompany the loss of skeletal muscle fibres. Increased levels of non-contractile entities, such as fibrous connective and fatty tissue, within the neuromuscular system are a key pathological factor in the dysregulation of skeletal muscle function. Myofibrosis often correlates with poor motor outcome in neuromuscular disorders, such as the progressive loss of muscle strength and concomitant endomysial changes in the Xlinked inherited disorder Duchenne muscular dystrophy [117]. In muscle pathology, changes in ECM components can be differentiated as being a consequence of a primary defect in the matrisome of muscles, such as the ECM diseases Collagen IV myopathy [118,119] and LAMA2 related congenital myopathy [120,121], or a secondary response in the form of reactive myofibrosis, as is seen in dystrophinopathies [122,123].

The systematic profiling of changes in ECM components in Collagen IV myopathy and Xlinked muscular dystrophy has resulted in interesting new findings in relation to primary ECM defects versus reactive fibrosis. Mutations in the genes encoding collagen isoform COL VI are the underlying cause of the severe UCMD type of Ullrich congenital muscular dystrophy and the milder BM type of Bethlem myopathy. Both disorders are characterized by skeletal muscle wasting, cycles of cellular degeneration and regeneration, and the substitution of contractile fibres with fat and connective tissue [119]. The cell biological and proteomic analysis of mouse models and biopsy material from patients afflicted with Collagen IV myopathy revealed metabolic dysregulation, enhanced cellular stress, autophagic impairment and alterations in mechano-transduction signalling pathways [124,125]. In the case of X-linked muscular dystrophy, a large number of proteomic studies have surveyed secondary changes down‐

An interesting non-physiological system is presented by external chronic low-frequency stimulation of fast muscles. This electro-stimulation method causes the complete activation of all affected motor units to a maximum extent [108]. During fast-to-slow transitions, skeletal muscles show a remarkable adaptation and transform physiologically and biochemically into motor units with an improved resistance to fatigue [109]. Chronic low-frequency stimulated fast muscles are characterized by decreased fibre calibres, an increase in the time-to-peak twitch tension, an increase in half-relaxation time and a significant elevation of aerobicoxidative capacity [110]. The proteomic analysis of continuous electro-stimulation at 10 Hz has demonstrated complex biochemical changes with a significant shift from glycolytic to more aerobic-oxidative metabolism [111,112]. The ECM of transforming skeletal muscle undergoes distinct changes and exhibits increased collagen levels [113,114]. In the stimulated *latissimus dorsi* model for testing the suitability of dynamic cardiomyoplasty to treat heart failure, the collagen content was shown to be significantly elevated in the paced muscle. Although the chronically electro-stimulated muscle increased the level of fatigue resistance, distal regions of the paced *latissimus dorsi* muscle were characterized by muscular atrophy and myofibrosis

A general myopathological parameter of a variety of acquired and inherited muscle diseases [115], as well as the gradual loss of contractile strength during the natural aging process [116], is the progressive accumulation of ECM components, especially collagens [7]. Inflammatory processes and tissue infiltration often accompany the loss of skeletal muscle fibres. Increased levels of non-contractile entities, such as fibrous connective and fatty tissue, within the neuromuscular system are a key pathological factor in the dysregulation of skeletal muscle function. Myofibrosis often correlates with poor motor outcome in neuromuscular disorders, such as the progressive loss of muscle strength and concomitant endomysial changes in the Xlinked inherited disorder Duchenne muscular dystrophy [117]. In muscle pathology, changes in ECM components can be differentiated as being a consequence of a primary defect in the matrisome of muscles, such as the ECM diseases Collagen IV myopathy [118,119] and LAMA2 related congenital myopathy [120,121], or a secondary response in the form of reactive

The systematic profiling of changes in ECM components in Collagen IV myopathy and Xlinked muscular dystrophy has resulted in interesting new findings in relation to primary ECM defects versus reactive fibrosis. Mutations in the genes encoding collagen isoform COL VI are the underlying cause of the severe UCMD type of Ullrich congenital muscular dystrophy and the milder BM type of Bethlem myopathy. Both disorders are characterized by skeletal muscle wasting, cycles of cellular degeneration and regeneration, and the substitution of contractile fibres with fat and connective tissue [119]. The cell biological and proteomic analysis of mouse models and biopsy material from patients afflicted with Collagen IV myopathy revealed metabolic dysregulation, enhanced cellular stress, autophagic impairment and alterations in mechano-transduction signalling pathways [124,125]. In the case of X-linked muscular dystrophy, a large number of proteomic studies have surveyed secondary changes down‐

**5. Neuromuscular disorders and the extracellular matrix**

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

myofibrosis, as is seen in dystrophinopathies [122,123].

[114].

**Figure 5.** Overview of reactive myofibrosis in X-linked muscular dystrophy. The transforming growth factor TGF-β and the activity of matrix metalloproteinases (MMP) and tissue inhibitors (TIMPs) play a crucial role in ECM activa‐ tion and modulation, which is characterized by increased levels of various collagens, proteoglycans, dermatopontin and periostin. Progressive ECM accumulation triggers a chronic replacement of muscle fibres by fibrotic scar tissue leading to a loss of muscle elasticity and contractile strength.

stream of the primary abnormality in the membrane cytoskeletal protein dystrophin [126]. Muscular dystrophy-related changes affect energy metabolism, cellular signalling, the excitation–contraction–relaxation cycle, the stress response, the cytoskeletal network and the ECM [127]. The recent proteomic profiling of established genetic animal models of dystro‐ phinopathy has revealed a drastic increase in various collagens, proteoglycans and matricel‐ lular proteins [83–86,128,129]. **Figure 5** outlines the histo- and pathobiochemical consequences of muscular changes and a hyperactive connective tissue in dystrophin-deficient skeletal muscles.

The simultaneous mass spectrometric analysis of dystrophin isoform Dp427 and collagen in moderately dystrophic *mdx-4cv* leg muscles revealed significant increases in collagens and associated ECM proteins, such as fibronectin, biglycan, asporin, decorin, prolargin, mimecan and lumican [85]. The pathoproteomic signature of the severely dystrophic *mdx-4cv* diaphragm included a significant increase in collagens and the related ECM proteins asporin, decorin, dermatopontin, prolargin and periostin [83]. Especially interesting was the proteomic identi‐ fication of dermatopontin and periostin [83,128,129]. Dermatopontin, also named tyrosine-rich acidic matrix protein TRAMP [130], is involved in matrix assembly and cell–matrix interactions [17] via interactions with decorin, TGF-β and fibronectin [131]. High levels of dermatopontin in the dystrophic *mdx* diaphragm most likely reflect an increased demand for collagen matrix organization within Dp427-deficient fibres [128]. Periostin is a crucial matricellular protein of 93 kDa [18] that is involved in the regulation of the biomechanical properties of connective tissues and collagen fibrillogenesis [132]. Normally periostin is only temporally expressed in the muscle ECM during cellular differentiation and regeneration processes [133], making its drastic up-regulation a characteristic feature of dystrophic muscles [83,86]. Of diagnostic and therapeutic importance is the fact that muscle biopsies from Duchenne patients exhibit an elevated concentration of periostin and that the deletion of periostin clearly reduces dystrophic symptoms and myofibrosis in mice by modulating the TGF-β pathway [134]. Interestingly, laminin-deficient muscular dystrophy also shows dysregulation of matricellular proteins as an early pathophysiological feature [120,135]. Therefore altered levels of periostin and related matricellular proteins are good biomarker candidates for the characterization of myofibrosis in relation to inherited muscular dystrophies [136]. Although the natural aging process is also associated with increased collagen levels [116], which were also shown by proteomics [137], the collagen accumulation is much less pronounced in senescent muscles as compared to muscular dystrophy.
