Extracellular Matrix Proteomics: The mdx-4cv Mouse Diaphragm as a Surrogate for Studying Myofibrosis in Dystrophinopathy

The progressive degeneration of the skeletal musculature in Duchenne muscular dystrophy is accompanied by reactive myofibrosis, fat substitution, and chronic inflammation. Fibrotic changes and reduced tissue elasticity correlate with the loss in motor function in this X-chromosomal disorder. Thus, although dystrophinopathies are due to primary abnormalities in the DMD gene causing the almost-complete absence of the cytoskeletal Dp427-M isoform of dystrophin in voluntary muscles, the excessive accumulation of extracellular matrix proteins presents a key histopathological hallmark of muscular dystrophy. Animal model research has been instrumental in the characterization of dystrophic muscles and has contributed to a better understanding of the complex pathogenesis of dystrophinopathies, the discovery of new disease biomarkers, and the testing of novel therapeutic strategies. In this article, we review how mass-spectrometry-based proteomics can be used to study changes in key components of the endomysium, perimysium, and epimysium, such as collagens, proteoglycans, matricellular proteins, and adhesion receptors. The mdx-4cv mouse diaphragm displays severe myofibrosis, making it an ideal model system for large-scale surveys of systematic alterations in the matrisome of dystrophic fibers. Novel biomarkers of myofibrosis can now be tested for their appropriateness in the preclinical and clinical setting as diagnostic, pharmacodynamic, prognostic, and/or therapeutic monitoring indicators.


Introduction
Animal model research plays an important role in the field of biomedicine and, particularly, in biomolecular investigations into the basic mechanisms that underlie the cellular pathogenesis of common human diseases [1]. In the case of monogenetic disorders, such as the X-chromosomally inherited muscle-wasting disease named Duchenne muscular dystrophy [2], the animal models should ideally (i) mimic a similar genotype that properly reflects the primary abnormality leading to the complex disease phenotype; (ii) show comparable patterns of disease onset, progression, and severity; (iii) display comparable secondary changes as observed in the human disorder such as multi-system alterations and pathobiochemical crosstalk; (iv) be characterized by complex pathophysiological features that resemble the disease in patients including myonecrosis and myofibrosis; (v) exhibit equivalent effects due to changes in energy metabolism, cellular signaling pathways, physiological adaptations, and natural repair mechanisms; (vi) trigger analogous responses of Figure 1. Overview of frequently used extracellular-matrix-enrichment-independent approaches for the bottom-up proteomic analysis of skeletal muscle specimens. Abbreviations used: ECM, extracellular matrix; FASP, filter-aided sample preparation; SCAD, surfactant-and-chaotropic-agent-assisted sequential extraction/on-pellet digestion; SDS, sodium dodecyl sulfate; SP3, single-pot solidphase-enhanced sample preparation; SPEED, detergent-free sample preparation by easy extraction and digestion; USP3, universal solid-phase protein preparation.  Overview of frequently used extracellular-matrix-enrichment-independent approaches for the bottom-up proteomic analysis of skeletal muscle specimens. Abbreviations used: ECM, extracellular matrix; FASP, filter-aided sample preparation; SCAD, surfactant-and-chaotropic-agent-assisted sequential extraction/on-pellet digestion; SDS, sodium dodecyl sulfate; SP3, single-pot solid-phase-enhanced sample preparation; SPEED, detergent-free sample preparation by easy extraction and digestion; USP3, universal solid-phase protein preparation.

Bottom-Up Proteomics versus Top-Down Proteomics
In principle, the main proteomic approaches used in the biological sciences can be categorized into two main types, bottom-up proteomics and top-down proteomics [77]. Bottom-up proteomics focuses on the large-scale identification of proteins using crude extracts as the starting material and peptide mass spectrometry for protein detection [78]. Top-down proteomics is usually based on the biochemical or gel electrophoretic separation of intact protein species which is then followed by the mass spectrometric analysis of intact proteoforms and their post-translational modifications [79]. An overview of the main techniques employed in skeletal muscle proteomics, including a description of the main analysis platforms, two-dimensional gel electrophoresis, liquid chromatography, antibody-based approaches, sample preparation, protein digestion protocols, mass spectrometry, data acquisition, single-cell analysis, and aptamer-based proteomics, has recently been published in the context of the proteomic profiling of the contractile apparatus of skeletal muscles [80]. Of note, single-cell proteomics is becoming increasingly important [81] and has been applied for studying individual muscle fibers [64,65]. Studying changes in specialized muscle fibers is crucial since fiber type shifting occurs in the skeletal musculature during physiological adaptations and in many neuromuscular disorders [82]. Figures 1 and 2 provide an overview of commonly used methods for the bottom-up or top-down proteomic analysis of crude protein extracts from total muscle tissue specimens without the prior usage of extensive enrichment processes [79,83].

Sample Handling and Protein Extraction for Gel-Free and Bottom-Up Proteomics
As summarized in Figure 1, efficient protein extraction for bottom-up muscle proteomics can be performed by various direct sample preparation techniques [83][84][85], such as the widely used detergent-based and filter-aided sample preparation (FASP) method [86,87], enhanced eFASP using alternative detergents [88], sample processing from cell lysis through the elution of purified peptides by the In-StageTip (iST) method [89,90], the

Sample Handling and Protein Extraction for Gel-Free and Bottom-Up Proteomics
As summarized in Figure 1, efficient protein extraction for bottom-up muscle proteomics can be performed by various direct sample preparation techniques [83][84][85], such as the widely used detergent-based and filter-aided sample preparation (FASP) method [86,87], enhanced eFASP using alternative detergents [88], sample processing from cell lysis through the elution of purified peptides by the In-StageTip (iST) method [89,90], the single-pot solid-phase-enhanced sample preparation (SP3) method [91], the universal solid-phase protein preparation (USP3) method [92], pressure-cycling technology (PCT) [93], surfactantand-chaotropic-agent-assisted sequential extraction/on-pellet digestion (SCAD) [94], or by detergent-free sample preparation by easy extraction and digestion (SPEED) [95]. A comprehensive description of a standardized bottom-up proteomic workflow, incorporating the FASP method, has recently been described in detail [96].

Sample Handling and Protein Extraction for Gel-Based and Top-Down Proteomics
Although the majority of mass spectrometric surveys are carried out with peptides and proteins that are isolated by gel-free systems, top-down proteomics routinely employs one-dimensional or two-dimensional gel electrophoretic techniques, or biochemical isolation methods such as affinity chromatography, for the preparation of intact proteoforms. As summarized in Figure 2, gel-based top-down proteomics can be based on GeLC-MS/MS using one-dimensional gradient gels in combination with liquid chromatography [97][98][99] or classical two-dimensional gel electrophoresis using isoelectric focusing in the first dimension and slab gel electrophoresis in the second dimension [100][101][102]. A gel-based method that has overcome the technical limitation of gel-to-gel variations is represented by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) [103][104][105]. This advanced gel technique employs differential proteome tagging with the CyDyes Cy2, Cy3, and Cy5 in combination with various software analysis packages to determine protein changes in comparative proteomics [79].

Extracellular-Matrix-Enrichment-Based Proteomic-Profiling Approaches
The main enrichment strategies for mass spectrometric studies of the isolated extracellular matrix are summarized in Figure 3, including both sequential depletion methods and a list of decellularization methods. The matrisome can be divided into components that belong to the insoluble versus the soluble extracellular matrix, often referred to as the iECM versus the sECM [74][75][76]. Sequential depletion approaches focus on the isolation of an insoluble pellet consisting of key extracellular matrix proteins via the systematic removal of soluble protein species by various washing steps. Commercially available 'Compartmental Protein Extraction' kits represent a convenient way to carry out the sequential solubilization and extraction of major subcellular fractions, such as the cytosol, nucleus, membranes, and cytoskeletal networks, and thereby enrich the iECM part of the matrisome as an insoluble pellet [75,106]. In contrast, the sECM can be prepared by a 'high salt' method that aims to isolate the chaotropic-agent-soluble matrisome. High-salt buffers are employed to remove the soluble protein fraction, followed by extracellular matrix extraction using chaotropic reagents [75,107]. Quantitative detergent solubility profiling (QDSP) is based on the sequential treatment with buffers of increasing detergent and salt concentration and harvesting of the matrisome as an insoluble pellet [75,108]. An interesting approach is the usage of the photo-cleavable surfactant 4-hexylphenylazosulfonate (Azo). Following treatment with the nonionic detergent Triton X-100 to produce an insoluble pellet, the Azo detergent is then used for the specific solubilization and isolation of the extracellular matrix fraction in association with the ultraviolet irradiation of Azo [75,109,110]. The 'three-step extraction' method is a sequential extraction process that is based on a high-salt step, a detergent solubilization step, and a final chaotropic-agent-based solubilization of the matrisome fraction [75,111].
Decellularization methods to treat tissue samples usually start with the disruption and removal of cellular material using various biochemical, chemical, or physical techniques, followed then by the isolation of the mostly insoluble extracellular matrix fraction [74][75][76]. Freeze-thaw cycles, agitation, pressurization, sonication, and osmotic pressure are typical methods used for cellular disruption. Biochemical approaches used for decellularization often use chemicals such as SDS, Triton X-100, and CHAPS, followed by the treatment of the insoluble matrisome with chaotropic agents, including high concentrations of urea, thiourea, or guanidine hydrochloride [111][112][113]. Prior to peptide mass spectrometry, the enriched extracellular matrix fraction is often digested by trypsin and LysC, but also with the help of cyanogen bromide or hydroxylamine [74][75][76].

Mass Spectrometry and Data Acquisition
The identification and characterization of individual proteoforms in crude tissue samples, subcellular fractions, enriched preparations of the matrisome, or isolated protein assemblies can be carried out by a variety of mass spectrometric methods [114][115][116][117], such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) [115,118] or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [114,119]. Both labelling and label-free approaches are routinely used for the detection of protein species. The bioanalytical advantages versus technical limitation of these frequently used methods have been reviewed in detail [120]. Stable heavy isotopes can be incorporated into peptides/proteins using chemical or metabolic labelling and then be used for the quantitation of protein abundance [121]. Examples of well-established proteomiclabelling methods are stable isotope labelling by amino acids in cell culture (SILAC) [122], the tandem mass tagging (TMT) technique [123], the isotope-coded affinity tagging (ICAT) approach [124], and the isobaric tagging for relative and absolute quantitation (iTRAQ) technique [125]. Alternatively, mass spectrometric analyses can be performed with labelfree quantitation techniques that focus on features from fragment-ion analysis (MS2), such as spectral counting, or measure the integrated peak intensity from the parent-ion mass analysis (MS1) [126,127]. Of note, the application of artificial intelligence (AI), machine Biomolecules 2023, 13, 1108 7 of 35 learning (ML), and deep learning (DL) algorithms has been increasingly used to evaluate complex spectral data [128].
Optimized data acquisition plays a key role in mass spectrometric analyses. This can be achieved by data-dependent acquisition (DDA) [129], data-independent acquisition (DIA) [130], or targeted data acquisition (TDA) [131]. In the context of DIA, the sequential window acquisition of all theoretical mass spectra mass spectrometry (SWATH-MS) method, which divides the mass range into small mass windows, has been successfully applied to studying extracellular matrix proteins [132]. Immunoprecipitation combined with SWATH-MS was successfully employed to investigate fukutin-related protein (FKRP)associated dystroglycanopathy [133]. This methodology revealed that the loss of FKRP function disrupts the localization and tethering of fibronectin to myosin-10, essential for fibronectin maturation and subsequent collagen binding. The evidence accumulated demonstrates the utility of combining SWATH-MS and enrichment methods to investigate distinct phenotypes associated with muscular disorders.
Important methods for focusing on the abundance of target peptides include selected/multiple reaction monitoring (SRM/MRM) [134] or parallel reaction monitoring (PRM) [135], which are based on the measurement of precursor peptides in predefined m/z ranges taking into account retention time. The MRM-based measurement of targeted peptides was recently applied for the detailed analysis of extracellular matrix proteins [136]. Importantly, the absolute quantification (AQUA) workflow involves the usage of stableisotope-labelled peptides that are spiked into the samples at predetermined concentrations and are then quantified simultaneously with the corresponding endogenous peptides. This greatly facilitates better accuracy, sensitivity, and reproducibility of protein detection [137].

The Extracellular Matrix of Skeletal Muscles
The extracellular matrix of skeletal muscle fibers plays a key role in the provision of force transmission and the external support for the continued stabilization, maintenance, and repair of contractile fibers [30][31][32], which is reflected by complex structure-function relationships [138,139]. Importantly, the processes that regulate and support cell adhesion and cell migration during development, wound healing, and regeneration involve extensive cell-matrix interactions, which warrants detailed omics-type studies into the composition of the matrisome. In the field of proteomics including extracellular matrix proteomics, it is important to stress the now generally accepted concept of the proteoform being the basic unit of the dynamic proteome [140,141].
Thus, the detailed biochemical information on individual proteoforms and their posttranslational modifications (PTMs), as detected by mass spectrometry, should be used to establish detailed proteomic maps of the matrisome. Importantly, PTM profiles of extracellular matrix proteins can provide important biochemical characteristics for detecting and understanding the complex pathways associated with skeletal muscle physiology and pathophysiology. Mounting evidence exists showing how the existence of various PTMs are essential for matrisomal protein secretion and functionality within the extracellular milieu. The thrombospondin type-1 domain (TSR) is a common molecular feature present in the thrombospondin family, where O-fucosylation has a crucial stabilizing effect on the TSR structure [142]. Methods for detecting hydroxylation and glycosylation sites in collagen IV have previously been documented [143]. Such methods are important steps for understanding the role of specific PTMs in health and disease, as hydroxylation is essential for the stability of collagen molecules, and associated fibril structures. Disulfide bonds, assembled through the action of protein disulfide isomerases (PDIs), are found in thrombospondins and fibronectin, playing important roles in the establishment of subunit stability [144]. Citrullination, in which the conversion of the amino acid arginine into the amino acid citrulline is established, is associated with several extracellular matrix proteins, including fibronectin, having important roles in protein clustering, focal adhesion stability, and signaling [145]. Table 1 lists the representative protein hit list using the standardized mass spectrometric identification of proteins belonging to the various layers of the extracellular matrix from wild-type mouse diaphragm muscle [37,146]. This proteomic analysis of matrisomal proteins was carried out by bottom-up proteomics [96] incorporating the detergent-based and filter-aided sample preparation (FASP) method [86,87]. The proteins listed in Table 1 cover proteoforms that are found throughout the endomysium, perimysium, and epimysium, as well as protein species that are enriched in the basal lamina, myotendinous junctions, tendons, and cartilage [147]. Major members of the proteoglycan family, matricellular proteins, and adhesion receptors of the sarcolemma were clearly identified by proteomics [17,37,146].  [96,146] with the help of the detergent-based and filter-aided sample preparation (FASP) method [86,87]. Proteins were selected on high-confidence amino acid sequence coverage with a peptide number of 2 and above for sequence recognition. Listed are the accession number of detected proteins, the names of identified extracellular matrix proteins, the corresponding gene name, the percentage sequence coverage, the number of identified peptides, and the predicted molecular mass in kDa.
Load bearing, fiber stretching, and continuous cycles of excitation, contraction, and relaxation put enormous physical strain on individual skeletal muscles. The surrounding layers of the extracellular matrix protect the muscular system from excess damage. The physical scaffolds provided by collagens and proteoglycans act as a supporting structure that embeds individual motor units with their motor neurons and corresponding muscle fibers, as well as capillaries and satellite cells [30][31][32]. Of special importance for the integrity of the sarcolemma is the linkage between the intracellular cytoskeleton and the extracellular basement membrane. Listed in Table 1 as a key component of the basal lamina is the heterotrimeric glycoprotein named laminin-211 [147], a multi-functional member of the large laminin family of basement membrane proteins [148]. Laminin-211, whose name refers to its composition consisting of the merosin alpha-2 (LAMA2) subunit, the beta-1 (LAMB1) subunit, and the gamma-1 (LAMC1) subunit, was shown to be involved in fiber stabilization and cell adhesion, as well as cell differentiation and proliferation mechanisms [148]. Laminin-211 can interact with collagens, integrins, the dystroglycan complex, nidogen, and heparan sulfate at the level of the plasma membrane [149] and also plays a central role in the organization of the neuromuscular junction [150].
An important binding partner for the laminin complex is the heparan sulfate proteoglycan named agrin, which mediates the aggregation of the nicotinic acetylcholine receptors at the post-synaptic structures during synaptogenesis [151]. This extracellular matrix component of the neuromuscular junction was identified by mass spectrometry in crude muscle extracts (accession number A2ASQ1-3; Agrn gene; 2 peptides; 1.6% sequence coverage; 198.2 kDa molecular mass) despite its low abundance. Additional basal-laminaenriched extracellular matrix proteins were detected as collagen isoforms IV (COL4A1), XV (COL15A1), and XVIII (COL18A1) [152], perlecan (HSPG-2) [153], and the nidogens named entactin (NID1) [154] and osteonidogen (NID2) [155], as listed in Table 1. Collagen IV is a major member of the muscle collagen family [156] and is highly enriched in the basal lamina [157]. Perlecan of 398 kDa is an abundant component of the basal lamina and also known as the basement-membrane-specific heparan sulfate proteoglycan core protein (HSPG-2) [153]. The nidogens are intrinsically involved in the assembly of the basal lamina and provide a linkage between the laminin complexes and the collagen IV system, as well as perlecan in the basement membrane [156,157].
Major sarcolemmal adhesion proteins that provide cell-matrix linkages are the alpha-7/beta-1 integrin (ITGA7, ITGB1) complex and the dystroglycan (alpha/beta-DAG1) complex, which are both involved in force transmission and signaling mechanisms. See Table 1 for the proteomic identification of these sarcolemmal protein receptors. The alpha-7/beta-1 integrins form an abundant trans-sarcolemmal adhesion site at costameres and myotendious junctions that links laminin and fibronectin of the extracellular matrix to the intracellular cytoskeleton [164]. The dystroglycans form the core of the sarcolemmal dystrophinglycoprotein complex and mediate the linkage between laminin and cortical actin [165][166][167] via tight interactions between the laminin-binding protein alpha-dystroglycan [168], the integral glycoprotein beta-dystroglycan [169], and the actin-binding domain of the Dp427-M isoform of the membrane cytoskeletal protein dystrophin [20]. The organization of the various layers of the extracellular matrix and the distribution of individual protein components is diagrammatically summarized in Figure 4.
Major sarcolemmal adhesion proteins that provide cell-matrix linkages are the alpha-7/beta-1 integrin (ITGA7, ITGB1) complex and the dystroglycan (alpha/beta-DAG1) complex, which are both involved in force transmission and signaling mechanisms. See Table 1 for the proteomic identification of these sarcolemmal protein receptors. The alpha-7/beta-1 integrins form an abundant trans-sarcolemmal adhesion site at costameres and myotendious junctions that links laminin and fibronectin of the extracellular matrix to the intracellular cytoskeleton [164]. The dystroglycans form the core of the sarcolemmal dystrophin-glycoprotein complex and mediate the linkage between laminin and cortical actin [165][166][167] via tight interactions between the laminin-binding protein alpha-dystroglycan [168], the integral glycoprotein beta-dystroglycan [169], and the actin-binding domain of the Dp427-M isoform of the membrane cytoskeletal protein dystrophin [20]. The organization of the various layers of the extracellular matrix and the distribution of individual protein components is diagrammatically summarized in Figure 4. Myotendinous-junction-enriched proteins listed in Table 1 are collagen XII (COL12A1) and FERM-domain-containing kindlin 2 (FERMT-2) [170][171][172], and the abundant collagens of the tendon are represented by the microfibrillar isoform collagen I (COL1A1, COL1A2) [173]. The group of cartilage-enriched proteins was detected in the The above-listed components of the extracellular matrix form a highly adaptable, dynamic, and acellular microenvironmental system that supports, embeds, and stabilizes skeletal muscle fibers and their physiological functions [177]. The complex meshwork of extracellular laminins, non-fibrillar collagens, collagen microfibrils, proteoglycans, matricellular proteins, and regulating enzymes, and their linkage to cell-associated adhesion receptors in the sarcolemma membrane, is involved in large numbers of cell biological processes in contractile tissues, including:

•
The stabilization of skeletal muscle fibers, and the provision of tissue strength and elasticity; • Structural support via physical scaffolding, and the provision of an embedding medium for motor units, capillaries, and pools of satellite cells; • The provision of neuromuscular homeostasis; • Mechanical force transduction from muscle fibers to the surrounding tissues; • The provision of cell-matrix interactions for the support of cell adhesion and cell migration during embryonic myogenesis, adult myogenesis, and tissue repair; • Cell-matrix support during cell differentiation, maturation, remodeling, fiber transitions, and muscle aging.
The bioinformatic STRING analysis [178] of the muscle matrisome is shown in Figure 5, which was carried out with the proteins listed in Table 1 above. The interaction map illustrates the close protein-protein linkage patterns within the extracellular matrix of skeletal muscles.
The above-listed components of the extracellular matrix form a highly adaptable, dynamic, and acellular microenvironmental system that supports, embeds, and stabilizes skeletal muscle fibers and their physiological functions [177]. The complex meshwork of extracellular laminins, non-fibrillar collagens, collagen microfibrils, proteoglycans, matricellular proteins, and regulating enzymes, and their linkage to cell-associated adhesion receptors in the sarcolemma membrane, is involved in large numbers of cell biological processes in contractile tissues, including: • The stabilization of skeletal muscle fibers, and the provision of tissue strength and elasticity; • Structural support via physical scaffolding, and the provision of an embedding medium for motor units, capillaries, and pools of satellite cells; • The provision of neuromuscular homeostasis; • Mechanical force transduction from muscle fibers to the surrounding tissues; • The provision of cell-matrix interactions for the support of cell adhesion and cell migration during embryonic myogenesis, adult myogenesis, and tissue repair; • Cell-matrix support during cell differentiation, maturation, remodeling, fiber transitions, and muscle aging.
The bioinformatic STRING analysis [178] of the muscle matrisome is shown in Figure  5, which was carried out with the proteins listed in Table 1 above. The interaction map illustrates the close protein-protein linkage patterns within the extracellular matrix of skeletal muscles.   with the following parameters: (i) full STRING network analysis where the edges indicate both functional and physical protein associations; (ii) full set of active interaction sources consisting of text mining, experiments, databases, co-expression, neighborhood, fusion, and co-occurrence; (iii) network edges using confidence whereby line thickness indicates the strength of data support; and (iv) network display mode with interactive scalable vector graphic. Abbreviations used: ECM, extracellular matrix.

Duchenne Muscular Dystrophy and Fibrosis
The most severe form of dystrophinopathy, Duchenne muscular dystrophy, is a primary-muscle-wasting disorder of early childhood [179] with a prevalence of approximately 1 in 5000 live male births [180], which is triggered by a variety of genetic abnormalities in the X-chromosomal DMD gene [181]. Initial clinical signs include developmental delays in Duchenne patients with proximal muscle weakness, temporal and spatial gait variations, decreased walking speed, and Gower's sign [182][183][184]. Characteristic patterns of toe walking and difficulties with climbing stairs progress at more advanced stages of the disease towards respiratory insufficiency, cardiomyopathy, scoliosis, and severe limitations in general mobility [185][186][187][188][189][190], which eventually results in the loss of unassisted ambulation and upper body weakness [191]. Approximately one-fifth of Duchenne patients suffer from intellectual developmental disorder that can be associated with learning difficulties, behavioral deficits, emotional problems, attention deficits, impaired language and speech development, cognitive deficiencies, and mental retardation [192][193][194]. Of note, secondary multi-system abnormalities in dystrophinopathy negatively affect whole-body homeostasis, including the proper functioning of the kidneys, the bladder, the gastrointestinal tract, and the liver [19].
Detailed descriptions of crucial aspects of dystrophinopathy can be found in extensive review articles that focus on the epidemiology [180], X-linked genetics [2], cellular pathogenesis [179], differential diagnosis [195], rehabilitation strategies [196], and emergency management [197] of Duchenne muscular dystrophy. Following the identification of the DMD gene and its involvement in X-linked Duchenne muscular dystrophy [198][199][200] and the discovery of the full-length muscle dystrophin protein (Dp427-M) and its many isoforms (Dp427-B, Dp427-P, Dp260-R, Dp140-B/K, Dp116-S, Dp71, and Dp45) [18,[201][202][203], detailed biochemical, biophysical, and physiological studies have established that full-length dystrophin acts as a sub-sarcolemmal anchoring node that is involved in cytoskeletal organization, the provision of lateral force transmission, the maintenance of muscle fiber stability, and the support and integration of cellular signaling mechanisms in skeletal muscles [20]. The loss of the dystrophin isoform Dp427-M destabilizes the entire dystrophin-associated glycoprotein complex and causes a drastic reduction in the core dystroglycan sub-complex at the sarcolemma membrane, as well as lower levels of sarcoglycans, sarcospan, dystrobrevins, and syntrophins [204]. The dystrophin-deficient muscle membrane system was shown to be more prone to contraction-induced rupturing and this, in turn, results in a pathophysiological Ca 2+ influx into muscle fibers. The chronically elevated Ca 2+ concentration increases the rate of calpain-mediated proteolysis and tissue destruction [22][23][24].
Dystrophic skeletal muscles are characterized by highly progressive myonecrosis [19] and chronic inflammation [27], which is accompanied by reactive myofibrosis [17,26,37,50]. On the histological level, the drastic decline in muscle strength is characterized by fiber branching, alterations in fiber diameter, roundly shaped myofibers, hypercontractility, a high level of central myonucleation, intra-muscular fatty accumulation, muscle infiltration by inflammatory cells, and high levels of collagen deposition. In general, muscledegeneration-induced myofibrosis is usually characterized by increased mechanical stiffness in contractile fibers with extensive levels of fibrillar disorganization and drastically increased deposition of collagens, as well as high levels of myofibroblasts and macrophages [25][26][27]205]. The histopathological changes that result in endomysial fibrosis reflect these changes and they correlate with the loss in motor function in Duchenne patients [54], making the degree of reactive myofibrosis a useful indicator of disease progression in dystrophinopathy [17]. A variety of muscle-associated cell types, including resident fibroblasts, the important category of fibro-adipogenic progenitors, inflammatory cells, and pericytes, can be activated to transform towards the myofibroblast phenotype [33][34][35]. This complex network of activated cells and drastically increased deposition of extracellular matrix components reflects the pathophysiological impact of reactive myofibrosis on the cellular pathogenesis of Duchenne muscular dystrophy [11]. This gives the development of novel anti-fibrotic pharmacotherapies a central position in the design of new treatment options to counteract dystrophinopathy-associated muscle pathology [28,[206][207][208][209].

Characterization of the mdx-4cv Mouse within the Context of Other Dystrophic Models
Both spontaneous and bioengineered mouse models that lack the dystrophin protein isoform Dp427-M have been instrumental in the detailed elucidation of the molecular and cellular pathogenesis of Duchenne muscular dystrophy, as well as the evaluation of experimental treatment strategies [210][211][212]. A comparative listing of major genetic mouse models of dystrophinopathy is provided in Table 2. This includes the original mdx-23 mouse compared to a variety of modified models including mdx/Dtna, mdx/Cmah, mdx/Utr, mdx/α7, mdx/Myod1, mdx-2cv, mdx-3cv, mdx-4cv, mdx-4cv/mTR-G2, mdx-5cv, mdx-52, mdx-βgeo, Dmd-null, hDMD/mdx-45, and hDMD/mdx-52. The table lists information on the genetic abnormalities that characterize the various mouse models and the observed severity of the dystrophic phenotype in individual models of Duchenne muscular dystrophy. Importantly, Table 2 gives an overview of the effect of mutations on the expression of distinct dystrophin isoforms ranging from Dp71 to Dp427. Depending on the genetic abnormality within the Dmd gene, impaired expression patterns might only cause the loss of the full-length dystrophins Dp427-M, Dp427-B, and Dp427-P, or also additional effects on the tissue-specific density of the shorter dystrophins Dp260-R, Dp140-B/K, Dp116-S, Dp71, and/or Dp45. Detailed comparisons of dystrophic mouse models have been carried out in recent reviews [213][214][215].
Compared to the onset of dystrophic changes in the skeletal musculature at approximately 1 month of age in the mdx-23 mouse and a maximum lifespan of approximately 2 years, the modified mdx-type mice differ considerably in the onset of histopathological changes, ranging from 2 to 8 weeks, and their longevity, ranging in lifespan from 1 to 23 months [216]. Both the spontaneously mutated mdx-23 mouse and the chemically mutated mdx-4cv mouse have comparable survival rates and similar onsets of moderate hindlimb degeneration, as judged by histopathology, at 3-4 weeks of age in association with physiological dysfunction such as reduced muscle force. The mdx-23 mouse is characterized by extensive cycles of degeneration and regeneration from 3 to 8 weeks, illustrated by high levels of central myonucleation, and some stabilization with age [38][39][40]. The mdx-4cv musculature shows large variation in skeletal muscle fiber sizes. In both model systems, the dystrophin-deficient diaphragm is severely affected starting at 3-4 weeks of age and shows high levels of reactive myofibrosis during aging [213][214][215]. Analogous to the skeletal musculature, the reduced presence of the cardiac dystrophin-glycoprotein complex causes sarcolemmal disintegration, myonecrosis, fatty tissue replacement, fibrotic scarring, and interstitial inflammation in the mdx-type heart [217]. The onset of cardiomyopathy is at approximately 6-9 months of age and associated with considerable proteome-wide changes during aging [218].
The range of modified mdx-type mouse models of dystrophinopathy with usually more severe phenotypes includes double mutants that also affect, besides the dystrophin levels, the expression of proteins such as the dystrophin-associated component dystrobrevin, the autosomal dystrophin homologue utrophin, myogenic differentiation protein MOD1, and α7-integrin [213][214][215][216]. In addition to the most frequently used murine models with their rapid disease progression, large animal models of Duchenne muscular dystrophy exist in the form of canine and porcine mutants [5,[219][220][221]. The Golden Retriever muscular dystrophy (GRMD) model exhibits a splice site mutation in intron-6, which causes the skipping of exon-7 and a resulting out-of-frame DMD transcript in exon-8 [220]. The complexity and progression of the clinical syndrome is severe in the GRMD model including cardiomyopathic complications, making dystrophic dogs good model systems for translational studies [222,223]. The porcine model of dystrophinopathy (DMD pig) is characterized by a deletion in exon-52 of the DMD gene and exhibits progressive cardiomyopathy [224]. In conjunction with mouse models, larger animals can be utilized to study pathophysiological mechanisms, validate the suitability and robustness of novel biomarker candidates, and be helpful during the preclinical testing phase of new therapeutic approaches such as gene editing [219,221]. Large disease-model animals are highly useful for facilitating the efficient translation of novel diagnostic methods to the clinical setting, such as imaging technology for the monitoring of myofibrosis [225]. Skeletal muscle fibrosis is seen in different mdx-type muscles to varying degrees [46][47][48]226], with the diaphragm being the most structurally affected and fibrotic muscle tissue [49][50][51][52]227,228]. However, one of the most widely employed dystrophic animal models, the mdx-23 mouse [38], exhibits a relatively high frequency of revertant fibers [39] and a relatively mild dystrophic phenotype [40]. In contrast, the chemically mutated mdx-4cv mouse which harbors a nonsense mutation in exon-53 [41][42][43] has a drastically reduced number of dystrophin-positive revertants [45]. Figure 6 provides a comparison of the main genetic, biochemical, physiological, and histological features of muscular abnormalities in Duchenne muscular dystrophy versus the widely used mdx-23 and mdx-4cv mouse models of dystrophinopathy. Although the mdx-4cv mouse with a nonsense point mutation that introduces a ature stop codon in exon-53 [41] does not reflect the genetic heterogeneity seen in cohorts of human Duchenne patients [2], consisting of a variety of splice site muta missense point mutations, nonsense point mutations, large deletions, large duplica small deletions, small duplications, and mid-intronic mutations [181], the pathobioc ical down-stream effects due to dystrophin deficiency are relatively comparable. T most-complete absence of the Dp427-M isoform of full-length dystrophin in skeletal cle fibers initiates the reduced anchoring of the alpha/beta-dystroglycan subcompl pha/beta/gamma/delta-sarcoglycans, sarcospan, syntrophins, and dystrobr [146,[229][230][231]. The collapse of the dystrophin-associated glycoprotein complex rende muscle plasma membrane more susceptible to contraction-induced rupturing follow the chronic influx of Ca 2+ ions both through the leaky sarcolemma and Ca 2+ -leak cha The resulting elevation of sarcosolic Ca 2+ levels and concomitant impairment of t minal Ca 2+ -buffering capacity of the sarcoplasmic reticulum negatively affect the fin ulation of the excitation-contraction coupling mechanism and overwhelm the Ca 2+sion system of damaged muscle fibers resulting in Ca 2+ -dependent proteolysis [22-24 histopathological consequences of the interplay between progressive fiber degene Figure 6. Comparative overview of the main features of Duchenne muscular dystrophy versus the mdx-23 and the mdx-4cv models of dystrophinopathy that are due to a spontaneous mutation versus being generated by chemical mutagenesis, respectively. Analogous to Duchenne patients, the skeletal musculature of both the mdx-23 and the mdx-4cv mouse harbor mutations in the Dmd gene, and exhibit progressive myonecrosis and chronic inflammation in combination with reactive myofibrosis, which is especially prominent in the aged diaphragm muscle. In contrast to the spontaneous mdx-23 mouse, the mdx-4cv skeletal musculature is characterized by a low frequency of dystrophin-positive revertant fibers. Abbreviations used: DMD, Duchenne muscular dystrophy; ECM, extracellular matrix; ENU, N-ethyl-N-nitrosourea.
Although the mdx-4cv mouse with a nonsense point mutation that introduces a premature stop codon in exon-53 [41] does not reflect the genetic heterogeneity seen in large cohorts of human Duchenne patients [2], consisting of a variety of splice site mutations, missense point mutations, nonsense point mutations, large deletions, large duplications, small deletions, small duplications, and mid-intronic mutations [181], the pathobiochemical down-stream effects due to dystrophin deficiency are relatively comparable. The almost-complete absence of the Dp427-M isoform of full-length dystrophin in skeletal muscle fibers initiates the reduced anchoring of the alpha/beta-dystroglycan subcomplex, alpha/beta/gamma/delta-sarcoglycans, sarcospan, syntrophins, and dystrobrevins [146,[229][230][231]. The collapse of the dystrophin-associated glycoprotein complex renders the muscle plasma membrane more susceptible to contraction-induced rupturing followed by the chronic influx of Ca 2+ ions both through the leaky sarcolemma and Ca 2+ -leak channels. The resulting elevation of sarcosolic Ca 2+ levels and concomitant impairment of the luminal Ca 2+ -buffering capacity of the sarcoplasmic reticulum negatively affect the fine regulation of the excitation-contraction coupling mechanism and overwhelm the Ca 2+extrusion system of damaged muscle fibers resulting in Ca 2+ -dependent proteolysis [22][23][24]. The histopathological consequences of the interplay between progressive fiber degeneration and abnormal ion homeostasis causes increased levels of inflammatory cells, impaired myoblast cell survival, central nucleation in mature fibers due to ongoing cycles of degeneration and regeneration, and increased levels of extracellular proteins [52,229,232]. One histopathological feature that is not observed in mdx-4cv muscles, compared to dystrophic skeletal muscles from both Duchenne and Becker's muscular dystrophy patients [233][234][235][236], are high levels of fat substitution.

Experimental Focus Bioanalytical Approach Major Findings References
(i) Generation, genotyping, and phenotyping of the mdx-4cv model Generation of mdx-4cv model Chemical mutagenesis with N-ethyl-N-nitrosourea (ENU) Induction of a C-to-T transition at position 7916 in exon-53 of the Dmd gene leading to premature translation termination [41] Genotyping of mdx-4cv model DNA sequencing, polymerase chain reaction analysis Protein-truncating nonsense mutation that introduces premature stop codon in exon-53 of Dmd gene [42][43][44]237] Characterization of mdx-4cv model Cell biological and biochemical analyses Less revertant fibers compared to spontaneous mdx-23 mouse; reduced level of dystrophin-glycoprotein complex [231] (ii) Evaluation of experimental therapies using the mdx-4cv model Exon-skipping therapy Antisense molecule-based skipping of defective exon Removal of exon-53 by-passed the protein-truncating mutation and restored the synthesis of semi-functional Dp427-M protein [238] Micro-dystrophin gene therapy Virus-or nano-carrier mediated delivery of micro-dystrophin gene Increased numbers of micro-dystrophin positive fibers using a variety of delivery mechanisms [239][240][241][242][243] Cell-based therapy Cell transplantation to introduce mini-dystrophin protein in dystrophic fibers High levels of mini-dystrophin expression, but no physiological improvements [244][245][246]

Histological and Biochemical Characterization of Fibrosis in the mdx-4cv Mouse Diaphragm
The diaphragm muscle is an important type of tissue for the focused analysis of pathophysiological changes since it is majorly affected in Duchenne patients that suffer from impaired cardiorespiratory function. A reduced thoracic cavity area and altered chest wall contraction patterns have been established to occur during cycles of inspiration and expiration in dystrophic respiratory muscles by imaging studies [276]. Severe diaphragmatic dysfunction, excessive fat infiltration in respiratory muscles, and cardiomyopathic complications occur mostly in the second decade of life in inherited muscular dystrophy, which frequently necessitates both the management of respiratory insufficiency by mechanical ventilatory support [277] and targeted drug therapy of the weakened and dystrophin-deficient heart muscle [185,278]. However, respiratory decline can already be observed during the early ambulatory phase in dystrophic boys [279] with diaphragmatic motions being drastically impaired following inspiration [280]. Analogous to the severely degenerative muscle phenotype seen in Duchenne patients, the aged mdx-4cv mouse diaphragm muscle shows the main histopathological hallmarks of dystrophinopathy [49,52]. This is summarized in the histological and immunofluorescence microscopical analysis depicted in Figure 7. dystrophin-deficient heart muscle [185,278]. However, respiratory decline can already be observed during the early ambulatory phase in dystrophic boys [279] with diaphragmatic motions being drastically impaired following inspiration [280]. Analogous to the severely degenerative muscle phenotype seen in Duchenne patients, the aged mdx-4cv mouse diaphragm muscle shows the main histopathological hallmarks of dystrophinopathy [49,52]. This is summarized in the histological and immunofluorescence microscopical analysis depicted in Figure 7. Histological and immunofluorescence microscopical analysis of dystrophic mdx-4cv mouse diaphragm muscle. Shown are transverse sections of 12-month-old diaphragm muscles from wildtype (wt) versus age-matched dystrophic mdx-4cv mice. Cryosections were stained with haematoxylin and eosin (H&E), as well as immuno-labelled with a monoclonal antibody to full-length dystrophin isoform Dp427-M. Hoechst-33342 staining was used to visualize myo-nuclei. Tissue preparation and immunofluorescence microscopy were carried out by optimized methods [146]. Bar equals 40 µm.
The illustration highlights myonecrosis, reactive myofibrosis, and zones of chronic inflammation. The dystrophic diaphragm exhibits more roundly shaped myofibers and a high frequency of central myo-nucleation, which is due to ongoing cycles of fiber degeneration and regeneration. These histopathological changes clearly correlate with skeletal muscle dysfunction and severe force deficits in the dystrophic mdx-type diaphragm [281]. However, one additional sign of muscular changes, which is often observed in human patients, is not present in dystrophic mdx-4cv mice, i.e., fat substitution. Immuno-labelling of dystrophin isoform Dp427-M confirmed the sarcolemmal localization of this membrane cytoskeletal protein in wild-type diaphragm and its almost-complete loss in the dystrophic and highly fibrotic mdx-4cv diaphragm. To complement microscopical studies, a comparative immunoblot analysis is routinely used to independently verify findings from proteomic studies. Western blotting surveys have clearly confirmed the drastic increase in many extracellular matrix proteins in muscular dystrophy, including various fibrosis markers such as collagens, fibronectin, dermatopontin, biglycan, and periostin in mdxtype muscles [282][283][284]. The glycoprotein fibronectin [285] is an exciting fibrosis-related biomarker candidate since its levels are also significantly increased in the serum of Figure 7. Histological and immunofluorescence microscopical analysis of dystrophic mdx-4cv mouse diaphragm muscle. Shown are transverse sections of 12-month-old diaphragm muscles from wildtype (wt) versus age-matched dystrophic mdx-4cv mice. Cryosections were stained with haematoxylin and eosin (H&E), as well as immuno-labelled with a monoclonal antibody to full-length dystrophin isoform Dp427-M. Hoechst-33342 staining was used to visualize myo-nuclei. Tissue preparation and immunofluorescence microscopy were carried out by optimized methods [146]. Bar equals 40 µm.
The illustration highlights myonecrosis, reactive myofibrosis, and zones of chronic inflammation. The dystrophic diaphragm exhibits more roundly shaped myofibers and a high frequency of central myo-nucleation, which is due to ongoing cycles of fiber degeneration and regeneration. These histopathological changes clearly correlate with skeletal muscle dysfunction and severe force deficits in the dystrophic mdx-type diaphragm [281]. However, one additional sign of muscular changes, which is often observed in human patients, is not present in dystrophic mdx-4cv mice, i.e., fat substitution. Immuno-labelling of dystrophin isoform Dp427-M confirmed the sarcolemmal localization of this membrane cytoskeletal protein in wild-type diaphragm and its almost-complete loss in the dystrophic and highly fibrotic mdx-4cv diaphragm. To complement microscopical studies, a comparative immunoblot analysis is routinely used to independently verify findings from proteomic studies. Western blotting surveys have clearly confirmed the drastic increase in many extracellular matrix proteins in muscular dystrophy, including various fibrosis markers such as collagens, fibronectin, dermatopontin, biglycan, and periostin in mdx-type muscles [282][283][284]. The glycoprotein fibronectin [285] is an exciting fibrosis-related biomarker candidate since its levels are also significantly increased in the serum of Duchenne patients compared to age-matched controls [286]. The immunoblots shown in Figure 8 illustrate the elevated levels of collagen isoform COL-VI, which, in muscle tissue, consists of three alpha chains [158], the annexin isoform ANXA2, and the matricellular protein periostin in the mdx-4cv diaphragm. It is not surprising that enhanced collagen deposition and a high concentration of fibronectin in fibrotic muscle tissue are closely related to increased dermatopontin levels since this tyrosine-rich acidic matrix protein plays a central role in cell-matrix interactions and matrix assembly [287][288][289].
Biomolecules 2023, 13, x FOR PEER REVIEW 22 Duchenne patients compared to age-matched controls [286]. The immunoblots show Figure 8 illustrate the elevated levels of collagen isoform COL-VI, which, in muscle ti consists of three alpha chains [158], the annexin isoform ANXA2, and the matricel protein periostin in the mdx-4cv diaphragm. It is not surprising that enhanced coll deposition and a high concentration of fibronectin in fibrotic muscle tissue are close lated to increased dermatopontin levels since this tyrosine-rich acidic matrix protein p a central role in cell-matrix interactions and matrix assembly [287][288][289]. Figure 8. Comparative immunoblot analysis of the dystrophic mdx-4cv mouse diaphragm m Shown is the immunoblotting of the glycolytic enzyme glyceraldehyde-3-phopshate dehydrog (GAPDH), used here as a house-keeping protein and loading control, alpha chain-containing gen VI (COL-VI) of the endomysium as an abundant marker of the extracellular matrix, the m cellular protein periostin (POSTN), and the membrane repair protein annexin A2 (ANXA2 electrophoretic separation was carried out with total protein extracts from aged wild-type (wt sus aged and dystrophic mdx-4cv diaphragm. Lanes 1 and 2 contain protein extracts from 15-m old wt and age-matched mdx-4cv muscle, respectively. On the left is shown a Coomassie Bri Blue-stained protein gel. The other images are identical immunoblots generated by electroph transfer and then labelled with monoclonal antibodies to GAPDH, COL-VI, POSTN, and AN as previously described in detail [55]. The position of immuno-labelled protein bands is indi by arrowheads. Molecular weight standards are marked on the left.

Proteomic Profiling of Fibrosis in the mdx-4cv Mouse Diaphragm
Because myofibrosis does not only directly impair tissue elasticity and the contra functions of skeletal muscle fibers but also has a negative impact on the regenerativ pacity of motor units following muscle degeneration and augments the susceptibilit muscular re-injury [10,[15][16][17], it is a major contributing factor to muscle weakness in trophinopathy [290]. It was therefore of interest to determine biological-systemchanges in X-linked muscular dystrophy using multi-omics approaches [291]. Prote profiling of dystrophic skeletal muscle has been carried out with a large number of p omic approaches using both patient biopsy specimens and a variety of animal mode dystrophinopathy [292][293][294][295][296][297][298]. Proteome-wide changes include alterations in the a dance of muscle-associated proteins that are involved in the regulation of excitationtraction coupling, ion homeostasis, cellular signaling, the contraction-relaxation cycl toskeletal networks, the extracellular matrix, bioenergetic pathways, and the cel stress response [299][300][301]. The extensive findings that have been generated by an model research underline the critical importance of preclinical studies for increasing Shown is the immunoblotting of the glycolytic enzyme glyceraldehyde-3-phopshate dehydrogenase (GAPDH), used here as a house-keeping protein and loading control, alpha chain-containing collagen VI (COL-VI) of the endomysium as an abundant marker of the extracellular matrix, the matricellular protein periostin (POSTN), and the membrane repair protein annexin A2 (ANXA2). Gel electrophoretic separation was carried out with total protein extracts from aged wild-type (wt) versus aged and dystrophic mdx-4cv diaphragm. Lanes 1 and 2 contain protein extracts from 15-monthold wt and age-matched mdx-4cv muscle, respectively. On the left is shown a Coomassie Brilliant Blue-stained protein gel. The other images are identical immunoblots generated by electrophoretic transfer and then labelled with monoclonal antibodies to GAPDH, COL-VI, POSTN, and ANXA2, as previously described in detail [55]. The position of immuno-labelled protein bands is indicated by arrowheads. Molecular weight standards are marked on the left.

Proteomic Profiling of Fibrosis in the mdx-4cv Mouse Diaphragm
Because myofibrosis does not only directly impair tissue elasticity and the contractile functions of skeletal muscle fibers but also has a negative impact on the regenerative capacity of motor units following muscle degeneration and augments the susceptibility for muscular re-injury [10,[15][16][17], it is a major contributing factor to muscle weakness in dystrophinopathy [290]. It was therefore of interest to determine biological-systemwide changes in X-linked muscular dystrophy using multi-omics approaches [291]. Proteomic profiling of dystrophic skeletal muscle has been carried out with a large number of proteomic approaches using both patient biopsy specimens and a variety of animal models of dystrophinopathy [292][293][294][295][296][297][298]. Proteome-wide changes include alterations in the abundance of muscle-associated proteins that are involved in the regulation of excitationcontraction coupling, ion homeostasis, cellular signaling, the contraction-relaxation cycle, cytoskeletal networks, the extracellular matrix, bioenergetic pathways, and the cellular stress response [299][300][301]. The extensive findings that have been generated by animal model research underline the critical importance of preclinical studies for increasing our mechanistic knowledge of muscle pathogenesis, establishing robust and reliable biomarker candidates, and developing novel treatment strategies for X-linked Duchenne muscular dystrophy [6,[302][303][304].
Myofibrosis-associated changes in dystrophin-deficient muscle fibers are diagrammatically summarized in Figure 9, which highlights characteristic alterations in distinct protein species at the level of the sarcolemma, basal lamina, endomysium, perimysium, and epimysium. The figure is based on the proteomic profiling of the dystrophic mdx-4cv diaphragm muscle [55] and relates to the information given in Figure 4 above on the distribution of key extracellular matrix proteins. The almost-complete loss of dystrophin isoform Dp427-M was shown to affect all layers of the matrisome, including the basal lamina with the significant reduction in the laminin-binding protein alpha-dystroglycan. The loss of the trans-sarcolemmal linkage via the dystrophin/dystroglycan sub-complex is the key trigger of membrane micro-rupturing in dystrophinopathies. The elevated levels of matrisomal proteins at the level of the basal lamina are characterized by the increased abundance of collagen IV and nidogen-2. Changes in the endomysium are reflected by an increased concentration of collagen V and collagen VI. Additional extracellular matrix proteins are found throughout the endomysium, perimysium, and epimysium. Fibrosisrelated increases were established for the matrisomal proteins fibronectin, vitronectin, and dermatopontin, as well as the non-structural matricellular protein periostin and the small leucine-rich proteoglycans asporin, biglycan, decorin, lumican, and mimecan/osteoglycin. Higher expression levels were also established for the extracellular-matrix-associated repair proteins annexin-2 and annexin-6 and the sarcolemmal integrin adhesion receptor subunits alpha-7 and beta-1.
Myofibrosis-associated changes in dystrophin-deficient muscle fibers are diagrammatically summarized in Figure 9, which highlights characteristic alterations in distinct protein species at the level of the sarcolemma, basal lamina, endomysium, perimysium, and epimysium. The figure is based on the proteomic profiling of the dystrophic mdx-4cv diaphragm muscle [55] and relates to the information given in Figure 4 above on the distribution of key extracellular matrix proteins. The almost-complete loss of dystrophin isoform Dp427-M was shown to affect all layers of the matrisome, including the basal lamina with the significant reduction in the laminin-binding protein alpha-dystroglycan. The loss of the trans-sarcolemmal linkage via the dystrophin/dystroglycan sub-complex is the key trigger of membrane micro-rupturing in dystrophinopathies. The elevated levels of matrisomal proteins at the level of the basal lamina are characterized by the increased abundance of collagen IV and nidogen-2. Changes in the endomysium are reflected by an increased concentration of collagen V and collagen VI. Additional extracellular matrix proteins are found throughout the endomysium, perimysium, and epimysium. Fibrosisrelated increases were established for the matrisomal proteins fibronectin, vitronectin, and dermatopontin, as well as the non-structural matricellular protein periostin and the small leucine-rich proteoglycans asporin, biglycan, decorin, lumican, and mimecan/osteoglycin. Higher expression levels were also established for the extracellular-matrix-associated repair proteins annexin-2 and annexin-6 and the sarcolemmal integrin adhesion receptor subunits alpha-7 and beta-1. Figure 9. Diagram of myofibrosis-related changes within the various layers of the extracellular matrix and associated structures in dystrophin-deficient skeletal muscle. Increased versus decreased protein isoforms are shown with a green versus red background, respectively. The collapse of the dystrophin-glycoprotein complex results in compensatory mechanisms, such as the activation of membrane repair mechanisms and reactive myofibrosis, which is characterized by drastic increases in various collagens, matricellular proteins, and proteoglycans.
Especially interesting is the drastic increase of periostin in the fibrotic mdx-4cv diaphragm, a matricellular protein that is involved in cellular signaling and the regulation of collagen fibrillogenesis [163,305]. In the extracellular matrix of skeletal muscles, periostin is only temporally expressed during regeneration and differentiation [306] but drastically Figure 9. Diagram of myofibrosis-related changes within the various layers of the extracellular matrix and associated structures in dystrophin-deficient skeletal muscle. Increased versus decreased protein isoforms are shown with a green versus red background, respectively. The collapse of the dystrophin-glycoprotein complex results in compensatory mechanisms, such as the activation of membrane repair mechanisms and reactive myofibrosis, which is characterized by drastic increases in various collagens, matricellular proteins, and proteoglycans.
Especially interesting is the drastic increase of periostin in the fibrotic mdx-4cv diaphragm, a matricellular protein that is involved in cellular signaling and the regulation of collagen fibrillogenesis [163,305]. In the extracellular matrix of skeletal muscles, periostin is only temporally expressed during regeneration and differentiation [306] but drastically increased during periods of fiber regeneration [307], including X-linked Duchenne muscular dystrophy [37,284,308]. This makes periostin a suitable fibrosis marker that can be used to judge the degree of myofibrotic changes in dystrophinopathy [284]. Changes in the annexin isoform ANAX2 [309][310][311] also have the potential to be highly useful for monitoring the level of membrane repair in fibrotic muscle fibers [312][313][314].
In summary, elevated levels of the below-listed major matrisomal marker proteins, which all play crucial roles in the extracellular matrix [73,[315][316][317], were identified to occur during extended periods of myofibrosis. The detection and characterization of these protein species were carried out by various mass-spectrometry-based proteomic techniques in combination with biochemical, cell biological, and physiological analyses.

Conclusions
The suitability of the severely dystrophic and fibrotic mdx-4cv diaphragm as a pathophysiological surrogate to study the progressive nature of dystrophinopathies has been outlined in this article. Histological, cell biological, and mass-spectrometry-based proteomic studies of the aged mdx-4cv mouse diaphragm have confirmed the presence of extensive reactive myofibrosis in this genetic mouse model of Duchenne muscular dystrophy. In conjunction with progressive myonecrosis, central myonuleation, and chronic inflammation, the fibrotic changes in the senescent mdx-4cv diaphragm make it an excellent model to study X-linked muscular dystrophy. The mdx-4cv mouse model was shown to be suitable for high-throughput surveys of complex changes in the expression levels of collagens, proteoglycans, matricellular proteins, regulatory enzymes, and adhesion receptors of the extracellular matrix. Following independent verification of their sensitivity, specificity, and robustness, proteomic biomarkers can now be used to improve differential diagnostics, prognostics, and therapeutic monitoring of X-linked Duchenne muscular dystrophy.

Data Availability Statement:
The mass spectrometric raw data from studies of the extracellular matrix from skeletal muscle shown in tables are available upon request.

Conflicts of Interest:
The authors declare no conflict of interest.