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

Abstract

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.

Proteome Profiling of the Dystrophic mdx Mice Diaphragm

“…One of the examples could be the differences in the expression of various collagen isoforms. A detailed study of their abundance (and other ECM proteins) in dystrophic muscles and the muscle-associated layers of connective tissue was summarized recently [22]. Based on the increase in collagen V and collagen VI in the endomysium, collagen IV, matrisomal proteins at the level of the basal lamina, myotendinous-junction-enriched collagen XII, and tendon-enriched collagen I, it was suggested that these be used as biomarkers of dystrophinopathy-associated myofibrosis.…”

“…Why the diaphragm is the most pathological muscle in dystrophin-deficient mdx mice is not really known. The reduced activation of the regenerative response in the diaphragm may result from the overproduction of extracellular matrix components and prominent myofibrosis, which inhibits regeneration (references in [22]).…”

Mass spectrometry-based proteomic characterization of the middle-aged mouse brain for animal model research of neuromuscular diseases

“… 16 Importantly, at this age the proteomic data are suitable for comparative studies of late-onset neuromuscular changes, such as reactive myofibrosis. 17 In general, mice of 3-6, 10-15 and 18-24 months of age are considered ‘mature adult’, ‘middle aged’ and ‘old’, respectively. The middle-aged group represents a phase in the life of mice during which early changes due to the natural aging process can be detected.…”

How Can Proteomics Help to Elucidate the Pathophysiological Crosstalk in Muscular Dystrophy and Associated Multi-System Dysfunction?

“…The main feature of the cellular pathogenesis of DMD is progressive myonecrosis, which is accompanied by chronic inflammation, reactive myofibrosis and an impaired regenerative capacity due to satellite cell dysfunction, as recently reviewed [ 258 ]. Skeletal muscles are characterized by the presence of extremely large proteins, i.e., giant proteins such as titin and nebulin [ 99 ], as well as many distinctly hydrophobic membrane proteins [ 50 ] and complex layers of mostly insoluble ECM components [ 259 ]. This makes proteomic studies of total tissue extracts technically challenging.…”