Concurrent Label-Free Mass Spectrometric Analysis of Dystrophin Isoform Dp427 and the Myofibrosis Marker Collagen in Crude Extracts from mdx-4cv Skeletal Muscles

Abstract

The full-length dystrophin protein isoform of 427 kDa (Dp427), the absence of which represents the principal abnormality in X-linked muscular dystrophy, is difficult to identify and characterize by routine proteomic screening approaches of crude tissue extracts. This is probably related to its large molecular size, its close association with the sarcolemmal membrane, and its existence within a heterogeneous glycoprotein complex. Here, we used a careful extraction procedure to isolate the total protein repertoire from normal versus dystrophic mdx-4cv skeletal muscles, in conjunction with label-free mass spectrometry, and successfully identified Dp427 by proteomic means. In contrast to a considerable number of previous comparative studies of the total skeletal muscle proteome, using whole tissue proteomics we show here for the first time that the reduced expression of this membrane cytoskeletal protein is the most significant alteration in dystrophinopathy. This agrees with the pathobiochemical concept that the almost complete absence of dystrophin is the main defect in Duchenne muscular dystrophy and that the mdx-4cv mouse model of dystrophinopathy exhibits only very few revertant fibers. Significant increases in collagens and associated fibrotic marker proteins, such as fibronectin, biglycan, asporin, decorin, prolargin, mimecan, and lumican were identified in dystrophin-deficient muscles. The up-regulation of collagen in mdx-4cv muscles was confirmed by immunofluorescence microscopy and immunoblotting. Thus, this is the first mass spectrometric study of crude tissue extracts that puts the proteomic identification of dystrophin in its proper pathophysiological context.

Multiomics Analysis of the mdx/mTR Mouse Model of Duchenne Muscular Dystrophy

“…Numerous proteoglycans such as biglycan (Bgn), decorin (Dcn), lumican (Lum), osteopontin (Spp1), tenascin C (Tnc), tenascin X (Tnxb), versican (Vcan), vinculin (Vcl), and vimentin (Vim) were upregulated at the transcript or protein level ( Figure 3D). These observations are consistent with previous findings in 3 month old mdx and 6 month old mdx 4cv animals, which are thought to have a more severe muscle phenotype from the original strain of mdx mice (32,33).…”

“…However, type Iα1 and type Iα2 collagen had a lower abundance in mdx/mTR muscles, while type VIα1 collagen protein was not different despite having an elevation in transcript ( Figure 3D). In 6 month old mdx 4cv animals, an elevation in type Iα1 and type VIα1 collagen protein was observed (33). It is possible that the differences between these results in collagen changes are due to an earlier degenerative stage of the mdx/mTR mice, prior to the onset of more severe fibrosis.…”

Comparative gel‐based proteomic analysis of chemically crosslinked complexes in dystrophic skeletal muscle

“…The trifunctional enzyme complex of the inner mitochondrial membrane system is an important component that mediates fatty acid utilization and altered enzymatic functionality has severe pathophysiological consequences . A previous survey of crude microsomal membranes by MS‐based proteomics has revealed elevated levels of this metabolic enzyme in dystrophin‐deficient skeletal muscle tissue . The 2‐oxoglutarate dehydrogenase complex is a multi‐enzyme assembly of the mitochondrial matrix that catalyses the overall conversion of 2‐oxoglutarate to succinyl‐CoA and CO 2 as a crucial part of the citric acid cycle in skeletal muscle .…”

Proteomics Unveils Fibroblast–Cardiomyocyte Lactate Shuttle and Hexokinase Paradox in Mouse Muscles

“…Label free proteomic approaches allowed studying molecular basis of muscle dystrophy [4][5][6] . A few recent studies have demonstrated that the filter aided sample preparation (FASP) 7 and multienzyme digestion FASP (MED-FASP) 8 methods facilitate proteomic analysis of muscles enabling creation of quantitative picture of the cellular organization and providing titers of individual proteins [9][10][11] Recently, we have described a simple analytical and computational approach to estimate titers of enzymes of basic metabolic pathways and proteins of the contractile machinery in skeletal muscles of mouse 10 .…”