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.
Highlights
Dystrophin proteins exist in a large number of isoforms with greatly differing tissue distributions and are encoded by the largest gene in the human genome within the Xp21 region of theX-chromosome [1,2,3]
X-linked muscular dystrophy is a primary muscle disease of the membrane cytoskeleton [72], i.e., the almost complete loss of the full-length dystrophin protein isoform of 427 kDa (Dp427) isoform of dystrophin is the underlying pathobiochemical defect that triggers progressive fiber degeneration [73], a crucial secondary alteration in dystrophinopathy is presented by extensive myofibrosis [74]
Since myofibrosis is an important clinical feature of Duchenne muscular dystrophy [77] and in order to correlate the loss of dystrophin with the dramatic increase in collagen, as demonstrated here by the label-free mass spectrometric analysis of total mdx-4cv skeletal muscle extracts, the molecular fate of collagen isoform COL-VI was evaluated by immunoblotting and immunofluorescence microscopy
Summary
X-chromosome [1,2,3]. The full-length muscle isoform of dystrophin, termed Dp427-M, functions as a membrane cytoskeletal actin-binding protein in contractile fibers [4,5,6]. A variety of changes in skeletal muscle-associated proteins involved in energy metabolism, excitationcontraction coupling, fiber contraction, ion homeostasis, the stress response, and cellular signaling have been identified by mass spectrometry, as reviewed by Dowling et al [21]. This included differential alterations in muscle-associated proteins, such as adenylate kinase isoform AK1 [22], the luminal Ca2+-binding protein calsequestrin [23], the cytosolic Ca2+-binding proteins regucalcin [24]. Since skeletal muscles from this animal model of dystrophinopathy are characterized by very few revertant fibers and exhibit myofibrosis [50,51,52,53], the proteomic analysis of total mdx-4cv muscle extracts was ideally suited to simultaneously study dystrophin deficiency and secondary fibrotic changes within the same analytical run
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