Résumé :
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Duchenne muscular dystrophy (DMD), the most common form of inherited neuromuscular disorder, is caused by null mutations in the dystrophin gene, leading to progressive and unrelenting muscle loss. Although the genetic basis of DMD is well resolved, the cellular mechanisms associated with the physiopathology remain largely unknown. In particular, little information is available regarding alterations in the proteome that may represent factors contributing to the disease progression. Here, using the clinically relevant Golden Retriever Muscular Dystrophy (GRMD) dog model, we employed two complementary proteomic screens, 2-D gel electrophoresis and isotope-coded affinity tag (ICAT) profiling, to compile quantitative changes in protein expression profiles of GRMD versus healthy dog muscles. We were able to identify for the first time global alterations in the dystrophic dog muscle proteome, namely the increased expression of 42 proteins and the decreased expression of 44 others. We found that the set of over-expressed proteins was composed of factors involved in apoptosis, calcium signaling and myoblast development/differentiation, a profile reflective of the inflammation and ongoing regeneration taking place within dystrophic muscle. The set of under-expressed proteins appeared primarily composed of metabolic proteins, many of which have been shown to be regulated by the transcriptional peroxisome proliferator-activated receptor-gamma co-activator 1 alpha (PGC- 1a). Immunofluorescence analysis showed a same expression profile of PGC-1a in subsarcolemmal as well as endomysial nuclei in healthy and GRMD dog muscles but with a lower intensity in the second one. In GRMD dog muscles, PGC-1a labeling was higher in cluster of small fibers. Next, the hypothesis of an altered transcriptional regulation of PGC-1a and its downstream putative targets was investigated by real-time RT-PCR. We validated the down-expression of the PGC-1a gene and four of its target genes. In addition, we noted that GRMD dog muscle displays a significant reduction in the abundance of DJ-1 which promotes the activity of PGC-1a . DJ-1 is described as a key regulator of the PTEN/Akt-dependent signal cascades which were previously shown to be deregulated in GRMD dog muscle. Collectively, our results provide novel insights into the pathophysiological processes of the clinically relevant DMD animal model, and emphasize the relevance of proteomic approaches to assess global changes such as those expected by the development of therapeutic strategies. In this context, we study the proteins with altered expression levels at different stages after systemic delivery of MuStem cells. In the future, a well-defined set of signature molecule could serve the evaluation of experimental therapy.
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