Résumé :
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Myostatin (MSTN), a growth factor member of the TGF-beta superfamily acts as a negative regulator of skeletal muscle growth and then contributes to muscle atrophy. The objective of this study* was to determine the interactions between this growth factor and calcium-dependent proteolytic system. The results indicate that overexpression of MSTN lead to a reduction in calpastatin expression (mRNA) and abundance (Proteins) in C2C12cells transfected with plasmid containing the MSTN cDNA compared to those transfected with empty plasmid as controls. In these conditions, we did not detect any change in the expression and abundance of calpains. In addition, cells transfected with MSTN exhibit a higher calpain activity as revealed by using t-BOC-LM-CMAC, a synthetic fluorescent calpain substrate which produces a blue fluorescence signal after cleavage. These data showed that MSTN acts indirectly on the calpain activity by regulation of both expression and abundance of their endogenous inhibitor: calpastatin, which decrease significantly (45%). Furthermore, using two-dimensional electrophoresis coupled to mass spectrometry, we performed proteomic profile to identify proteins that were altered in C2C12 transfected with MSTN and their controls. This analysis revealed 10 differentially abundant protein spots (6 up- and 4 down-abundant). Over-expression of MSTN resulted in a regulation of proteins implicated in muscle energetic metabolism with a decreased abundance of Pdia3 and increased abundance of ATP5b and PHGDH. Moreover, proteins involved in structural and functional organization of muscle cell (CapZa, TBB5, _-actin) was revealed to be increased in response to MSTN over-expression. Abundance of two heat shock proteins HSPd1 (or HSP60) and HSP9a and of nuclear envelop protein (LMNA) and splicing-factor proline- and glutamine-rich (SFPQ) revealed to be increased. In agreement with literature data, some of these protein targets of MSTN are also known to be altered in muscle atrophy and others identified as substrates (CapZa, TBB5, SFPQ) or calpain regulators (HSPd1, HSP9a). All together, these results are in favour of muscle atrophy. This advance suggests an establishment of interactions between MSTN and calpain-dependent proteolytic system and present a progress in the understanding of molecular network involved in the atrophy process.* MYOTROPHY, projet ANR BLAN08-2_309209
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