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We have previously shown that calpain promotes myoblast fusion by acting on protein kinase C-alpha and the cytosolic phosphorylated form of MARCKS. In other cell types, various isoforms of calpain, PKC alpha and MARCKS were found associated with caveolae. These vesicular invaginations of the plasma membrane are essential for myoblast fusion and differentiation. We have isolated caveolae from myoblasts and studied the presence of calpain isoforms and their possible effects on signalling mediated by caveolae-associated PKC. Our results show that milli-calpain co-localizes with myoblast caveolae. Futhermore we provide evidence, using a calcium ionophore and a specific inhibitor of calpains (calpastatin peptide), that milli-calpain reduces the PKC alpha and MARCKS content in these structures. Purified milli-calpain causes the appearance of the active catalytic fragment of PKC alpha (PKM), without having an effect on MARCKS. Addition of phorbol myristate acetate, an activator of PKC, induces tranlocation of PKC alpha towards caveolae and results in a significant reduction of MARCKS associated with caveolae. This phenomenon is not observed when a PKC alpha inhibitor is added at the same time. We conclude that the presence of biologically active milli-calpain within myoblast caveolae induces, in a PKC alpha-dependent manner, MARCKS translocation towards the cytosol. Such a localised signalling event may be essential for myoblast fusion and differentiation.

Several studies have already demonstrated that micro- and milli-calpains (CAPN 1-CAPN 2), calcium-dependent intracellular cysteine-proteases are involved in many biological phenomenon including muscle growth and development. More particularly, recent studies have demonstrated that milli-calpain is implicated in myoblast fusion. Moreover, in primary muscle cells, these proteases do not appear simultaneously throughout muscle cell differentiation. Because micro- and milli-calpains do not have the same intracellular localization, it appears likely that these two calcium-dependent proteases have different biological roles during muscle cell differentiation. The goal of this study is to determine the role of micro-calpain. We therefore, have developed a muscle cell line in which micro-calpain is over-expressed, using the inducible Tet Regulated Expression System. The outcome is observed by following the behavior of different proteins, considered to be potential substrates of the protease. The present study shows important decreases in the expression level of ezrin (68%), vimentin (64%) and caveolin 3 (76%) whereas many other cytoskeletal proteins remain remarkably stable. Concerning the myogenic transcription factors, only the level of myogenin decreased (59%) after the over-expression of micro-calpain. Ultra structural studies have shown that the myofibrils formed near the cell periphery are normally oriented, lying along the longitudinal axis. This regularity is lost progressively towards the cell center where the cytoskeleton presented an increasing disorganization. All these results indicate that micro-calpain is involved in regulation pathway of myogenesis via at least its action on ezrin, vimentin, caveolin 3 and myogenin, a muscle transcription factor.