Involvement of micro-calpain (CAPN 1) in muscle cell 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.