Regulation of the M-cadherin-β-catenin complex by calpain 3 during terminal stages of myogenic differentiation

Calpain 3 (CAPN3) belongs to a family of Ca-dependent, nonlysosomal, cysteine proteases that includes both ubiquitously expressed and tissue-specific members (19). CAPN3 (with a molecular mass of 94 kDa) is the major calpain isoform expressed in adult skeletal muscles. Mutations in CAPN3 cause limb girdle muscular dystrophy 2A (LGMD2A), an autosomal recessive muscle disease characterized by progressive atrophy and weakness of the proximal limb muscles (40). We generated CAPN3 knockout (C3KO) mice and showed that these mice develop moderate muscle atrophy and growth deficiencies which are consistent with the human phenotype of LGMD2A. Examination of muscles from adult C3KO mice revealed abnormal sarcomere structure. Furthermore, studies on primary myoblast cultures revealed severe abnormalities during the terminal steps of myogenesis. C3KO myoblasts were able to fuse to form myotubes, but myofibrillogenesis was inhibited (or delayed) as was shown by ultrastructural and biochemical analyses (30). Mechanisms occurring during the final steps of myogenic differentiation (i.e., myoblast fusion and sarcomere assembly) are not well understood in mammals. However, several classes of transmembrane molecules, including members of the cadherin and immunoglobulin families, have been shown to play a role in the fusion process (26). It is likely that the mammalian fusion complex comprises many components with redundant functions, a feature that makes its analysis very difficult. Numerous observations indicate that M-cadherin is involved in the fusion of myogenic cells. For example, synthetic peptides that bind to the extracellular domain of M-cadherin and block homophilic interactions were able to block myoblast fusion in a dose-dependent manner (48). Recently, the importance of M-cadherin for fusion of cultured myoblasts was confirmed by the RNA interference method (11). Moreover, changes in M-cadherin levels led to changes in fusion: downregulation of M-cadherin caused inhibition, while upregulation of M-cadherin caused enhancement of the fusion process (11, 46). Although these data show that M-cadherin is involved in myoblast fusion, mice lacking M-cadherin develop normal skeletal musculature and M-cadherin knockout myogenic cells can fuse normally, suggesting that other molecules can largely compensate for the lack of M-cadherin in vivo (23). Thus, these observations indicate that, even though M-cadherin loss of function does not prevent fusion, perturbation of M-cadherin levels modulates fusion of myogenic cells in vitro, suggesting that M-cadherin plays a role in mediating the fusion process. One of the intracellular components of the cadherin complex is β-catenin, a protein that has been shown to play a dual role in myogenesis. First, a cytoplasmic pool of β-catenin plays a role in Wnt signaling that regulates myogenic fate specification during both embryonic development (14) and adult muscle regeneration (38, 41). β-Catenin also plays a role in later stages of myogenic differentiation as a component of the cadherin cell adhesion complex (32). Shortly after myoblasts are transferred to differentiation medium, β-catenin levels increase and β-catenin is recruited to the cadherin complex. This process has been shown to be crucial for expression of myogenin, a transcription factor that regulates progression towards the terminal stages of myogenic differentiation (18). However, perplexingly, overexpression of β-catenin had an inhibitory effect on myogenesis (18), suggesting that there is a mechanism for regulating the β-catenin pool not only in the cytoplasm, which is a well-known phenomenon in Wnt signaling, but also at the membrane. Recently, a muscle-specific ubiquitin ligase, called Ozz-E3 that regulates specific degradation of β-catenin associated with the cell membrane, was identified (36). Interestingly, accumulation of β-catenin due to null mutation of Ozz-E3 or due to overexpression of a β-catenin fragment that competes for binding to Ozz leads to severe defects in myofibrillogenesis, resulting in the absence of visible sarcomere formation in mutant myotubes. In spite of this in vitro phenotype, Ozz null mice form well-developed muscles, albeit with sarcomere abnormalities. These in vitro and in vivo characteristics are very similar to those which we observed in C3KO mice (30). In this study we further examined the biochemical and morphological features of C3KO myotubes to elucidate a role for CAPN3 in myogenesis. In particular, we investigated the relationship between CAPN3 and the cadherin complex. This study revealed that in addition to defects in myofibrillogenesis, some C3KO myotubes were morphologically abnormal and contained big clusters of myonuclei. Overall, the myonuclear indices of C3KO myotubes and adult myofibers were higher than those in wild-type (WT) myotubes and myofibers. We found that both β-catenin and M-cadherin are substrates for CAPN3 protease and that CAPN3 specifically controls the level of membrane-associated β-catenin and M-cadherin during myogenesis. We hypothesize that persistence of the M-cadherin-β-catenin complex at the membranes of C3KO myotubes causes excessive fusion and delayed myofibrillogenesis in the absence of CAPN3.