Regulation of the M-Cadherin-β-Catenin Complex by Calpain 3 during Terminal Stages of Myogenic Differentiation

Abstract

The cysteine protease calpain 3 (CAPN3) is essential for normal muscle function, since mutations in CAPN3 cause limb girdle muscular dystrophy type 2A. Previously, we showed that myoblasts isolated from CAPN3 knockout (C3KO) mice were able to fuse to myotubes; however, sarcomere formation was disrupted. In this study we further characterized morphological and biochemical features of C3KO myotubes in order to elucidate a role for CAPN3 during myogenesis. We showed that cell cycle withdrawal occurred normally in C3KO cultures, but C3KO myotubes have an increased number of myonuclei per myotube. We found that CAPN3 acts during myogenesis to specifically control levels of membrane-associated but not cytoplasmic ␤-catenin and M-cadherin. CAPN3 was able to cleave both proteins, and in the absence of CAPN3, M-cadherin and ␤-catenin abnormally accumulated at the membranes of myotubes. Given the role of M-cadherin in myoblast fusion, this finding suggests that the excessive myonuclear index of C3KO myotubes was due to enhanced fusion. Postfusion events, such as ␤1D integrin expression and myofibrillogenesis, were suppressed in C3KO myotubes. These data suggest that the persistence of fusion observed in C3KO cells inhibits subsequent steps of differentiation, such as integrin complex rearrangements and sarcomere assembly.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...

Thromboxane Governs the Differentiation of Adipose-Derived Stromal Cells Toward Endothelial Cells In Vitro and In Vivo

“…Calpain, a member of the family of intracellular Ca 2+ -dependent cysteine proteases, 68 has been reported to mediate β-catenin degradation. 36,37,69,70 Using a C-terminal-specific antibody, we detected a substantial reduction in cleaved fragments of β-catenin in TP-KO ASCs. Accordingly, calpain activity was also suppressed in TP-KO ASCs, and re-expression of TP in TP-KO ASCs restored calpain activity and increased β-catenin fragmentation.…”

Satellite cell senescence underlies myopathy in a mouse model of limb-girdle muscular dystrophy 2H

“…Isolated single fibers with associated satellite cells were incubated on plates coated with ECL Cell Attachment Matrix (Millipore). Proliferating cells were maintained as described previously (81). Myoblast fusion was induced by placing the cells in differentiation medium: DMEM supplemented with insulin-transferrin-selenium (1:100; Invitrogen) for indicated amount of time.…”

Functional interaction between TRPC1 channel and connexin-43 protein: a novel pathway underlying S1P action on skeletal myogenesis

“…In particular, we sought to define the role played by S1P-mediated TRPC1 activation on the expression and activity of m-calpain, a Ca 2? -sensitive protease, whose levels increase during early myogenic differentiation and after mechanical stimulation [30][31][32][33]. We also explored the functional repercussion of TRPC1-mediated m-calpain activation in the regulation of classical PKCs, Cx43 remodeling and myoblast differentiation and fusion.…”