Abstract
Myoblast migration is a key step in myogenesis and regeneration. It allows myoblast alignment and their fusion into myotubes. The process has been shown to involve m-calpain or mu-calpain, two Ca(2+)-dependent cysteine proteases. Here we measure calpain activity in cultured cells and show a peak of activity at the beginning of the differentiation process. We also observed a concomitant and transient increase of the influx of Ca(2+) and expression of TRPC1 protein. Calpains are specifically activated by a store-operated entry of Ca(2+) in adult skeletal muscle fibres. We therefore repressed the expression of TRPC1 in myoblasts and studied the effects on Ca(2+) fluxes and on differentiation. TRPC1-depleted myoblasts presented a largely reduced store-operated entry of Ca(2+) and a significantly diminished transient influx of Ca(2+) at the beginning of differentiation. The concomitant peak of calpain activity was abolished. TRPC1-knockdown myoblasts also accumulated myristoylated alanine-rich C-kinase substrate (MARCKS), an actin-binding protein and substrate of calpain. Their fusion into myotubes was significantly slowed down as a result of the reduced speed of cell migration. Accordingly, migration of control myoblasts was inhibited by 2-5 microM GsMTx4 toxin, an inhibitor of TRP channels or by 50 microM Z-Leu-Leu, an inhibitor of calpain. By contrast, stimulation of control myoblasts with IGF-1 increased the basal influx of Ca(2+), activated calpain and accelerated migration. These effects were not observed in TRPC1-knockdown cells. We therefore suggest that entry of Ca(2+) through TRPC1 channels induces a transient activation of calpain and subsequent proteolysis of MARCKS, which allows in turn, myoblast migration and fusion.
Highlights
During myogenesis and muscle regeneration, myogenic stem cells proliferate as myoblasts
We previously showed that in adult skeletal muscle fibres, there was a Ca2+ leak channel that could be activated by store depletion or by membrane stretching; this channel is abnormally open in dystrophin-deficient fibres and seems to be constituted of TRPC1 and/or TRPC4 isoforms (Vandebrouck et al, 2002)
Western blot analysis showed an important decrease of the expression of TRPC1 protein (Fig. 1B); pooling the results for both TRPC1 siRNAs, we measured a repression of 70.5±11%, n=6
Summary
During myogenesis and muscle regeneration, myogenic stem cells (called satellite cells) proliferate as myoblasts. During the initial phase of differentiation, Ca2+ activates several myogenic transcription factors such as myogenin and MEF2 (Black and Olson, 1998; Molkentin and Olson, 1996), triggering the expression of muscle-specific genes The best candidate molecules seem to belong to the superfamily of transient receptor potential (TRP) channels (for reviews, see Owsianik et al, 2006; Venkatachalam and Montell, 2007). These TRP channels present a similarity of structure (six transmembrane domains) and some sequence homology. The remaining two subgroups, muclopins (TRPML) and polycystins (TRPP) are only distantly related to the other subfamilies
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