TRPC1 regulates skeletal myoblast migration and differentiation

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 μ-calpain, two Ca2+-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 Ca2+ and expression of TRPC1 protein. Calpains are specifically activated by a store-operated entry of Ca2+ in adult skeletal muscle fibres. We therefore repressed the expression of TRPC1 in myoblasts and studied the effects on Ca2+ fluxes and on differentiation. TRPC1-depleted myoblasts presented a largely reduced store-operated entry of Ca2+ and a significantly diminished transient influx of Ca2+ 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 μM GsMTx4 toxin, an inhibitor of TRP channels or by 50 μM Z-Leu-Leu, an inhibitor of calpain. By contrast, stimulation of control myoblasts with IGF-1 increased the basal influx of Ca2+, activated calpain and accelerated migration. These effects were not observed in TRPC1-knockdown cells. We therefore suggest that entry of Ca2+ through TRPC1 channels induces a transient activation of calpain and subsequent proteolysis of MARCKS, which allows in turn, myoblast migration and fusion.