TRPC1 regulates skeletal myoblast migration and differentiation.
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.
Louis
Magali
M
Université catholique de Louvain, Institute of Neuroscience, Laboratory of Cell Physiology, 55/40 avenue Hippocrate, 1200 Brussels, Belgium.
Zanou
Nadège
N
Van Schoor
Monique
M
Gailly
Philippe
P
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D032448:Myoblasts, Skeletal / Q000166:cytology* / Q000378:metabolism; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D050052:TRPC Cation Channels / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014162:Transfection
D047908:Intracellular Signaling Peptides and Proteins; C000619711:Marcks protein, mouse; D008565:Membrane Proteins; D050052:TRPC Cation Channels; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D007334:Insulin-Like Growth Factor I; D002154:Calpain; D002118:Calcium