Elevation of NO Increases Ca2+ Entry and Resting Ca2+ and Na+ Concentrations in Skeletal Muscle Cells

Abstract

Protein S-nitrosylation is a reversible post-translational modification and can have profound effects on protein function in skeletal muscle. S-nitrosylation has been proposed to exert regulatory effects on ion channels such as RyR1 and TRPCs, by modifying the redox state of critical thiols. Increased nitric oxide (NO) levels may play a crucial role in the alteration of intracellular resting [Ca2+]r and [Na+]r homeostasis in skeletal muscle. The present work aimed to characterize the contribution of NO using the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) on intracellular Ca2+ and Na+ homeostasis in Wt myotubes. In quiescent Wt myotubes 100 µM SNAP increased Ca2+ entry, [Ca2+]r and [Na+]r. These effects could be blocked with either Gd3+ or BTP-2 and partially reversed by DTT, a known inhibitor of S nitrosylation, suggesting that the effect of SNAP was through a reversible increase in the TRPC channel activity. Furthermore, we found that RyRs are not the principal target for S-nitrosylation because SNAP incubation increases [Ca2+]r and [Na+]r in RyR1/RYR3 null myotubes. It is known that NO production is elevated in mdx muscle. Here we found that DTT or BTP-2 significantly reduced [Ca2+]r in mdx myotubes and DTT was able to restore a normal Ca2+ entry. We then looked at the effects of SNAP on K+ induced Ca2+ release and SR Ca2+ content in WT and mdx myotubes. SNAP did not significantly decrease sarcoplasmic reticulum Ca2+ content in either WT or mdx myotubes but reduced the magnitude of the Ca2+ transient induced by K+ in Wt but not in mdx myotubes. These results suggest that nitrosative modifications play a key role regulating TRPC activity in muscular dystrophy and their role in the Ca2+ and Na+ overload seen in dystrophic muscles.