Nitric oxide facilitates NFAT-dependent transcription in mouse myotubes

Intracellular calcium transients in skeletal muscle cells initiate phenotypic adaptations via activation of calcineurin and its effector nuclear factor of activated t- cells ( NFAT). Furthermore, endogenous production of nitric oxide ( NO) via calcium- calmodulin- dependent NO synthase ( NOS) is involved in skeletal muscle phenotypic plasticity. Here, we provide evidence that NO enhances calcium- dependent nuclear accumulation and transcriptional activity of NFAT and induces phosphorylation of glycogen synthase kinase- 3 beta ( GSK- 3 beta) in C2C12 myotubes. The calcium ionophore A23187 ( 1 mu M for 9 h) or thapsigargin ( 2 mu M for 4 h) increased NFAT transcriptional activity by seven- and fourfold, respectively, in myotubes transiently transfected with an NFAT- dependent reporter plasmid ( pNFAT- luc, Stratagene). Cotreatment with the NOS- inhibitor N-G- nitro- L- arginine methyl ester ( L- NAME; 5 mM) or the guanylate cyclase inhibitor 1H[ 1,2,4] oxadiazolo[ 4,3- a] quinoxalin- 1- one ( ODQ; 10 mu M) prevented the calcium effects on NFAT activity. The NO donor diethylenetriamine-NONO ( DETA- NO; 10 mu M) augmented the effects of A23187 on NFAT- dependent transcription. Similarly, A23187 ( 0.4 mu M for 4 h) caused nuclear accumulation of NFAT and increased phosphorylation ( i. e., inactivation) of GSK- 3 beta, whereas cotreatment with L- NAME or ODQ inhibited these responses. Finally, the NO donor 3-( 2- hydroxy- 2- nitroso- 1- propylhydrazino)- 1- propanamine ( PAPA-NO; 1 mu M for 1 h) increased phosphorylation of GSK- 3 beta in a manner dependent on guanylate cyclase activity. We conclude that NOS activity mediates calcium- induced phosphorylation of GSK- 3 beta and activation of NFAT- dependent transcription in myotubes. Furthermore, these effects of NO are guanylate cyclase- dependent.