Nitric oxide facilitates NFAT-dependent transcription in mouse myotubes

Center for Exercise Science, Department of Applied Physiology and Kinesiology, University of Florida, Gainesville, Florida Submitted 12 December 2007 ; accepted in final form 7 February 2008 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-3β (GSK-3β) in C2C12 myotubes. The calcium ionophore A23187 [GenBank] (1 µM for 9 h) or thapsigargin (2 µM 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 µM) prevented the calcium effects on NFAT activity. The NO donor diethylenetriamine-NONO (DETA-NO; 10 µM) augmented the effects of A23187 [GenBank] on NFAT-dependent transcription. Similarly, A23187 [GenBank] (0.4 µM for 4 h) caused nuclear accumulation of NFAT and increased phosphorylation (i.e., inactivation) of GSK-3β, 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 µM for 1 h) increased phosphorylation of GSK-3β in a manner dependent on guanylate cyclase activity. We conclude that NOS activity mediates calcium-induced phosphorylation of GSK-3β and activation of NFAT-dependent transcription in myotubes. Furthermore, these effects of NO are guanylate cyclase-dependent. nitric oxide synthase; nuclear factor of activated t-cells; glycogen synthase kinase-3β; soluble guanylate cyclase; myosin heavy chain I/β Address for reprint requests and other correspondence: D. Criswell, PO Box 118206, Center for Exercise Science, Univ. of Florida, Gainesville, FL 32611 (e-mail: dcriswell{at}hhp.ufl.edu )