Endothelial nitric oxide synthase is involved in calcium-induced Akt signaling in mouse skeletal muscle

Abstract

We hypothesized that targeted mutation of the endothelial nitric oxide synthase (eNOS) gene would reduce Akt-related signaling events in skeletal muscle cells, compared to wild type (WT) controls. Results show that slow myosin heavy chain (type I/β) expression and the abundance of slow-twitch fibers are reduced in plantaris muscle of eNOS −/− mice, compared to WT. Further, basal phosphorylation of Akt (p-Akt (Ser-473)/total Akt) and GSK-3β (GSK-3β (Ser-9)/total GSK-3β) are reduced 60–70% in primary myotubes from eNOS −/− mice. Treatment with the calcium ionophore, A23187 (0.4 μM, 1 h), increased phosphorylation of Akt and GSK-3β by ∼2-fold ( P < 0.05) in myotubes from WT mice, but had no effect on phosphorylation of these proteins in eNOS −/− myotubes. Additionally, A23187 treatment failed to induce nuclear translocation of the transcription factor, NFATc1, in eNOS −/− myotubes. Treatment with the nitric oxide donor, propylamine propylamine NONOate (PAPA-NO; 1 μM for 1 h) increased Akt and GSK-3β phosphorylation, and induced NFATc1 nuclear translocation in WT and eNOS −/− myotubes, and eliminated differences from WT in the NOS knockout cultures. Parallel experiments in C2C12 myotubes found that Akt phosphorylation induced by NO or the guanylate cyclase activator, YC-1, is prevented by co-treatment with either a guanylate cyclase or PI3K inhibitor (10 μM ODQ or 25 μM LY2904002, respectively). These data suggest that eNOS activity is necessary for calcium-induced activation of the Akt pathway, and that nitric oxide is sufficient to elevate Akt activity in primary myotubes. NO appears to influence Akt signaling through a cGMP, PI3K-dependent pathway.