Nitric oxide (NO) induces mitochondrial biogenesis in skeletal muscle cells via upregulation of the peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α). Further, we have shown that nitric oxide interacts with the metabolic sensor enzyme, AMPK. Therefore, we tested the hypothesis that nitric oxide and AMPK act synergistically to upregulate PGC-1α mRNA expression and stimulate mitochondrial biogenesis in culture. L6 myotubes treated with nitric oxide donors, S-nitroso-N -penicillamine (SNAP, 25 μm) or diethylenetriamine-NONO (DETA-NO, 50 μm), exhibited elevated AMPK phosphorylation, PGC-1α mRNA and protein, and basal and uncoupled mitochondrial respiration (P < 0.05). Pre-treatment of cultures with the AMPK inhibitor, Compound C, prevented these effects. Knockdown of AMPKα1 in L6 myotubes using siRNA reduced AMPKα protein content and prevented upregulation of PGC-1α mRNA by DETA-NO. Meanwhile, siRNA knockdown of AMPKα2 had no effect on total AMPKα protein content or PGC-1α mRNA. These results suggest that NO effects on PGC-1α expression are mediated by AMPKα1. Paradoxically, we found that the AMPK-activating compound, AICAR, induced NO release from L6 myotubes, and that AICAR-induced upregulation of PGC-1α mRNA was prevented by inhibition of NOS with N G -nitro-l-arginine methyl ester (l-NAME, 1 mm). Additionally, incubation of isolated mouse extensor digitorum longus (EDL) muscles with 2 mm AICAR for 20 min or electrical stimulation (10 Hz, 13 V) for 10 min induced phosphorylation of AMPKα (P < 0.05), which was completely prevented by pre-treatment with the NOS inhibitor, l-N G -monomethyl arginine (l-NMMA, 1 mm). These data identify the AMPKα1 isoform as the mediator of NO-induced effects in skeletal muscle cells. Further, this study supports a proposed model of synergistic interaction between AMPK and NOS that is critical for maintenance of metabolic function in skeletal muscle cells.

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Laser therapy at the 3-W (360-J) dose level was an effective treatment modality to increase blood flow in the soft tissues.

Mechanical stretch of skeletal muscle activates nitric oxide (NO) production and is an important stimulator of satellite cell proliferation. Further, cyclooxygenase (COX) activity has been shown to promote satellite cell proliferation in response to stretch. Since COX-2 expression in skeletal muscle can be regulated by NO we sought to determine if NO is required for stretch-induced myoblast proliferation and whether supplemental NO can counter the effects of COX-2 and NF-kappaB inhibitors. C2C12 myoblasts were cultured for 24 h, then switched to medium containing either the NOS inhibitor, L-NAME (200 microM), the COX-2 specific inhibitor NS-398 (100 microM), the NF-kappaB inhibiting antioxidant, PDTC (5 mM), the nitric oxide donor, DETA-NONOate (10-100 microM) or no supplement (control) for 24 h. Subgroups of each treatment were exposed to 1 h of 15% cyclic stretch (1 Hz), and were then allowed to proliferate for 24 h before fixing. Proliferation was measured by BrdU incorporation during the last hour before fixing, and DAPI stain. Stretch induced a twofold increase in nuclear number compared to control, and this effect was completely inhibited by L-NAME, NS-398 or PDTC (P < 0.05). Although DETA-NONOate (10 microM) did not affect basal proliferation, the NO-donor augmented the stretch-induced increase in proliferation and rescued stretch-induced proliferation in NS-398-treated cells, but not in PDTC-treated cells. In conclusion, NO, COX-2, and NF-kappaB are necessary for stretch-induced proliferation of myoblasts. Although COX-2 and NF-kappaB are both involved in basal proliferation, NO does not affect basal growth. Thus, NO requires the synergistic effect of stretch in order to induce muscle cell proliferation.

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