Multi-therapeutic strategy targeting Akt-mTOR and FoxO1 pathway to counteract skeletal muscle atrophy consecutive to hypoxia.

Chronic oxygen deprivation, whether due to high altitude or certain diseases as cardiorespiratory pathologies, leads to muscle atrophy. To limit muscle loss, counteracting programs rely on only one therapeutic approach: return to sea level altitude, physical activity or nutritional supplementation; however, little effects are noticed on muscle mass of subjects presenting a severe hypoxemia. We hypothesized that the combination of several treatments (electrical stimulation and/or nutritional supplementation and/or oxygenation) would improve anabolic responses, counteracting thus efficiently hypoxia-induced muscle atrophy. In C2C12 myotubes submitted to hypoxia, we aim at testing several treatments based on the combination of electrical stimulation, amino acids supplementation and/or an oxygenation period. In comparison to untreated muscle cells under hypoxia, all treatments had an anabolic impact on myotubes morphology (myogenic fusion index, diameter and density of myotubes), on proteosynthesis pathway (Akt, mTOR, GSK-3β, 4E-BP1 and P70S6K), on proteolysis pathway (FoxO1, myostatin, ubiquitin-proteasome system) and on hypoxia marker (REDD1) protein level. Electrical stimulation alone resulted in hyperphosphorylation of Akt and FoxO1 while its combination with amino acids supplementation alleviated atrophy exemplified by fusion index and myotubes diameter increase up to 48 hours post-application. Electrical stimulation followed by a period of oxygenation of hypoxic muscle cells strongly increased the activation status of 4E-BP1 and P70S6K. Lastly, simultaneous application of all treatments (electrical stimulation, amino acids supplementation and oxygenation) was the only condition resulted in activation of mTOR concomitantly to myostatin level decrease. These results support that the activation of the mTOR pathway through the combined application of electrical stimulation and BCAAs is strongly influenced by oxygen availability, and that oxygen plays a critical role in optimizing protein synthesis pathway in hypoxic skeletal muscle cells.