Acute simulated hypoxia and ischemia in cultured C2C12 myotubes : decreased phosphatidylinositol 3-kinase (PI3K)/Akt activity and its consequences for cell survival

[摘要] Cells are equipped with an array of adaptive mechanisms to contest the undesirable effects ofischemia and the associated hypoxia. Indeed, many studies have suggested that there is anincrease in the PI3K/Akt pathway activation during hypoxia and ischemia. Damaged muscle canbe regenerated by recruiting myogenic satellite cells which undergo differentiation andultimately lead to the regeneration of myofibres. The C2C12 murine myogenic cell line ispopular for studying myogenesis in vitro, and has been used in many studies of ischemicmicroenvironments. PI3K/Akt pathway activity is increased during C2C12 myogenesis and thisis known to produce an apoptosis resistant phenotype. In this study, we provide evidence thathigh basal levels of PI3K activity exist in C2C12 myotubes on day ten post-differentiation.Ischemia is characterized by depleted oxygen and other vital nutrients, and ischemic cell death isbelieved to be associated with an increasingly harsh environment where pH levels decrease andpotassium levels increase. By employing a model that mimics these changes in skeletal muscleculture, we show that both acute simulated ischemia and acute hypoxia cause decreases inendogenous levels of the p85 and p110 subunits of PI3K and a consequent reduction in PI3Kactivity. Supplementing skeletal muscle cultures with inhibitors of the PI3K pathway providesevidence that the protective effect of PI3K/Akt is subsequently lost in these conditions. UsingWestern blot analysis, a PI3K ELISA assay as well as known inhibitors of the PI3K pathway inconjunction with the MTT assay we are able to demonstrate that the activation of downstream effectors of PI3K, including Akt, are concurrently decreased during acute simulated ischemiaand acute hypoxia in a manner that is independent of PDK-1 and PTEN and that the decreases inthe PI3K/Akt pathway activity produce a knock-on effect to the downstream signalling oftranscription factors, such as Fox01 and Fox04, in our model. We proceed to provide compellingevidence that the apoptotic resistance of C2C12s is at least partially lost due to these decreases inPI3K/Akt pathway activity, by showing increased caspase-3 and PARP cleavage. Then, usingvital staining techniques and a DNA fragmentation assay, we demonstrate increased cellmembrane impairment, cell death and apoptosis after three hours of simulated ischemia andhypoxia in cultured C2C12 myotubes. In addition to the main findings, we produce evidence ofdecreased flux through the mTOR pathway, by showing decreased Akt-dependantphosphorylation at the level of TSC2 and mTOR during simulated ischemia and hypoxia.Finally, we present preliminary findings indicating increased levels of HIF1α and REDD-1,representing a possible oxygen sensing mechanism in our model. Therefore, we show that thereis in fact a rapid decrease in PI3K/Akt activity during severe, acute simulated ischemia andhypoxia in C2C12 myotubes on day ten post-differentiation, and this causes a concomitant downregulation in cell survival pathways and increased activity of cell death machinery. Thereafter,we propose a possible mechanism of action and provide a platform for future studies.

[发布机构] Stellenbosch University