| Oxygen plays an important role in energy metabolization and homeostasis as well as a variety of life processes. Changes of oxygen concentration, as an important physiological and pathological regulator, might influence the whole life span, from embryogenesis and development to the maintenance of normal function, dysfunction, disease and aging. The concentration of oxygen in atmosphere is 21%, whereas the level of oxygen at the tissue in vivo is significantly less. Arterial blood is about 12% oxygen, and the mean tissue level of oxygen is about 3%, the partial saturation of oxygen found in mature skeletal muscle is reported to be between 1 to 10%. A few muscle stem cells exist in adult mammalian skeletal muscle. Muscle stem cells can be activated to initiate proliferation and give rise to spindly myoblasts, which undergo differentiation and cell fusion into multinucleated myotubes. Muscle satellite cells isolated from skeletal muscle and cultured in vitro are called myoblasts. It is obvious that the tissue-specific myoblasts are required for maintenance and repair of postnatal skeletal muscle. So, physiological hypoxia is actually optimum condition for myogenesis. There have been few studies on effects of hypoxia on proliferation and differentiation of myoblasts up to now. In the present study we observed effects of 3% O2 on myogenic differentiation of C2C12 myoblasts and probed possible mechanisms involved in the inhibition of myogenic differentiation by hypoxia.We firstly observed the differentiation of C2C12 myoblasts in normoxia or 3% O2 by using immunofluorescence and immunocytochemistry analysis. The results showed that C2C12 myoblasts cultured in DM under 3% O2 conditions hardly fused into myotubes and expressed MHC protein compared with cells cultured under normoxic conditions, which was also confirmed by Western Blot analysis. RT-PCR and Western Blot analysis detected the expression of MRFs in C2C12 myoblasts, and hypoxia down-regulated the expression of MRFs at both mRNA and protein levels.The above data indicated that 3% O2 inhibited myogenic differentiation of myoblasts. After the phenomenon of the inhibition of myogenic differentiation by hypoxia was demonstrated, we further probed possible mechanisms involved in the inhibition of myogenic differentiation by hypoxia. HIF-1 is an important transcription factor expressed in response to hypoxic conditions. To investigate whether HIF-1|áwas involved in the inhibition of myogenic differentiation by hypoxia, RT-PCR and Western Blot analysis detected the expression of HIF-1|áat both mRNA and protein levels in C2C12 myoblasts cultured in DM under hypoxic conditions. The results showed that HIF-1|áprotein in C2C12 myoblasts was stable and not degraded under normoxic conditions, and the expression of HIF-1|áwas hardly influenced by oxygen conditions at both protein level and transcriptional level. Meanwhile, we detected the location of HIF-1|áprotein in differentiating C2C12 myoblasts and found that hypoxia did not influence the location of HIF-1|áprotein. These above results suggest that the inhibition of myogenic differentiation by hypoxia might be independent of HIF-1 signaling.We found that the expression level of Smad3 protein decreased during the inhibition of myogenic differentiation by hypoxia. However, the forced expression of exogenous Smad3 did not rescue the inhibition of myogenic differentiation by hypoxia. These data indicated that Smad3 protein did not play a vital role during the inhibition of myogenic differentiation by hypoxia. In addition, down-regulation of Smad3 protein might be a concomitant event during the inhibition of myogenic differentiation by hypoxia. Subsequently, we investigated the possible cause of Smad3 protein decrease and found that the expression level of phospho-Smad3 protein deceased during the inhibition of myogenic differentiation by hypoxia, which was paralleled by a decrease in Smad3 protein under hypoxic conditions. It was reported that the eventual fate of receptor-activated Smads was controlled by ubiquitin-mediated proteolysis. We added MG-132, the inhibitor of the 26S proteasome, into cultures and found that MG-132 induced accumulation of phospho-Smad3 in C2C12 myoblasts cultured in hypoxia. These results suggest that hypoxia-induced loss of phospho-Smad3 protein involve its degradation by the ubiquitin-proteasome pathway.ERK1/2 MAPK pathway plays a role in myogenesis. Further investigation on the mechanism involved in the inhibition of myogenic differentiation by hypoxia showed that ERK1/2 were phosphorylated and activated in differentiating C2C12 myoblasts, however, ERK1/2 activation was suppressed during the inhibition of myogenic differentiation by hypoxia. To investigate whether ERK1/2 MAPK pathway was involved in the inhibition of myogenic differentiation by hypoxia, we transiently transfected a constitutively active MEK1(E) construct, which phosphorylated and activated ERK1/2 in C2C12 myoblasts. Western Blot analysis showed that the level of MHC expression in C2C12 myoblasts transfected with MEK1(E) was higher than that in cells transfected with empty vector control under hypoxic conditions, which was also confirmed by immunofluorescence analysis. These results indicated that increasing ERK1/2 activity by forced expression of MEK1(E) could partially reverse the inhibition of myogenic differentiation by hypoxia. These suggest that ERK1/2 MAPK pathway might be involved in the inhibition of myogenic differentiation by hypoxia.Taken together, combining with our previous laboratory results that 3% O2 promoted proliferation of myoblasts, our findings indicated a new effect of hypoxia on skeletal muscle development that physiological hypoxia (3% O2) promoted proliferation of myoblasts and inhibited myogenic differentiation of myoblasts. During investigating possible mechanisms involved in the inhibition of myogenic differentiation by hypoxia, we found a possible mechanism that ERK1/2 MAPK pathway might be involved in hypoxia-mediated inhibition of myogenic differentiation.
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