## 4 Discussion
The data indicate that under hypoxia, a failure of cell withdrawal from the cell cycle results in a reduction in the cell population committed for terminal differentiation. Consistent with our data, previous studies showed inhibited myogenic differentiation under physiological to severe hypoxia (\(\leq\)3% O\({}_{2}\)) for C2C12 myoblasts as well as primary murine satellite cells [39; 40; 41; 72]. Cell cycle withdrawal plays an important role in terminal differentiation [39]. In the same line, the morphometric analysis results followed a common trend in terms of the reduction in the myotube formation parameters under hypoxia indicative of terminal differentiation, suggesting impaired myogenic differentiation. One possible interpretation is that differentiation under hypoxia reduces the number of myoblasts committed to cell fusion and myotube formation. Another justification of our data is that hypoxia causes a short-term deacetylation of histones related to MyoD promoters, leading to decreased MyoD expression and delayed myogenin expression, with a subsequent reduction in MHC production and myotube formation [72]. Along this line, it was reported that hypoxia activates the transcription factor Bhlhe40 via the p53 signaling pathway, which reduces the binding capacity between MyoD and myogenin, resulting in an inhibition of myogenin expression [41]. A recent study demonstrated that hypoxia suppresses myoblast function through an epigenetic modulation of autophagy as well as the activation of histone deacetylase 9, resulting in an inactivation of canonical Wnt signaling, which impairs MyoD and myogenin expression [43]. The analysis revealed upregulated VEGF expression in the myogenic differentiated cells under hypoxia compared to non-induced cells, suggesting the induction of an angiogenic response to compensate for the low O\({}_{2}\) concentration. A similar report showed increased VEGF mRNA expression under hypoxia [46; 73; 74]. In contrast to hypoxia, an upregulated mTOR expression was detected under normoxia, indicative of increased protein synthesis and justifying the impaired myotube formation shown under hypoxia. Unexpectedly, the upregulation of MHC1 and MHC7 was detected after day 7 under hypoxia without an increase in mTOR expression, suggesting impaired myogenic differentiation. In agreement with our results, a study revealed that hypoxia converts the myogenic potential of insulin growth factors (IGFs) into a mitogenic effect by suppressing the IGF-induced Akt/mTOR and p38 signaling pathway [42]. The inhibition of the Akt/mTOR signaling pathway was referred to as a hypoxia-related reduction in IGF-1 receptor sensitivity [75]. Similarly, it was found that the activation of HIF1\(\alpha\) under hypoxia suppresses myogenic differentiation, on the one hand by activating the Notch signaling pathway and on the other hand by inhibiting the canonical Wnt signaling pathway [44; 76]. The transcription factor HIF1\(\alpha\) plays for a crucial role in maintaining oxygen homeostasis and regulating cellular adaptations under hypoxia [29]. Our results indicate that there was a variability in HIF1\(\alpha\) expression for both experimental conditions at 3 h, 72 h, and day 7, but this was not statistically significant. The data suggest that the inconsistency in HIF1\(\alpha\) expression may be due to its short lifespan and rapid adjustment of mRNA expression. Consistent with previous studies, our results demonstrate that the gene expression of HIF1\(\alpha\) remains unchanged or even reduced under 1% O\({}_{2}\) concentration in myoblasts [46; 73; 74]. Similarly, a study found that the increase in HIF1\(\alpha\) expression in vivo under hypoxia is only temporary or non-existent in many cell lines [29]. On the other hand, HIF1\(\alpha\) seems to play a role in myogenic differentiation under normoxic conditions, and it was reported that under normoxic conditions, constant HIF1\(\alpha\) expression was observed in regenerating myotubes during the course of myogenic differentiation [77; 78].