## 3 Results
### Morphometric Assessment of Myogenic Differentiation under Hypoxia  
Under both culture conditions, an incipient formation of myotubes indicative of myogenic differentiation could be detected. The initial observation demonstrated fewer small myotubes being visible under hypoxic conditions compared to predominantly short and wide myotubes under normoxia. To evaluate whether a low O\({}_{2}\) concentration modulates myotube formation, morphometric measurements of the myotubes after day 7 were carried out. The data analysis showed a reduction in the count and the size (\(\mu\)m\({}^{2}\)) of the myotubes under hypoxia (\(p<0.05\)) compared to the cells cultivated in normoxic conditions (Figure 6a,b,g,h). In contrast, by measuring the length of the individual myotubes, the data showed increases in the length of the myotubes under hypoxia (\(p<0.05\)) compared to normoxic conditions (Figure 6c). To assess the fusion capacity of myoblasts, the percentage  
Figure 5: The effect of hypoxia on myogenic relative markers expression after 3 h, 72 h and 7 days under normoxic (NX) and hypoxic (HX) conditions. (**a**) Relative MyoD expression. (**b**) Relative myogenin expression. (**c**) Relative nitric oxide synthase 1 (NOS1) expression. (**d**) Relative vascular endothelial growth factor (VEGF) expression. (**e**) Relative mammalian target of rapamycin (mTOR) expression (**f**) Relative myosin heavy chain 7 (MHC7) expression. Non-induced cells processed in parallel in basal medium served as a negative control (NC). Data are presented as means \(\pm\) SEM. * = \(p<0.05\), ** = \(p<0.01\).  
of nuclei within the myotubes relative to the total nuclei in the microscopic field was quantified and referred to as the fusion index. The data analysis revealed a decrease in the fusion index of the myotubes under hypoxia (\(p<0.01\)) compared to the same measurement under normoxia (Figure 6d). Similarly, a reduction in the number of nuclei per myotube (n) was observed under hypoxia compared to normoxia (Figure 6e). The analysis of the nuclear to cytoplasmic ratio (N/C) for the individual myotubes by dividing the myotube size (\(\upmu\)m\({}^{2}\)) by the number of nuclei within the myotube was performed. The analysis revealed increases in these values under hypoxia (\(p<0.01\)) compared to normoxic conditions (Figure 6f-h). It is demonstrated that differentiation under hypoxia reduces the fusion index as well as increasing the nuclear-cytoplasmic ratio compared to cells under normoxia.  
Figure 6: Morphometric assessment of myogenic differentiation under hypoxic (HX) compared to normoxic (NX) conditions (\(n=6\)). (**a**) Number of myotubes. (**b**) Size of myotubes (\(\upmu\)m\({}^{2}\)). (**c**) Length of myotube (\(\upmu\)m). (**d**) Myoblast fusion index (%). The number of nuclei in myotubes was divided by the total number of nuclei within a microscopic field. (**e**) Number of nuclei per individual myotube. (**f**) Quantification of nuclear to cytoplasmic ratio (N/C). The area of the myotube was divided by the number of nuclei within the myotube. (**g**,**h**) Immunofluorescence shows myosin heavy chain-positive myotubes (red) after 7 days under NX and HX conditions, respectively. Data are presented as means \(\pm\) SEM. * = \(p<0.05\), **= \(p<0.01\)**. DAPI (blue) was used to visualize the nuclei. Scale bar = 20 \(\upmu\)M.