High Throughput Screening of Mitochondrial Bioenergetics in Myoblasts and Differentiated Myotubes

HIF-1α/MMP-9 Axis Is Required in the Early Phases of Skeletal Myoblast Differentiation under Normoxia Condition In Vitro

“…Accordingly, C2C12 cells were cultured in PM for 24 h and then shifted to DM different lengths of time (24, 48, 72 h and 5 days) to observe the differentiation, al maintaining physiological oxygen levels. We observed that C2C12 cells after 24 h of ture in differentiation medium (DM, DMEM + 2% HS) exhibited nuclear expression o Western blot analyses of SDH-B and LDH-A suggested that the cells underwent a metabolic shift during differentiation, from anaerobic glycolysis to the oxidative pathway, confirming previously published data [7,8] and contributing to validating our cell myogenic differentiation model. Indeed, we observed a decrease in the expression of LDH-A over time (Figure 2B) concomitant to an increase in SDH-B expression (Figure 2A).…”

“…Western blot analyses of SDH-B and LDH-A suggested that the cells metabolic shift during differentiation, from anaerobic glycolysis to the oxida confirming previously published data [7,8] and contributing to validating o genic differentiation model. Indeed, we observed a decrease in the expressi over time (Figure 2B) concomitant to an increase in SDH-B expression (Figu In line with these observations, the cells also exhibited an increase in th of PGC-1α, which is recognized as a key regulator of mitochondrial bioge 2C-H), which reached the maximum expression level in the myotubes (Figu Subsequently, we investigated the expression and subcellular localizati during C2C12 myoblast differentiation by RT-PCR, Western blotting and co nofluorescence analyses (Figure 3).…”

“…However, in accordance with our findings and other previous research [19,28], these papers [21,29] demonstrated the nuclear expression of HIF-1α in the early phase (induction) of C2C12 cell myogenesis. In myoblasts the nuclear HIF-1α expression may be consistent with the functional induction of the genes encoding glycolytic enzymes required to satisfy the cell energy demand in the early phases of the myogenic differentiation process [7][8][9]19,[50][51][52]. Along these lines, we have confirmed, in our experimental myogenesis model, that the cells undergo metabolic reprogramming during the differentiation process; in fact, proliferating myoblasts mostly use glycolysis to produce energy whereas myotubes show a more developed aerobic capacity, as assessed by the analyses of the expression of LDH-A and SDH-B.…”

“…Similarly to other stem cells, SCs undergo an energy metabolic reprogramming during differentiation, consisting of a shift in the use of metabolic substrate. In particular, proliferating myoblasts produce energy mainly through glycolysis, which is different to myotubes which exhibit a major aerobic capacity [7][8][9]. Several papers highlighted the hypoxia-inducible factor (HIF)-1α as a regulator of myogenesis and SCs/myoblasts' homeostasis in vitro and in vivo [10][11][12][13][14][15][16][17][18][19][20][21][22].…”