Figure 3.

Increased fatty acid β-oxidation by high CO₂ impairs C2C12 myoblasts differentiation into myotubes. (A) C2C12 myoblasts were exposed to normal (N) or high CO₂ conditions (H) for 3 days, harvested, lysated, and protein expression analyzed by Western Blot. Upper panel: graph showing relative values of phosphorylated‐ vs. total-AMP-activated Kinase (AMPK) and Acetyl-CoA carboxylase (ACC; n = 6–10). Lower panel: representative Western blots. (B–E) Mitochondrial respiration was measured using Seahorse technology to detect the OCR values in C2C12 myoblasts exposed for 3 days to normal (N) or high CO₂ (H) conditions in the absence or presence of 10 μM of the fatty acid import inhibitor etomoxir (ETO). (B) Graph shows a representative OCR after treatment with oligomycin, FCCP and rotenone. (C) Graph shows basal mitochondrial respiration. (D) Graph shows coupled mitochondrial respirationb. (E) Graph shows maximal mitochondrial respiration. (F) C2C12 myoblasts were exposed to normal (N) or high CO₂ conditions (H) for 3 days in the absence or presence of 10 μM ETO and allowed to differentiate in normal CO₂ conditions up to 4 days. Upper panel: graph showing fusion index (n = 4–5). Lower panel: representative immunofluoresecent images from C2C12 myoblasts at 4 days of differentiation showing MHC (green) expression and nuclei (blue). Scale bar: 100 μm. Data from (A) was analyzed by unpaired t-test. Data from (C–F) were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; n.s: not significant.