Exposure to oxidative insult (H2O2) or cigarette smoke extract (CSE) results in a similar degree of wasting in C2C12 myotubes without cellular inflammation
To further dissect the importance of oxidative stress on muscle dysfunction, we compared the direct impact of a ROS, H2O2and CSE on C2C12 myotubes. Both H2O2 and CSE exposure resulted in a similar dose-dependent reduction in myotube size (Figure 3A–B). Unlike that of the higher dose, the reduction in myotube size induced by low doses of H2O2 (5 μM) or CSE (10%) did not impact cell viability (Figure 3C and H). In line with the presence of oxidative stress, exposure to H2O2(10 and 100 μM) elicited a robust expression of Nox2 which is involved in the formation of ROS in muscle (Figure 3D) (Sakellariou et al., 2013). Unlike that of H2O2, no significant induction of Nox2 expression was observed under sub-maximal dosages of CSE (Figure 3I). Meanwhile, the expression of glutathione peroxidase 1 (Gpx1 ), a detoxifying enzyme that scavenges H2O2, was unaltered by either of the stimuli (Figure 3E and J). Noteworthy, the maximal concentration of either H2O2 or CSE was capable of eliciting a cellular inflammatory response evidenced by the increasedIl-6 gene expression (Figure 3F and K) and release of IL-6 (Figure 3G and L), but not at their respective sub-maximal concentrations. This suggests oxidative stress accounts for some of the deleterious effects of CSE and this oxidative stress-driven myofiber wasting can occur without any detectable cellular inflammation recapitulating that of our in vivo model.