Electrical Pulse Stimulation and N-Acetylcysteine Protect Against Oxidative Stress-Induced Mitochondrial Dysfunction in Muscle Cells

Chronic alcohol exposure is known to induce oxidative stress, impairing mitochondrial function in skeletal muscle, which contributes to metabolic dysfunction and reduced muscle performance. Effective interventions, such as exercise and antioxidant supplementation with N-acetylcysteine (NAC), have shown potential in enhancing mitochondrial function and skeletal muscle health. In this study, we hypothesize that the combined intervention of electrical pulse stimulation (EPS), as a simulation of exercise, and N-acetylcysteine (NAC) will mitigate tert-butyl hydroperoxide (TBHP)-induced mitochondrial dysfunction in differentiated skeletal muscle myotubes. C2C12 mouse myoblasts were differentiated into myotubes and subjected to TBHP (400 µM, 24 hours) to induce oxidative stress. Myotubes underwent EPS (30V, 1Hz, 2ms pulses, 24 hours) simulating exercise, with or without concurrent NAC treatment (1 mM) in the presence or absence of TBHP. Mitochondrial function was evaluated using the Seahorse XF Pro Analyzer Mito Stress Test. Mitochondrial content and reactive oxygen species (ROS) levels were assessed with live-cell imaging using MitoTracker Green (100 nM) and MitoSOX Red (500 nM), respectively. Samples were stained for 30 minutes at 37°C, visualized via the Keyence BZ-X810 microscope, and analyzed using two-way ANOVA with Tukey’s post-hoc test. The results demonstrated that TBHP treatment significantly elevated ROS levels compared to controls (p<0.01), indicating increased oxidative stress. Both, EPS and NAC independently reduced ROS levels (p<0.05), while their combination was most effective, bringing ROS levels close to control values. TBHP exposure also caused notable mitochondrial dysfunction, as evidenced by decreased maximal respiration and spare respiratory capacity. Interventions with EPS and NAC showed trends toward restoring mitochondrial function, and the combined approach significantly improved mitochondrial health scores, approaching those of the control group. This study demonstrates that exercise and NAC supplementation independently and combined mitigate mitochondrial dysfunction caused by TBHP-induced oxidative stress. These findings highlight the potential for combined antioxidant and exercise interventions to promote skeletal muscle health under oxidative stress conditions. This research was supported by NIH/NIAAA T32AA007577, P60AA009803 (PEM) and R21AA030869 (LS). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.