Assessment of Exercise-Like Electrical Pulse Stimulation on SIRT1 Signaling and Mitochondrial Function in Ethanol-Treated Myotubes

Chronic alcohol exposure profoundly impairs skeletal muscle metabolic health by disrupting mitochondrial redox homeostasis and suppressing key deacetylation pathways that regulate mitochondrial quality and biogenesis. Our previous work demonstrated that alcohol impairs myoblast differentiation and muscle regeneration. Exercise has been shown to enhance myogenic function and improve mitochondrial health, yet how it directly modulates myogenic and mitochondrial regulatory pathways remains unclear. The goal of this study was to determine whether electrical pulse stimulation (EPS), as an exercise mimic, can mitigate ethanol-induced mitochondrial dysfunction in differentiated skeletal muscle myotubes. C2C12 myoblasts were differentiated into myotubes for 7 days and exposed to ethanol (50 mM, 72 h). During differentiation, cells received exercise-like EPS (25 Hz, 1-ms pulses every 5 s for 1 h/day) in the presence or absence of ethanol. Gene expression of mitochondrial regulators was quantified by RT-qPCR, and SIRT1/SIRT3 protein expression and PGC-1α acetylation were evaluated by Western blot and immunoprecipitation assays. Mitochondrial function was assessed using the Seahorse XF Pro Mito Stress Test, while mitochondrial content and reactive oxygen species (ROS) were measured by live-cell imaging with MitoTracker Green (100 nM) and MitoSOX Red (500 nM). Samples were stained for 30 min at 37°C, imaged on a Keyence BZ-X810 microscope, and analyzed by two-way ANOVA with Tukey’s post-hoc test. Ethanol significantly reduced SIRT1 (p< 0.001) and SIRT3 (p< 0.05) protein expression and increased PGC-1α acetylation, consistent with suppressed SIRT1 deacetylase activity and impaired mitochondrial biogenesis signaling. Ethanol also impaired mitochondrial respiration, lowering maximal respiration (p< 0.05) and spare respiratory capacity (p< 0.01), while increasing mitochondrial ROS and reducing mitochondrial content (p< 0.05). EPS improved mitochondrial function across groups, enhancing oxygen consumption rate and restoring spare respiratory capacity (p< 0.05). Notably, EPS-treated ethanol cultures exhibited mitochondrial profiles approaching those of untreated controls. Together, these findings demonstrate that chronic EPS rescues both regulatory (SIRT1/PGC-1α) and functional mitochondrial deficits caused by ethanol exposure. Ongoing studies will define additional molecular mechanisms through which exercise-like stimulation promotes mitochondrial resilience and myogenic capacity under alcohol-induced stress. This work was supported by F32AA031901 (KMRG), P60AA009803 (PEM), and R21AA030869 (LS). This abstract was presented at the American Physiology Summit 2026 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.