Extracellular Vesicles and Nanoparticles Released from Electrically Stimulated C2C12 Myotubes Protect H9c2 Cardiomyocytes from Oxidative Stress-Induced Damage

While the benefits of exercise are well-ackowledged, the underlying molecular mechanisms remain to be fully understood. Previous research in our laboratory demonstrated that electrical pulse simulation (EPS) of cultured C2C12 myotubes activates the Nrf2 pathway, resulting in an upregulation of key antioxidant enzymes that protect the myotubes from oxidative stress-induced cytotoxicity. In the current study, we hypothesized that these upregulated antioxidant enzymes could be secreted via extracellular vesicles (EVs) and non-vesicular extracellular nanoparticles (nv-ENPs). Additionally, we hypothesized that these particles could protect H9c2, a rat cardiac cell line, from hydrogen peroxide (H 2 O 2 )-induced damage. Using differential ultracentrifugation (dUN), EVs and nvENPs were isolated from the conditioned media of EPS-treated C2C12 cells (one hr/day for six consecutive days) and compared to those from non-stimulated cells. Transmission electron microscopy (TEM) revealed that the isolated particles displayed a characteristic morphology, with EVs showing a cup-shaped structure and lipid bilayer membrane, while nvENPs exhibit a pill-like and amembranous structure with high electron density. Western blot analysis demonstrated that EVs/nvENPs from EPS-C2C12 myotubes contained higher levels of antioxidant proteins downstream of the Nrf2 pathway (NQO1 and GSTA2) than those isolated from non-stimulated myotubes. Using a CCK-8 cell viability assay, we found that H9c2 cells pretreated with EPS-C2C12-derived EVs/nvENPs showed significantly higher survival rates following H 2 O 2 challenge compared to cells treated with particles from non-stimulated C2C12 cells (p < 0.05, n = 3/group). Our findings suggest that electrically stimulated C2C12 myotubes release antioxidant-enriched EVs/nvENPs that protect cardiomyocytes from oxidative stress. This reveals a potential novel mechanism by which exercise benefis cardiovascular health, wherein contracting skeletal muscles release antioxidant-enriched EVs/nvENPs into the bloodstream, which are then taken up by the heart to enhance its antioxidant defenses and mitigate oxidative stress-related damage, such as that seen in ischemia/reperfusion injury. R01 HL160820 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.