Influence of heat therapy on myotube fusion and markers of mitochondrial dynamics in TNF α–induced insulin resistant myotubes

Background: As the primary site of insulin-mediated glucose uptake, functional skeletal muscle (SKM) mass is critical for preventing insulin resistance (IR) and type 2 diabetes (T2D). Heat therapy has been shown to improve glycemic control in people with T2D, likely supporting improvements in SKM insulin sensitivity, but the underlying mechanisms remain unclear. Possible mechanistic pathways include improved SKM differentiation capacity and mitochondrial dynamics favoring fusion, which contribute to SKM repair, growth, and glucose metabolism. Purpose: To determine whether heat therapy alters differentiation capacity and markers of mitochondrial dynamics in C2C12 myotubes using TNFα treatment as an in vitro model of IR. Methods: After 3 days of proliferation, C2C12 myoblasts were differentiated in media containing TNFα (0, 5, or 10 ng/mL; T0, T5, or T10) for 8 days to induce IR. Differentiating myoblasts were assigned to control (NH) or heat therapy (HT) groups (N = 5 per group and TNFα concentration). Both groups were kept at 37°C. Beginning on day 1 of differentiation, cells assigned to HT were exposed to 40°C for 60 min daily using a 4 day on/1 day off/3 day on protocol. 24 hours after the final treatment, cells were either methanol-fixed or harvested, pelleted, and stored at -80°C. Fixed myotubes were HEMA3 stained and myotube density assessed as an index of differentiation capacity. Mitochondrial fission (DRP1, pDRP1S637) and fusion (OPA1, MFN2) proteins vs β actin (housekeeping) were quantified by Western blotting of total cell lysates. Results: Main effects of TNFα were found for myotube density, pDRP1S637, MFN2, and OPA1 (p< 0.001, 0.004, < 0.001, and 0.035, respectively). Myotube density was lower in T10 vs T5 and T0 (both p< 0.001) and in T0 vs T5 (p=0.01). pDRPS637 was lower in T10 vs T0 (p=0.013) and T5 (p=0.022). MFN2 was lower in T10 vs T5 and T0 (both p=0.001) and in T5 vs T0 (p=0.036). OPA1 was lower in T10 vs T5 (p=0.048). TNFα treatment did not alter total DRP1. Heat therapy did not have a significant effect on myotube density or levels of any measured markers for mitochondrial dynamics. Conclusions: Despite evidence that heat therapy protects against IR and T2D, the present study did not find heat therapy sufficient to protect against the effects high TNFα treatment on myotube differentiation or markers of mitochondrial dynamics in C2C12 myotubes. Interestingly, moderate TNFα treatment increased differentiation while preserving expression levels of mitochondrial markers measured in untreated C2C12 myotubes. Moderate TNFα likely induces hormetic stress, with a slight reduction to outer mitochondrial membrane fusion (MFN2) while maintaining inner mitochondrial membrane fusion (OPA1). Studies are underway to examine heat therapy on mitochondrial respiratory function, mitochondrial content, oxidative stress, and intracellular calcium trafficking in IR myotubes. ACKNOWLEDGMENTS: Funding: NIH/NIA R01AG084597 (mPIs: HYL, DEL) 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.