Regulation of mitochondrial oxidative proteins and content markers in response to chronic heat in TNFα-treated C2C12 myotubes

Mitochondrial bioenergetic dysfunction in skeletal muscle (SKM) is a recognized metabolic feature associated with insulin resistance (IR) and type 2 diabetes. Possible contributing mechanisms include decreased mitochondrial content and expression of electron transport chain (ETC) complex proteins. Additionally, improving SKM mitochondrial function is a promising strategy for addressing IR. One such strategy is mild heat stress, evidenced by reports that repeated intermittent heat increases expression of ETC proteins and promotes mitochondrial biogenesis in healthy myotubes. However, the impact of chronic intermittent heat on markers of mitochondrial bioenergetic function in IR myotubes remains unexplored. HYPOTHESIS: We hypothesized that chronic intermittent heat (HC) would preserve mitochondrial content and ETC protein in IR myotubes. METHODS: C2C12 myoblasts were cultured in growth media at 37°C for 3 days. Then, growth media was replaced with differentiation media containing 0 (vehicle), 5, or 10 ng/ml tumor necrosis factor-alpha (TNFα) as an established method of inducing IR in myotubes. Myotubes were assigned to HC or a no heat control (NC) group (N=5 per group and TNFα concentration). HC myotubes were exposed to heat stress from day 1 of differentiation (40°C, 8 days, for 60 min/day) with a one-day (day 5) rest from heat. NC myotubes were maintained at 37°C (i.e., no heat exposure). Cells were harvested 24 hrs after the final heating session, and expression of ETC complex proteins (NDUFB8, complex I; COXIV, complex IV) and mitochondrial content markers (TOM20, VDAC) were assessed by Western blotting. RESULTS: For NDUFB8, there was a main effect of TNFα (p< 0.001) where NDUFB8 expression was significantly lower in myotubes treated with 10 ng/ml TNFα than 0 (p=0.015) or 5 (p=0.002) ng/ml TNFα regardless of heat. For COXIV, there was a TNFα × heat interaction effect (p=0.035). In NC, COXIV expression was lower in myotubes treated with 10 vs 5 ng/mL TNFα (p=0.036). In HC, COXIV was lower in myotubes treated with 10 vs 0 ng/ml (p=0.018) and 5 (p=0.01) ng/ml TNFα. For TOM20, there was a TNFα × heat interaction effect (p=0.041). In NC, TOM20 expression increased in myotubes treated with 10 ng/mL TNFα than 0 (p=0.043) or 5 (p=0.049) ng/ml TNFα, but HC prevented this increase. VDAC expression remained unchanged across groups. CONCLUSION: Contrary to our hypothesis, HC did not prevent a decrease in expression of selected ETC proteins in IR myotubes. However, HC prevented an increase in TOM20 in IR myotubes, which likely reflects more stable mitochondrial content in heat-exposed myotubes across TNFα treatments. It is possible that the increase in mitochondrial content in IR myotubes in the NC group was due to mitohormesis in response to increased mitochondrial stress. Therefore, the lack of change in mitochondrial content with HC suggests an improved stress response (e.g., antioxidant capacity) and mitochondrial function in IR myotubes, but this remains to be tested. 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.