Heat stress elevates intracellular H₂O₂ and induces Tiron-attenuated gene expression in mouse skeletal muscle

Changes in intracellular hydrogen peroxide concentration ([H₂O₂]_i) serve as a key signal regulating cellular adaptation. In response to heat stress, increased ROS production and upregulation of antioxidant-related genes have been observed in skeletal muscle in vitro. However, the specific temperature dependence of [H₂O₂]_i changes induced by heat, as well as the quantitative relationship with induced gene expression, remain unclear. This investigation tested the hypothesis that the increase in [H₂O₂]_i during heat stress regulates antioxidant-related gene expression. To measure [H₂O₂]_i, the highly H₂O₂-sensitive HyPer7 fluorescent protein was expressed in the tibialis anterior (TA) muscle of male C57BL/6J mice. The temperature dependence of [H₂O₂]_i was determined via in vivo imaging during a 30-min heating protocol from 30°C to 40°C. Furthermore, one hour after maintaining the muscle temperature at 40°C for 30 minutes, the TA was excised, and gene expression was evaluated using transcriptomic analysis. It was found that raising the temperature of the TA from 30°C to 40°C for 20 minutes resulted in a 28% increase in [H₂O₂]_i (p < 0.001). This [H₂O₂]_i increase was significantly suppressed by the antioxidant Tiron (i.e., 14%). Transcriptome analysis revealed that 22 heat-induced genes were attenuated by Tiron; these were strongly enriched in Gene Ontology terms related to "unfolded protein binding". Transcription factor predictions identified HSF1 as a candidate upstream regulator of this Tiron-attenuated gene set. In conclusion, heat stress in vivo increases [H₂O₂]_i in a temperature-dependent manner, and this redox change contributes to the induction of cell protection-related genes.