Cooling-induced changes in intracellular hydrogen peroxide and gene expression in mouse skeletal muscle in vivo

Changes in intracellular hydrogen peroxide concentration ([H₂O₂]) constitute an important signal-controlling cellular adaptations. In response to cooling, decreases in [H₂O₂] and changes in antioxidant-related gene expression have been observed in skeletal muscle. However, the specific temperature dependence of cooling-induced [H₂O₂] changes and their quantitative relationship to induced gene expression are unknown. This investigation tested the hypothesis that differences in muscle cytosolic and mitochondrial [H₂O₂] changes during cooling/rewarming determine the pattern of H₂O₂-related gene expression. H₂O₂-sensitive cytosolic (HyPer7) and mitochondrial (MLS-HyPer7) fluorescent proteins were expressed into tibialis anterior (TA) muscle of male C57BL/6J mice. The temperature dependence of [H₂O₂] was determined via in vivo imaging during a 3-min cooling protocol from 35°C to 0°C. Two cooling patterns [6 bouts of intermittent cooling (I-Cool) vs. sustained cooling (S-Cool); both to 13°C] were applied over 60 min. Three hours after cooling, the muscles were removed, and gene expression was evaluated using real-time PCR. The decrease in [H₂O₂] was observed in both cytosolic and mitochondrial compartments from 35°C to 13°C but was of greater magnitude in the cytosol; in contrast, further cooling from 12°C to 0°C induced a rebound increase especially in cytosolic [H₂O₂]. I-Cool increased the mRNA level of Nrf2 (+15%, <i>P</i> < 0.001). S-Cool decreased the mRNA levels of Sod2, Cat, and Ucp3 (i.e., -20, -23, and -30%, respectively, <i>P</i> < 0.05). In conclusion, the greatest decrease in temperature-dependent [H₂O₂] occurred at 13°C in the cytosolic and mitochondrial compartments of muscle fibers, and I-Cool increased Nrf2 mRNA expression, whereas S-Cool decreased several antioxidant-related genes.<b>NEW & NOTEWORTHY</b> This in vivo model successfully characterized the effects of cooling on cytosolic and mitochondrial [H₂O₂] in mouse tibialis anterior skeletal muscle. Cooling decreased [H₂O₂] down to ∼13°C, but the effect was reversed at still lower temperatures. Sustained cooling decreased mRNA levels of antioxidant-related genes (Sod2, Cat, and Ucp3), whereas intermittent cooling increased Nrf mRNA expression. These results help elucidate the mechanistic bases for skeletal muscle adaptation to cooling.