Repeated heat exposure upregulates skeletal muscle aquaporin-4 expression in male mice.

Heat acclimation involves coordinated physiological adaptations, yet the role of skeletal muscle water channels remains insufficiently defined. We investigated how repeated heat exposure affects aquaporin-4 (AQP4) in skeletal muscle using both in vivo and in vitro models. Male C57BL/6NJ mice were exposed to daily passive heating (45°C for 30 min/day, 14 days). Repeated heat exposure reduced body-weight gain and markedly attenuated Hspa1a induction following an acute heat challenge, confirming systemic heat acclimation. Immunofluorescence analysis revealed an approximately 1.3-fold increase in AQP4 protein in fast-twitch muscles without changes in its subcellular localization. In vitro, C2C12 myocytes were subjected to repeated heat bouts (42°C, 3 h/day for 1-5 days). Heat-acclimated cells exhibited blunted Hspa1a induction in response to acute heat stress and progressively increased AQP4 protein levels (approximately 1.6-fold), indicating that heat stress directly promotes AQP4 upregulation in muscle cells. Together, these findings identify AQP4 as a heat-responsive component of skeletal muscle and suggest that AQP4 may contribute to peripheral osmotic regulation during heat acclimation.