Sex-dependent calcium dynamics in mouse skeletal muscle: Responses to cooling and caffeine

Intracellular calcium ion concentration ([Ca²⁺]i) regulation in skeletal muscle may vary with sex and muscle fiber type, but the precise nature of its response to temperature changes and pharmacological caffeine stimulation is not fully understood. This study aimed to elucidate sex-dependent and muscle fiber type-specific characteristics of muscle cooling, caffeine stimulation, and their combined effects. We investigated the effects of cooling (30 °C to 0 °C) and caffeine stimulation (1.25-80 mM) separately and in combination (cooling + 1.25 mM caffeine) in fast-twitch (plantaris, PLA) and slow-twitch (soleus, SOL) muscles of male and female C57BL/6J mice. [Ca²⁺]i dynamics were analyzed using in vivo Fura-2 bioimaging under isoflurane anesthesia. The temperature threshold for the onset of [Ca²⁺]i accumulation was significantly higher in SOL than in PLA, with no significant difference between sexes (males: PLA 2.3 ± 0.9 °C, SOL 4.5 ± 2.2 °C; females: PLA 2.3 ± 0.8 °C, SOL 4.3 ± 1.3 °C). Conversely, the [Ca²⁺]i response to caffeine was significantly higher in females than in males at high concentrations (80 mM). Furthermore, the combined stimulation of cooling and caffeine had a greater effect on females than on males. Our findings also indicate that the phosphorylation response of ryanodine receptors to caffeine was significantly higher in females than in males. In conclusion, while no sex differences were observed in the [Ca²⁺]i response to cooling, clear sex-dependent differences (males < females) were observed in the response to caffeine.