Thermal acclimation results in dramatic changes in the fractional volume of mitochondria within skeletal muscle of teleost fish. We investigated the hypothesis that changes in mitochondrial volume represent a compensatory response to temperature-induced changes in intracellular diffusion coefficients (D) of the high-energy phosphate compounds ATP and creatine phosphate (PCr). Using 31P nuclear magnetic resonance spectroscopy, we determined DPCr and DATP in goldfish (Carassius auratus) skeletal muscle at 25 °C and 5 °C: DPCr was 3.28±0.18×10−6 cm2 s−1 at 25 °C and 2.00±0.09×10−6 cm2 s−1 at 5 °C; DATP was 2.13±0.16×10−6 cm2 s−1 at 25 °C and was estimated to be 1.30×10−6 cm2 s−1 at 5 °C. There was no evidence for an effect of acclimation temperature or fiber type on DATP or DPCr. A mathematical reaction–diffusion model was used to calculate profiles of [ATP], [PCr] and the free energy of ATP hydrolysis (ΔGATP) in activated goldfish muscle fibers at 5 °C and 25 °C. The results showed spatial and temporal constancy of [ATP], [PCr] and ΔGATP in red fibers at both temperatures, regardless of changes in acclimation temperature or mitochondrial density. The model also showed spatial and temporal constancy of [ATP] in white fibers at 5 °C and 25 °C, but gradients in [PCr] and ΔGATP developed in white fibers under all conditions of temperature and acclimation temperature. These gradients were attenuated in cold-acclimated animals by cold-induced increases in mitochondrial density. However, the model shows that the proximal stimulus for temperature-induced changes in mitochondrial volume density in muscle is not a disruption in intracellular diffusion of high-energy phosphates.

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