Maintaining mitochondrial NAD+ homeostasis is key for heat-induced skeletal muscle injury prevention despite presence of intracellular cation alterations.

Abstract

Mitochondrial dysfunction is implicated in heat-induced skeletal muscle (SKM) injury and its underlying mechanisms remain unclear. Evidence suggests that cellular ions and molecules, including divalent cations and adenine nucleotides, are involved in the regulation of mitochondrial function. In this study, we examined Ca²⁺, Mg²⁺, and NAD⁺ levels in mouse C2C12 myoblasts and SKM in response to heat exposure. During heat exposure, mitochondrial Ca²⁺ levels increased significantly, whereas cytosolic Ca²⁺ levels remained unaltered. The mitochondrial Ca²⁺ levels in the SKM of heat-exposed mice were 28% higher compared to control mice. No changes in cytosolic Ca²⁺ were detected between the two groups. Following heat exposure, cytosolic and mitochondrial Mg²⁺ levels were reduced by 47% and 23% in C2C12 myoblasts, and by 51% and 44% in mouse SKMs, respectively. In addition, heat exposure decreased mitochondrial NAD⁺ levels by 32% and 26% in C2C12 myoblasts and mouse SKMs, respectively. Treatment with the NAD⁺ precursor nicotinamide riboside (NR) partially prevented heat-induced depletion of NAD⁺. Additionally, NR significantly reduced heat-increased mitochondrial fission, mitochondrial depolarization, and apoptosis in C2C12 myoblasts and mouse SKMs. No effects of NR on heat-induced changes in intracellular Ca²⁺ and Mg²⁺ levels were observed. This study provides in vitro and in vivo evidence that acute heat stress causes alterations in mitochondrial Ca²⁺, Mg²⁺, and NAD⁺ homeostasis. Our results suggest mitochondrial NAD⁺ homeostasis as a therapeutic target for the prevention of heat-induced SKM injury.