Rapamycin maintains NAD⁺/NADH redox homeostasis in muscle cells

Rapamycin delays multiple age-related conditions and extends lifespan in organisms ranging from yeast to mice. However, the mechanisms by which rapamycin influences longevity are incompletely understood. The objective of this study was to investigate the effect of rapamycin on NAD⁺/NADH redox balance. We report that the NAD⁺/NADH ratio of C2C12 myoblasts or differentiated myotubes significantly decreases over time in culture, and that rapamycin prevents this effect. Despite lowering the NADH available to support ATP generation, rapamycin increases ATP availability, consistent with lowering energetic demand. Although rapamycin did not change the NAD⁺/NADH ratio or steady-state ATP concentration in the livers, kidneys, or muscles of young mice, optical redox imaging revealed that rapamycin caused a substantial decline in the NADH content and an increase in the optical redox ratio (a surrogate of NAD⁺/NADH redox ratio) in muscles from aged mice. Collectively, these data suggest that rapamycin favors a more oxidized NAD⁺/NADH ratio in aged muscle, which may influence metabolism and the activity of NAD⁺-dependent enzymes. This study provides new insight into the mechanisms by which rapamycin might influence the aging process to improve health and longevity among the aging population.