Dietary nitrate supplementation mitigates age‐related changes at the neuromuscular junction in mice

In ageing, denervation and neuromuscular junction (NMJ) instability occur alongside mitochondrial alterations and redox unbalance, potentially playing a significant role in the process. Moreover, the synthetic pathway was shown to be critical for proper innervation and NMJ stability. Nitric oxide (NO) modulates redox status, mitochondrial function and the synthetic pathway. Its bioavailability declines with age. We hypothesize that nitrate supplementation could counteract age-related neuromuscular alterations. We compared young (Y) (7 months old), old (O) (24 months old) and old mice supplemented daily with 1.5 mm inorganic NaNO₃ dissolved in drinking water for 8 weeks (ON) (24 months old). Compared to Y, O mice displayed impaired NO signalling and transport (lower phosphorylated-neuronal NO synthase and sialin content); greater nitrosative and oxidative stress (higher 3-nitrotyrosine levels and protein carbonylation); lower glutathione peroxidase (GPX antioxidant enzyme); smaller muscle fibres; and larger muscle fibrosis. NMJ integrity was impaired, exhibiting age-related alterations such as larger fragmentation, lower overlap, larger endplate areas and lower compactness. Consistently, greater expression of denervation-associated markers (Gadd45α, MyoG, RUNX1, AChRγ and NCAM1) and higher NCAM1+ fibres percentage suggested denervation. Importantly, mitochondrial content, dynamics and function were unchanged. Compared to O, ON mice showed improved NO bioavailability in muscle (higher nitrate-nitrite concentration); lower fibrosis and improved muscle fibre size; higher phosphorylation of P70S6K and S6, downstream factors of Akt/mammalian target of rapamycin synthetic pathway; lower oxidative stress (lower carbonylated proteins and mitochondrial hydrogen peroxide production, higher GPX protein levels); reverted age-related alterations of NMJ morphology; and lower percentage of NCAM1+ fibres. Nitrate supplementation could be a therapeutic strategy to counteract muscle decline with ageing. KEY POINTS: Ageing leads to instability at the neuromuscular junction (NMJ), which is crucial for muscle size and function, ultimately giving rise to denervation and muscle fibres loss. Mitochondrial function, redox status and activation of synthetic pathway are critical processes for proper muscle innervation and stability of the NMJ. Nitric oxide was shown to modulate intracellular processes involved in NMJ stability such as balance of reactive oxygen species, mitochondrial function and protein synthesis. Its bioavailability decreases with ageing. Our study shows that nitrate supplementation in old mice improved redox balance, enhanced the anabolic pathway and stabilized nerve-muscle interactions, suggesting a potential strategy to mitigate the neuromuscular decline associated with ageing.