Introduction
Skeletal muscle disuse reduces muscle protein synthesis rates, induces atrophy, and lowers mitochondrial function. Overexpression of mitochondrial calcium transporter can attenuate denervation-induced muscle atrophy, suggesting a relationship between mitochondria, calcium, and protein metabolism. Since dietary nitrate alters calcium homeostasis, we examined the impact of nitrate supplementation on disuse-induced changes in protein synthesis rates and mitochondrial function in mice.

Methods
Female C57Bl/6N mice were subjected to unilateral hindlimb casting and consumed standard drinking water (H₂O) or water supplemented with 1mM sodium nitrate (NO₃). All water throughout immobilization (3- or 7-days) contained 4% deuterated water. Mitochondrial respiration, protein content, and myofibrillar protein synthesis rates were measured in red gastrocnemius. Subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria were isolated from vastus lateralis to measure mitochondrial protein synthesis rates. Data were analyzed using paired Student’s t-tests within each group (control vs. immobilized limb) and expressed as mean±SD.

Results
Compared to the control limb (C), immobilization (I) induced muscle atrophy (3- and 7-days), decreased myofibrillar protein synthesis rates (3- and 7-days), and reduced mitochondrial protein synthesis rates (7-days, p=0.05). Dietary nitrate did not prevent these impairments. However, nitrate prevented the immobilization-induced reductions in mitochondrial content and respiration (3- and 7-days). In addition, a protective effect of nitrate on mitochondrial protein synthesis rates occurred at 3-days. While mitochondrial protein synthesis rates were ~30% lower following 3-days of immobilization in H₂O-consuming mice, nitrate prevented this decline in both SS (NO₃ C: 3.79±0.73 vs. NO₃ I: 3.49±0.84 %/day, p=0.44) and IMF (NO₃ C: 2.69±0.62 vs. NO₃ I: 2.34±0.61 %/day, p=0.11) mitochondria. 

Conclusion
Dietary nitrate mitigated the immobilization-induced impairments in mitochondrial bioenergetics (3- and 7-days) and mitochondrial protein synthesis rates (3-days) in female mice. Therefore, while nitrate supplementation was not sufficient to prevent muscle atrophy, nitrate may represent a promising therapeutic strategy to preserve mitochondrial function during muscle disuse.