Nitrate, an inorganic anion abundant in vegetables, is converted in vivo to bioactive nitrogen oxides including NO. We recently demonstrated that dietary nitrate reduces oxygen cost during physical exercise, but the mechanism remains unknown. In a double-blind crossover trial we studied the effects of a dietary intervention with inorganic nitrate on basal mitochondrial function and whole-body oxygen consumption in healthy volunteers. Skeletal muscle mitochondria harvested after nitrate supplementation displayed an improvement in oxidative phosphorylation efficiency (P/O ratio) and a decrease in state 4 respiration with and without atractyloside and respiration without adenylates. The improved mitochondrial P/O ratio correlated to the reduction in oxygen cost during exercise. Mechanistically, nitrate reduced the expression of ATP/ADP translocase, a protein involved in proton conductance. We conclude that dietary nitrate has profound effects on basal mitochondrial function. These findings may have implications for exercise physiology- and lifestyle-related disorders that involve dysfunctional mitochondria.

Key points Dietary supplementation with inorganic nitrate has beneficial effects on skeletal muscle responses to exercise. Both mitochondrial and extra‐mitochondrial explanations have been proposed. Contractile force of fast‐twitch muscles was enhanced in mice supplemented with 1 mm NaNO3 in drinking water for 7 days. Myoplasmic free [Ca2+] during tetanic stimulation was increased in fast‐twitch muscles of nitrate‐supplemented mice and this was accompanied by increased expression of calsequestrin 1 and the dihydropyridine receptor. These results provide a new mechanism by which nitrate exerts beneficial effects on muscle function with applications to sports performance and a potential therapeutic role in conditions with muscle weakness. Abstract  Dietary inorganic nitrate has profound effects on health and physiological responses to exercise. Here, we examined if nitrate, in doses readily achievable via a normal diet, could improve Ca2+ handling and contractile function using fast‐ and slow‐twitch skeletal muscles from C57bl/6 male mice given 1 mm sodium nitrate in water for 7 days. Age matched controls were provided water without added nitrate. In fast‐twitch muscle fibres dissected from nitrate treated mice, myoplasmic free [Ca2+] was significantly greater than in Control fibres at stimulation frequencies from 20 to 150 Hz, which resulted in a major increase in contractile force at ≤50 Hz. At 100 Hz stimulation, the rate of force development was ∼35% faster in the nitrate group. These changes in nitrate treated mice were accompanied by increased expression of the Ca2+ handling proteins calsequestrin 1 and the dihydropyridine receptor. No changes in force or calsequestrin 1 and dihydropyridine receptor expression were measured in slow‐twitch muscles. In conclusion, these results show a striking effect of nitrate supplementation on intracellular Ca2+ handling in fast‐twitch muscle resulting in increased force production. A new mechanism is revealed by which nitrate can exert effects on muscle function with applications to performance and a potential therapeutic role in conditions with muscle weakness.

Sexual dimorphism is apparent in humans, however, to date no studies have investigated mitochondrial function focusing on intrinsic mitochondrial respiration (i.e., mitochondrial respiration for a given amount of mitochondrial protein) and mitochondrial oxygen affinity (p50mito) in relation to biological sex in human. A skeletal muscle biopsy was donated by nine active women, and ten men matched for maximal oxygen consumption (VO2max) and by nine endurance trained men. Intrinsic mitochondrial respiration, assessed in isolated mitochondria, was higher in women compared to men when activating complex I (CIP) and complex I+II (CI+IIP) (p < 0.05), and was similar to trained men (CIP, p = 0.053; CI+IIP, p = 0.066). Proton leak and p50mito were higher in women compared to men independent of VO2max. In conclusion, significant novel differences in mitochondrial oxidative function, intrinsic mitochondrial respiration and p50mito exist between women and men. These findings may represent an adaptation in the oxygen cascade in women to optimize muscle oxygen uptake to compensate for a lower oxygen delivery during exercise.

Mitochondrial potassium channels have been implicated in myocardial protection mediated through pre-/postconditioning. Compounds that open the Ca2+- and voltage-activated potassium channel of big-conductance (BK) have a pre-conditioning-like effect on survival of cardiomyocytes after ischemia/reperfusion injury. Recently, mitochondrial BK channels (mitoBKs) in cardiomyocytes were implicated as infarct-limiting factors that derive directly from the KCNMA1 gene encoding for canonical BKs usually present at the plasma membrane of cells. However, some studies challenged these cardio-protective roles of mitoBKs. Herein, we present electrophysiological evidence for paxilline- and NS11021-sensitive BK-mediated currents of 190 pS conductance in mitoplasts from wild-type but not BK−/− cardiomyocytes. Transmission electron microscopy of BK−/− ventricular muscles fibres showed normal ultra-structures and matrix dimension, but oxidative phosphorylation capacities at normoxia and upon re-oxygenation after anoxia were significantly attenuated in BK−/− permeabilized cardiomyocytes. In the absence of BK, post-anoxic reactive oxygen species (ROS) production from cardiomyocyte mitochondria was elevated indicating that mitoBK fine-tune the oxidative state at hypoxia and re-oxygenation. Because ROS and the capacity of the myocardium for oxidative metabolism are important determinants of cellular survival, we tested BK−/− hearts for their response in an ex-vivo model of ischemia/reperfusion (I/R) injury. Infarct areas, coronary flow and heart rates were not different between wild-type and BK−/− hearts upon I/R injury in the absence of ischemic pre-conditioning (IP), but differed upon IP. While the area of infarction comprised 28±3% of the area at risk in wild-type, it was increased to 58±5% in BK−/− hearts suggesting that BK mediates the beneficial effects of IP. These findings suggest that cardiac BK channels are important for proper oxidative energy supply of cardiomyocytes at normoxia and upon re-oxygenation after prolonged anoxia and that IP might indeed favor survival of the myocardium upon I/R injury in a BK-dependent mode stemming from both mitochondrial post-anoxic ROS modulation and non-mitochondrial localizations.

Intense exercise training is a powerful stimulus that activates mitochondrial biogenesis pathways and thus increases mitochondrial density and oxidative capacity. Moderate levels of reactive oxygen species (ROS) during exercise are considered vital in the adaptive response, but high ROS production is a serious threat to cellular homeostasis. Although biochemical markers of the transition from adaptive to maladaptive ROS stress are lacking, it is likely mediated by redox sensitive enzymes involved in oxidative metabolism. One potential enzyme mediating such redox sensitivity is the citric acid cycle enzyme aconitase. In this study, we examined biopsy specimens of vastus lateralis and triceps brachii in healthy volunteers, together with primary human myotubes. An intense exercise regimen inactivated aconitase by 55-72%, resulting in inhibition of mitochondrial respiration by 50-65%. In the vastus, the mitochondrial dysfunction was compensated for by a 15-72% increase in mitochondrial proteins, whereas H2O2 emission was unchanged. In parallel with the inactivation of aconitase, the intermediary metabolite citrate accumulated and played an integral part in cellular protection against oxidative stress. In contrast, the triceps failed to increase mitochondrial density, and citrate did not accumulate. Instead, mitochondrial H2O2 emission was decreased to 40% of the pretraining levels, together with a 6-fold increase in protein abundance of catalase. In this study, a novel mitochondrial stress response was highlighted where accumulation of citrate acted to preserve the redox status of the cell during periods of intense exercise.