Dietary nitrate increases tetanic [Ca²⁺]_i and contractile force in mouse fast‐twitch muscle

Abstract

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.

Dietary nitrate supplementation enhances high-intensity running performance in moderate normobaric hypoxia, independent of aerobic fitness

“…It was suggested that the decreased ATP turnover occurring after nitrate supplementation might be related to a reduced ATP requirement for actin-myosin cross-bridge cycling and/or calcium handling, given previous reports that NO slows actin-myosin cross-bridge cycling [59,60] and inhibits calcium-ATPase activity [61,62]. Interestingly, a recent murine model investigation reported greater force production, and increased expression of the calcium handling protein calsoquestrin 1 and the dihydropyridine receptor in type II muscle fibres following nitrate supplementation [9]. It was speculated that such effects in humans may allow muscle activation at a lower frequency for an equivalent force production, lowering motor unit recruitment and concomitantly the ATP cost of exercise.…”

“…Nitrate supplementation had no effect on severe-intensity cycling TTE in normoxia, but improved TTE by ~ 8.6 % in hypoxia relative to placebo. Further, there is evidence from murine model investigations that nitrate supplementation may enhance tissue blood flow [41,42] and muscle contractile function [9] preferentially in type II muscle fibres. It is therefore possible that nitrate supplementation is more effective during shorter more high-intensity exercise, during which these muscle fibres are more heavily recruited [9,[43][44][45]64], or else in individuals with greater distribution of type II muscle fibres [43].…”

“…Further, there is evidence from murine model investigations that nitrate supplementation may enhance tissue blood flow [41,42] and muscle contractile function [9] preferentially in type II muscle fibres. It is therefore possible that nitrate supplementation is more effective during shorter more high-intensity exercise, during which these muscle fibres are more heavily recruited [9,[43][44][45]64], or else in individuals with greater distribution of type II muscle fibres [43]. The time-trial duration employed in this study (< 6 minutes) was considerably less than other investigations (~ 17 -48 minutes) which have not observed an ergogenic effect of nitrate supplementation in hypoxia [38][39][40], and may be important.…”

“…These include, but are not restricted to: lower blood pressure [1][2][3], reduced O2 consumption (V O2) during steady-state exercise [4][5][6], attenuated muscle metabolic perturbations [7,8], and enhanced muscle force production [9,10]. These effects are not directly attributable to the nitrate anion, which is relatively inert [11].…”

“…Evidence from murine models suggests that the effects of dietary nitrate supplementation may be fibre type specific, increasing muscle perfusion and oxygenation [41,42], and enhancing calcium handling and force production [9] in type II muscle fibres only, as may be recruited during high-intensity exercise (for review, see Jones et al [43]). Moreover, nitrate supplementation has been demonstrated to elicit beneficial effects when the contribution of type II muscle towards force generation is high, including at fast but not slow muscle contraction frequencies [44], and during the transition from moderate to severe but not unloaded to moderate intensity exercise [45].…”