Incubation with sodium nitrite attenuates fatigue development in intact single mouse fibres at physiological $P_{{\text{O}}_2}$

Abstract

Dietary nitrate (NO₃⁻) supplementation, which increases plasma nitrite (NO₂⁻) concentration, has been reported to attenuate skeletal muscle fatigue development. Sarcoplasmic reticulum (SR) calcium (Ca²⁺) release is enhanced in isolated single skeletal muscle fibres following NO₃⁻ supplementation or NO₂⁻ incubation at a supra-physiological $P_{{\text{O}}_2}$ but it is unclear whether NO₂⁻ incubation can alter Ca²⁺ handling and fatigue development at a near-physiological $P_{{\text{O}}_2}$. We hypothesised that NO₂⁻ treatment would improve Ca²⁺ handling. and delay fatigue at a physiological $P_{{\text{O}}_2}$ in intact single mouse skeletal muscle fibres. Each muscle fibre was perfused with Tyrode solution pre-equilibrated with either 20% ($P_{{\text{O}}_2}$ = 150 Torr) or 2% O₂ ($P_{{\text{O}}_2}$ = 15.6 Torr) in the absence and presence of 100 μM NaNO₂. At supra-physiological $P_{{\text{O}}_2}$ (i.e. 20% O₂), time to fatigue was lowered by 34% with NaNO₂ (control: 257 ± 94 vs. NaNO₂: 159 ± 46 s, Cohen’s d = 1.63, P < 0.05), but extended by 21% with NaNO₂ at 2% O₂ (control: 308 ± 217 vs. NaNO₂: 368 ± 242 s, d = 1.14, P < 0.01). During the fatiguing contraction protocol completed with NaNO₂ at 2% O₂, peak cytosolic Ca²⁺ concentration ([Ca²⁺]_c) was not different (P > 0.05) but [Ca²⁺]_c accumulation between contractions was lower, concomitant with a greater SR Ca²⁺ pumping rate (P < 0.05) compared to the control condition. These results demonstrate that increased exposure to NO₂⁻ blunts fatigue development at near-physiological, but not at supra-physiological, $P_{{\text{O}}_2}$ through enhancing SR Ca²⁺ pumping rate in single skeletal muscle fibres. These findings extend our understanding of the mechanisms by which increased NO₂⁻ exposure can mitigate skeletal muscle fatigue development.

Introduction

The gaseous signalling molecule nitric oxide (NO) was first recognized as an endothelium-derived smooth muscle relaxant (Murad et al. 1978; Ignarro et al. 1987). However, it is now appreciated that NO can also impact a wide array of physiological processes in skeletal muscle (Stamler & Meissner, 2001; Suhr et al. 2013). It is well established that NO can be produced by the NO synthase (NOS) enzymes, which catalyse the five-electron oxidation of L-arginine to NO and L-citrulline (Moncada & Higgs, 1991). More recently, an alternative, O₂-independent pathway for NO generation has been identified in which inorganic nitrate (NO₃⁻) can be sequentially reduced to nitrite(NO₂⁻)and then to NO (Lundberg & Weitzberg, 2009, 2010; Clanton, 2019). Importantly, dietary supplementation with NO₃⁻, which increases circulating plasma [NO₂⁻], has been shown to improve skeletal muscle perfusion (Ferguson et al. 2013, 2015), contractile and metabolic efficiency (Bailey et al. 2010; Larsen et al. 2011; Vanhatalo et al. 2011; Fulford et al. 2013) and contractility (Hernández et al. 2012; Haider & Folland, 2014; Coggan et al. 2015a; Whitfield et al. 2017), and to blunt the development of skeletal muscle fatigue (Bailey et al. 2009, 2010; Wylie et al. 2013; Porcelli et al. 2015).

The chemical reduction of NO₂⁻ to NO is increased as $P_{{\text{O}}_2}$ (Castello et al. 2006) and pH (Modin et al. 2001) decline. There is evidence that dietary NO₃⁻ supplementation is more effective at improving skeletal muscle oxygenation and metabolism and delaying fatigue in hypoxia compared to normoxia (Vanhatalo et al. 2011; Kelly et al. 2014). Moreover, NO₃⁻ supplementation increases force production (Hernández et al. 2012) and perfusion (Ferguson et al. 2013) of fast-twitch (type II) skeletal muscle, which exhibits a lower pH (Tanaka et al. 2016) and $P_{{\text{O}}_2}$ (McDonough et al. 2005) during contractions, compared to slow-twitch (type I) skeletal muscle. Therefore, the existing evidence suggests that the potential for NO₃⁻ supplementation to improve skeletal muscle function may depend on intramuscular pH and $P_{{\text{O}}_2}$.

Although several studies have reported improved exercise economy and/or performance after short-term (3–7 days) (e.g. Larsen et al. 2007, 2011; Bailey et al. 2009, 2010; Porcelli et al. 2015; Whitfield et al. 2016) and acute (Wylie et al. 2013) dietary NO₃⁻ supplementation, the mechanisms that underlie these effects remain controversial. For example, short-term NO₃⁻ supplementation has been reported to improve exercise economy in association with (Larsen et al. 2011), or independently of (Whitfield et al. 2016), improved efficiency of mitochondrial oxidative phosphorylation (i.e. a higher P/O ratio). Alternatively, an attenuated high-energy phosphate cost of sub-maximal (Bailey et al. 2010) and maximal (Fulford et al. 2013) skeletal muscle force production has been observed following short-term NO₃⁻ supplementation. It is well documented that a significant portion of the energy liberated from high-energy phosphate metabolism is coupled to skeletal muscle Ca²⁺ handling (Walsh et al. 2006; Barclay, 2015). Accordingly, alterations in skeletal muscle Ca²⁺ handling might play an important role in improving skeletal muscle function after NO₃⁻ ingestion. In line with this postulate, increased tetanic contractile force and cytosolic [Ca²⁺] ([Ca²⁺]_c) have been observed in single mouse flexor digitorum brevis (FDB) myocytes after short-term (i.e. 7 days) in vivo NaNO₃ supplementation (Hernández et al. 2012). Moreover, NaNO₃ supplementation increased tetanic contractile force (Hernández et al. 2012) and the content of the Ca²⁺ handling proteins, calsequestrin 1 (CASQ1) and the dihydropyridine receptor (DHPR) in type II extensor digitorum longus muscle, but not in type I soleus muscle (Hernández et al. 2012; Ivarsson et al. 2017; cf. Whitfield et al. 2017). Acute exposure to NO₂⁻ has also been reported to increase [Ca²⁺]_c in isolated skeletal muscle fibres during tetanic contractions (Andrade et al. 1998b). Therefore, alterations in skeletal muscle Ca²⁺ handling appear to play an important role in the improvement in skeletal muscle function after acute and short-term NO₃⁻ supplementation.

Given that skeletal muscle fatigue and perturbations to Ca²⁺ handling appear to develop in synchrony (Westerblad & Allen, 1994; Allen et al. 2008), improved Ca²⁺ handling after NO₃⁻ or NO₂⁻ treatment (Andrade et al. 1998b; Hernández et al. 2012) might be expected to delay skeletal muscle fatigue. However, it has yet to be determined whether acutely exposing skeletal muscle fibres to NO₂⁻ can abate fatigue development, and to what extent this might be attributable to alterations in Ca²⁺ handling, during repeated tetanic contractions. A limitation of previous experiments assessing the effects of NO₃⁻ and NO₂⁻ administration on skeletal muscle force production and Ca²⁺ dynamics is the high experimental $P_{{\text{O}}_2}$ (95% O₂) of the perfusate employed in these studies (Andrade et al. 1998b; Hernández et al. 2012). This is important because the intracellular $P_{{\text{O}}_2}$ in muscle fibres during intense skeletal muscle contractions is lowered from ~5% O₂ (~30 Torr) to 0.5% O₂ (~2–5 Torr) (Richardson et al. 1995; Hirai et al. 2018), and there is evidence that the effects of NO on skeletal muscle contractility and [Ca²⁺]_c are influenced by the $P_{{\text{O}}_2}$ (Eu et al. 2003). Accordingly, further research is required to assess the effects of NO₂⁻ administration on skeletal muscle contractility, fatigue and [Ca²⁺]_c at a $P_{{\text{O}}_2}$ that better reflects that which is manifest in vivo during intense contractions.

The purpose of the present study was to assess the effects of acute NO₂⁻ administration on Ca²⁺ handling, evoked force and fatigue resistance in single intact mouse FDB myocytes at both a near-physiological $P_{{\text{O}}_2}$ and the more commonly used supra-physiological $P_{{\text{O}}_2}$. The single muscle fibre model utilised in the current study also allowed us to isolate the effects of NO₂⁻ administration on intramyocyte processes in the absence of altered extramyocyte processes such as perfusion. We hypothesized that skeletal muscle Ca²⁺ handling and contractility would be improved, and fatigue development would be delayed, in FDB fibres after NO₂⁻ incubation at a physiological, but not a supra-physiological, $P_{{\text{O}}_2}$.

Methods

Ethical approval

All procedures were approved by the University of California, San Diego Institutional Animal Care and Use Committee (UCSD-IACUC). The experiments in the present investigation comply with The Journal of Physiology policy for animal studies, as described by Drummond (2009). Adult male mice (12–16 weeks old; C57BL/6J; The Jackson Laboratory, Bar Harbor, ME, USA; a total of 22 mice were utilized in the present study) were allowed access to water and food ad libitum, and were killed by an intraperitoneal overdose of sodium pentobarbital (Sleepaway; 150 mg/kg). Death was confirmed by absence of movement, heartbeat, and response to toe pinching followed by rapid cervical dislocation to ensure killing.

Single mouse fibre isolation

After killing, the FDB muscles from both posterior feet were quickly excised and individual, intact single muscle fibres (total of 28 fibres used in this study) were micro-dissected from the whole muscle and transferred to an intact muscle fibre system (model 1500A with force transducer model 403A, Aurora Scientific Inc., Aurora, ON, Canada), as described previously (Nogueira et al. 2018). The intact muscle fibre system was placed on the stage of a Nikon inverted microscope with a 40× long distance Fluor objective. During the experimental procedures, fibres were superfused with Tyrode solution (in mM: 121 NaCl, 5 KCl, 1.8 CaCl₂, 0.5 MgCl₂, 0.4 NaH₂PO₄, 24 NaHCO₃, 5.5 glucose and 0.1 K₂EGTA, constantly bubbled with 5% CO₂ (for solution pH 7.4) and either 20% O₂ or 2% O₂ as described below). All experimental procedures were performed at 22°C.

Isometric force measurements and experimental protocol

Isolated single mouse fibres were electrically stimulated using a Grass S88X stimulator (Quincy, MA, USA) and signal was acquired and analysed as described previously (Gandra et al. 2018). Force development (in mN) was normalized to the cross-sectional area (in mm²) determined from the diameter of the fibre (32.8 ± 6.6 μm diameter; n = 22 fibres). After being mounted into an experimental chamber, fibres were loaded or injected with the respective fluorescent probe (BCECF-AM or FURA-2) or not treated with any fluorescent probe (as described above). All fibres underwent 30 min of constant superperfusion with Tyrode solution bubbled with 20% O₂–5% CO₂ (for extracellular pH 7.4), and N₂ balance, followed by electrical stimulations to evoke contractions. Fibre length was then adjusted to achieve optimal isometric tetanic force at 100 Hz (350 ms trains, 0.5 ms pulses, 8 V; L₀). Thereafter, fibres rested for a further 30 min with constant superperfusion with Tyrode solution bubbled with either 20% O₂ (for extracellular $P_{{\text{O}}_2}$ ~156 Torr) or 2% O₂ (for extracellular $P_{{\text{O}}_2}$ of 15.6 ± 0.05 Torr). The chamber was sealed with a glass cover slip in order to maintain the oxygen tension for the experimental protocol. The oxygen tension of the solution was measured using a needle-type housing fibre optic oxygen microsensor (OXYMICRO, World Precision Instruments, Sarasota, FL,USA)immersed in the experimental chamber solution.

Initially, all fibres were electrically stimulated at different frequencies of pulse stimulation(force-frequency curve; FF; 1–150 Hz; 100 s rest between trains, FF 1). After 5 min of rest, fibres completed a fatigue-inducing contraction protocol (Fatigue 1) comprising a series of 100 Hz trains with the stimulation frequency increased every 2 min (0.25, 0.3, 0.36, 0.43, 0.52, 0.62, 0.75, 0.9, 1.1 trains s⁻¹) until task failure, which was defined as the time required for force to decrease by 50%. Immediately following task failure (control), the fibre was super-perfused with a 100 μM NaNO_{2 s}olution solubilized in Tyrode (or modified Tyrode) solution when pH_i was measured; see below) and allowed to recover for 60 min. Subsequently, the FF and fatiguing contraction protocols described above were repeated (FF 2 and Fatigue 2, respectively). A schematic diagram of the experimental protocol is presented in Fig. 1. In experiments performed on fibres microinjected with FURA-2, the chamber was switched to a Tyrode solution containing 10 mM caffeine immediately following the last train of the FF curve and fatigue to evoke a single 120 Hz train and 100 Hz trains, respectively. The concentration of NaNO₂ was chosen based on the study of Andrade et al. 1998bb, who reported alterations in skeletal muscle force and calcium handling in intact single mouse fibres at non-fatiguing conditions in hyperoxia.

Figure 1.

Schematic diagram of the experimental protocol

Intracellular Ca²⁺ and pH assessment during contraction

Cytosolic calcium concentration ([Ca²⁺]_c) and intracellular pH (pH_i) changes were obtained by fluorescence spectroscopy using a Photon Technology International illumination and detection system (DeltaScan model) (Nogueira et al. 2018).

[Ca²⁺]_c measurements.

Single muscle fibres (n = 5) were pressure injected using a micropipette filled with the ratiometric compound, FURA-2 (Life technologies, Carlsbad, CA, USA; 12 mM, diluted in 150 mM KCl and 10 mM HEPES pH 7.0), followed by 1 h of rest, and subsequent measurement of [Ca²⁺]_c as described previously (Gandra et al. 2018). Fluorescence excitation ratio (340/380 nm; R) was converted to [Ca²⁺]_c according to eqn (1).

$${\left[{\text{Ca}}^{2+}\right]}_{\text{c}}=K_{\text{D}}\beta \left[\left(R-R_{\text{min}}\right)/\left(R_{\text{max}}-R\right)\right].$$ (1)

From eqn (1), K_D is the dissociation constant for Ca²⁺-FURA-2, which was set to 224 nM (Westerblad & Allen, 1991); β (4.51 ± 1.43; n = 5 fibres) is the fluorescence ratio between high and no [Ca²⁺]_c at 380 nm and was determined for each of the contracting fibres as described previously (Bakker et al. 1993; Andrade et al. 1998a); R_min (0.24 ± 0.04) and R_max (5.03 ± 1.88) are the fluorescence ratios at no cytosolic Ca²⁺ and high Ca²⁺, respectively, and were determined using an internal in vivo calibration described by Gandra et al. (2018).

The contraction-induced [Ca²⁺]_c was calculated by averaging the [Ca²⁺]_c signal in the final 100 ms of stimulation and subsequently used to determine peak [Ca²⁺]_c. The [Ca²⁺]_c before each contraction (i.e. basal [Ca²⁺]_c) was calculated by averaging the signal in the 100 ms preceding each stimulation. To determine myofilament Ca²⁺ sensitivity, force development data at each peak [Ca²⁺]_c during the force-frequency (FF) curves were fitted with a sigmoidal equation (Gandra et al. 2018) described in eqn (2):

$$P=P_{\text{min}}+\left[\left(P_0{\left[{\text{Ca}}^{2+}\right]}_c^n\right)/\left(C{a}^{2+}{50}^n+{\left[{\text{Ca}}^{2+}\right]}_c^n\right)\right].$$ (2)

From eqn (2), P is the force developed at different [Ca²⁺]_c, P₀ is the maximal force development, P_min is the minimum force developed, Ca²⁺₅₀ is the midpoint of the force-[Ca²⁺]_c curve, and n is n_H, the Hill coefficient.

To determine whether fatiguing contractions altered myofilament Ca²⁺ sensitivity, force development data during the fatigue-inducing contraction protocol were plotted at each peak [Ca²⁺]_c and fitted with eqn (2) to determine Ca²⁺₅₀ during fatigue. However, the first 40 s of contractions were not used to determine Ca²⁺₅₀ since they represent the phase 1 of fatigue (Westerblad & Allen, 1991).

During the contraction protocols, SR Ca²⁺ pumping was measured using the procedures described by (Nogueira et al. 2018). Briefly, a SR Ca²⁺ pumping curve was obtained by plotting the rate of [Ca²⁺]_c decline (−d[Ca²⁺]_c/dt) during the elevated long tail of [Ca²⁺]_c decay (from 100 ms to 3 s after the stimulation period) versus [Ca²⁺]_c (eqn (3)).

$$-\text{d}{\left[{\text{Ca}}^{2+}\right]}_{\text{c}}/\text{d}t=A{\left[{\text{Ca}}^{2+}\right]}_{\text{c}}^N-L.$$ (3)

From eqn (3), A, Nand Lare three adjustable parameters representingtherateofSRCa²⁺ pumping(in μM^N−1 s⁻¹), the power function, and the SR Ca²⁺ leak, respectively. In order to directly compare SR Ca²⁺ pumping(in μM⁻³ s⁻¹) between the control and NaNO₂ conditions at the same time points of the fatigue protocols (first contraction, 80 s of contractions, and the point of task failure), the curves were fitted with N and L fixed at 4 and 30, respectively. These values were based on mean values of 4.4 ± 0.4 for N and 32 ± 9 and for L obtained in individual experiments (n = 5 fibres), with a maximum increase in least-square error of 15% (Nogueira et al. 2018).

pH_i measurements.

Single muscle fibres (n = 5) were loaded with 2′, 7′-bis-(2-carboxyethyl)-5-(and −6)-carboxyfluorescein (BCECF-AM; Life Technologies, Carlsbad, CA, USA; prepared as stock solution of 10 mM in 100% ethanol and diluted in Tyrode solution to a final concentration of 10 μM BCECF-AM and 0.1% ethanol) for 30 min at room temperature. After the incubation period, excess BCECF-AM was washed out twice with 10 ml Tyrode solution, followed by a 30 min resting period with constant superperfusion with Tyrode. Intracellular pH (pH_i) changes during contractions were performed as described previously (Nogueira et al. 2013). After completing the experimental protocol, each fibre loaded with BCECF-AM underwent an in vivo calibration procedure, involving 20 min incubation in two different pH buffered solutions (175 mM KCl, 1.2 mM KH₂PO₄, 0.5 mM MgCl₂, 10 mM HEPES, 10 μM nigericin), previously titrated with KOH to pH 5.0 and 9.0 as the fluorescence signal was detected. To determine the logarithmic dissociation constant(pK_A), another group of fibres (n = 3) were loaded with BCECF-AM, incubated for 20 min in each of the 12 different pH buffered solutions (ranging from pH 5.0 to pH 9.0), and the fluorescence signal was detected (Nogueira et al. 2013). To convert the fluorescence signals to pH_i, fluorescence excitation ratio (440/490 nm; F) was then converted to pH_i according to eqn (4).

$${\text{pH}}_{\text{i}}=\text{p}{K}_{\text{A}}-\text{log }\left[\left(F-F_{\text{b}}\right)/\left(F_{\text{a}}-F\right)\right]-\text{log }\left(S_{\text{b}}/S_{\text{a}}\right).$$ (4)

From eqn (4), pK_A was set to 7.14 (7.14 ± 0.05, n = 3 fibres). S_b and S_a are the fluorescence signals from 440 nm excitation when BCECF is H⁺-free (reached at pH 9.0) and H⁺-bound (reached at pH 5.0), respectively. The fluorescence ratio of S_b/S_a was determined as 0.45 ± 0.28 (total of n = 8 fibres). F_a and F_b are the fluorescence ratios at H⁺-bound and H⁺-free states, respectively, and were determined as 2.56 ± 0.62 for F_a and 8.63 ± 0.99 for F_b (total of n = 8 fibres).

When pH_i was measured during the contractile protocols, a modified Tyrode solution was applied where 20 mM NaCl was substituted with sodium lactate (101 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 0.5 mM MgCl₂, 0.4 mM NaH₂PO₄, 24 mM NaHCO₃, 5.5 mM glucose, 0.1 mM EGTA, 0.2% FBS and 20 mM sodium lactate). This solution was applied in order to produce contractile-induced changes in intracellular pH in single mouse fibres (Westerblad & Allen, 1992).

Statistics

The experimental results are presented as means ± standard deviation (SD). For comparison between two groups, a paired Student’s t test was used and effect size was calculated using Cohen’s d. For multiple comparisons, a one-way ANOVA followed by the Tukey test or a two-way ANOVA followed by the Bonferroni test was used, as indicated. All the analyses were conducted using GraphPad Prism version 4.00 for Windows (San Diego, CA, USA). Statistical significance was accepted when P < 0.05.

Results

Influence of $P_{{\text{O}}_2}$ and NaNO₂ administration on single fibre fatigue development

To assess the influence of NaNO₂ incubation on fatigue development and the potential $P_{{\text{O}}_2}$ dependence of this effect, single muscle fibres were exposed to either a supra-physiological or a near-physiological $P_{{\text{O}}_2}$ [i.e. with 20% O₂ (~150 Torr) or 2% O₂ (~15.6 Torr), respectively] in the absence of any injection or loading with fluorescent probes. At 20% O₂, time to task failure following NaNO₂ administration was 34 ± 19% shorter compared to standard Tyrode solution (control: 257 ± 94 vs. NaNO₂: 159 ± 46 s; Cohen’s d = 1.63, P < 0.05, Fig. 2A, n = 4 fibres). There were no differences in initial isometric force between the control and NaNO₂ fatigue runs at 20% O₂ (control: 413 ± 63 vs. NaNO₂: 410 ± 69 kPa; P > 0.05). However, when fibres were superfused with 2% O₂, which was the smallest extracellular $P_{{\text{O}}_2}$ that did not lower time to task failure compared to 20% O₂ (data not shown), NaNO₂ treatment increased time to task failure by 19 ± 18% (control: 538 ± 286 vs. NaNO₂: 607 ± 304s, d=1.83, P<0.05, Fig.2B, n=4fibres). There was also no difference in initial isometric force between the control and NaNO₂ fatigue runs at 2% O₂ (control: 495 ± 111 vs. NaNO₂: 477 ± 98 kPa; P > 0.05). There was no difference in time to task failure between the two fatigue runs at 2% O₂ when NaNO₂ was absent prior to and during the second fatigue-inducing contraction protocols (first fatigue run: 330 ± 131 vs. second fatigue run: 288 ± 143 s; P > 0.05, n = 4 fibres). When single muscle fibres were microinjected with FURA-2 to measure [Ca²⁺]_c responses during and between contractions, NaNO₂ treatment at near-physiological $P_{{\text{O}}_2}$ did not increase time to task failure compared to the control condition (control: 142 ± 33 vs. NaNO₂: 165 ± 73 s, d = 0.51, P > 0.05, n = 5 fibres). However, in fibres loaded with BCECF and perfused with a modified Tyrode solution (i.e. with 20 mM sodium lactate) to measure pH_i, time to task failure was increased with NaNO₂ administration (control: 291 ± 52 vs. NaNO₂: 379 ± 82 s; 31 ± 18% increase; d = 1.62, P < 0.05; n = 5 fibres). When the fibres from all experiments at near-physiological $P_{{\text{O}}_2}$ were pooled, time to task failure was enhanced by 21 ± 22% with NaNO₂ treatment compared to preceding control condition (control: 308 ± 217 vs. NaNO₂: 368 ± 242 s; d = 1.14, P < 0.01, Fig. 2C, n = 14 fibres).

Figure 2. Effect of sodium nitrite (NaNO₂) incubation on fatigue development at supra-physiological and near-physiological oxygen tensions in single skeletal muscle fibres.

A, time to task failure during repeated tetanic contractions performed in fibres not loaded with fluorescent probes at a supra-physiological $P_{{\text{O}}_2}$ (20% O₂, ~156 Torr) in the absence (Fatigue 1; control) or presence of 100 μM NaNO₂ (fatigue 2; n = 4 fibres). B, time to task failure in fibres not loaded with fluorescent probes performed at near-physiological $P_{{\text{O}}_2}$ (2% O₂, ~15 Torr) in the absence and presence of 100 μM NaNO₂ (n = 4 fibres). C, time to task failure in all fibres from this study (not loaded and loaded with fluorescent probes) that were performed at near-physiological $P_{{\text{O}}_2}$ (2% O₂, ~15 Torr) in the absence (Fatigue 1; control) or presence of 100 μM NaNO₂ (Fatigue 2; n = 14 fibres). The bars represent group mean data, with the lines representing responses in individual muscle fibres. Data are presented as means ± SD. *P < 0.05 vs. Fatigue 1.

Influence of NaNO₂ on force and intracellular Ca²⁺ responses during single non-fatiguing contractions

During the single contraction non-fatiguing FF curves, maximal tetanic force was not different (P > 0.05), but sub-maximal force during 20–50 Hz contractions was lowered in the NaNO₂ condition compared to the control condition (P < 0.01, n = 5 fibres; Fig. 3A). For example, evoked force at 30 Hz was 42 ± 20% lower in the NaNO₂ condition (P < 0.01). Peak [Ca²⁺]_c was lowered in the NaNO₂ condition at sub-maximal (e.g. by 21 ± 11% at 30 Hz, P < 0.01) and maximal (e.g. by 22 ± 15% at 150 Hz, P < 0.05) force development, as well as during 120 Hz contractions evoked in the presence of 10 mM caffeine (by 32 ± 21%), compared to the control condition (P < 0.05). In fibres that were superfused with 2% O₂ but not treated with NaNO₂, maximal and submaximal forces were not different between the first and the second FF curves (data not shown, n = 4, P > 0.05). When isometric force development (shown in Fig. 3A) was plotted against [Ca²⁺]_c (shown in Fig. 3B) across the FF curve, there were no differences between the control and NaNO₂ conditions (Fig. 3C). Indeed, the midpoints of the force-[Ca²⁺]_c curves were not different between the control and NaNO₂ conditions (e.g. Ca²⁺₅₀ was 474 ± 154 nM vs. 472 ± 122 nM for control and NaNO₂, respectively, P > 0.05, n = 5 fibres).

Figure 3. Sodium nitrite (NaNO₂) incubation at a near-physiological oxygen tension (2% O₂, ~15 Torr) does not alter myofibrillar calcium (Ca²⁺) sensitivity during single evoked non-fatiguing contractions.

A, isometric force development in single skeletal muscle fibres evoked by different frequencies of pulse stimulation before Fatigue 1 (FF 1) and before Fatigue 2 (FF 2; after 1 h incubation with 100 μM NaNO₂). B, intracellular cytosolic Ca²⁺ concentration ([Ca²⁺]_c) at rest, contractions at different pulse-frequencies, and at 120 Hz stimulation in the presence of 10 mM caffeine. C, plot of force development against [Ca²⁺]_c at each pulse-frequency. *P < 0.05 vs. control. Data (n = 5 fibres) are presented as means ± SD.

Influence of NaNO₂ on intracellular Ca²⁺ responses during repeated fatigue-inducing contractions

There was no difference in peak [Ca²⁺]_c achieved during the evoked contractions between the control and NaNO₂ conditions throughout the fatigue-inducing contraction protocols (P > 0.05, Fig. 4A). However, after 100 s of the fatigue-inducing contraction protocol, basal [Ca²⁺]_c followingtheevokedcontractionswaslowerintheNaNO₂ condition (P < 0.05). Moreover, basal [Ca²⁺]_c at the point of task failure was also lower in the NaNO₂ condition (109 ± 20 nM) compared to the control condition (136 ± 28 nM, d = 1.33, P < 0.05, Fig. 4B). The midpoint of the force-[Ca²⁺]_c curves (Ca²⁺₅₀), an index for predicting the myofilament Ca²⁺ sensitivity, was determined during the fatigue-inducing contractions and compared with the data obtained in the FF curves. While the Ca²⁺₅₀ increased during the fatigue-inducing contraction protocol compared to the value obtained from the FF curve in the control condition (from 474 ± 154 nM to 601 ± 141 nM, P < 0.05), the Ca²⁺₅₀ was not different between the fatigue-inducing contraction protocol and the FF curve in the NaNO₂ condition (from 472 ± 122 nM to 566 ± 95 nM, P > 0.05, Fig. 4C).

Figure 4. Isometric force development and intracellular cytosolic calcium concentration ([Ca²⁺]_c) responses during a repeated fatigue-inducing contraction protocol completed in the absence and presence of sodium nitrite (NaNO₂) at a near-physiological oxygen tension (2% O₂, ~15 Torr).

A, peak [Ca²⁺]_c responses up to the time of task failure. B, basal [Ca²⁺]_c data, obtained from the 100 ms period before each contraction, up to the time of task failure (*P < 0.05 vs. control). Note the blunted increase in basal [Ca²⁺]_c in the NaNO₂ condition. C, change (Δ) in basal [Ca²⁺]_c from rest to task failure. D, Ca²⁺₅₀ before and during fatiguing contractions, with group mean responses as bars and individual responses as lines (*P < 0.05 vs. FF 1). Data (n = 5 fibres) are presented as means ± SD.

Influence of NaNO₂ on sarcoplasmic reticulum Ca²⁺ pumping during fatiguing contractions

There were no differences between the control and NaNO₂ conditions in the plot of [Ca²⁺]_c decay rate for each [Ca²⁺]_c following the first contraction of the repeated fatigue-inducing contraction protocol (Fig. 5A). Similarly, the rate of SR Ca²⁺ pumping following the first contraction was not different between the control (3323 ± 2209 μM⁻³ s⁻¹) and NaNO₂ (3069 ± 2276 μM⁻³ s⁻¹) conditions (P > 0.05, Fig. 4D). During the fatigue-inducing contraction protocol, the rate of SR Ca²⁺ pumping was slower after 80 s (534 ± 5436 μM⁻³ s⁻¹) compared to the first contraction (P < 0.05) and slower at the time of task failure (200 ± 2176 μM⁻³ s⁻¹) compared to both the first contraction and after 80 s of the fatigue-inducing contraction protocol in the control condition (P < 0.05). Likewise, the rate of SR Ca²⁺ pumping was progressively slowed from the first contraction, after 80 s (955 ± 8546 μM⁻³ s⁻¹) and at the time of task failure (486 ± 3806 μM⁻³ s⁻¹) during the fatigue-inducing contraction protocol in the NaNO₂ condition (P < 0.05). After 80 s of contractions (Fig. 5B) and at the time of task failure (Fig. 5C), the plots of [Ca²⁺]_c decay rate for each [Ca²⁺]_c were left-shifted in the NaNO₂ condition compared to the control condition. Moreover, compared to the control condition, the rate of SR Ca²⁺ pumping was 110 ± 78% (d = 0.63) and 212 ± 105% (d = 0.97) higher with NaNO₂ treatment after 80 s of contractions (Fig. 5E) and at the time of task failure (Fig. 5F), respectively, during the fatigue-inducing contraction protocol (P < 0.05).

Figure 5. Analysis of sarcoplasmic reticulum calcium (Ca²⁺) pumping at different time-points during the fatigue-inducing contraction protocols run in the absence and presence of sodium nitrite (NaNO₂) at a near-physiological oxygen tension (2% O₂, ~15 Torr).

The intracellular cytosolic Ca²⁺ concentration ([Ca²⁺]_c) dependence of the rate of [Ca²⁺]_c decay (−d[Ca²⁺]_c/dt) during the ‘tail’ of [Ca²⁺]_c decay is illustrated after the first contraction (A), after 80 s of contractions (B) and the contraction at the time of task failure (C). Note the left shifting of the NaNO₂ curve compared to the control curve after 80 s of contractions and at the time of task failure, but not after the first contraction. The changes in SR Ca²⁺ pumping rate at these time points are presented in D (first contraction), E (after 80 s of contractions), and F (time of task failure). *P < 0.05 vs. control. Data (n = 5 fibres) are presented as means ± SD.

Intracellular pH changes during fatiguing contractions

Compared to the resting values, pH_i at task failure was lower in both the control (Pre: 7.46 ± 0.23 vs. Post: 7.34 ± 0.22) and NaNO₂ (Pre: 7.46 ± 0.24 vs. Post: 7.25 ± 0.21) conditions (P < 0.0001, n = 5 fibres; Fig. 6). There were no differences in pH_i between the control and NaNO₂ conditions over the first 300 s of the fatigue-inducing contraction protocol (P > 0.05). However, the change in pH_i from the start to the end of the fatigue-inducing contraction protocol was greater in the NaNO₂ condition (−0.20 ± 0.04) compared to the control condition (−0.12 ± 0.05, P < 0.05).

Figure 6. Intracellular pH (pH_i) responses during the fatigue-inducing contraction protocols run in the absence and presence of sodium nitrite (NaNO₂) at a near-physiological oxygen tension (2% O₂, ~15 Torr).

pH_i changes during the repeated fatiguing contractions. *P < 0.05 vs. control. †P < 0.05 vs. initial contraction. Data (n = 5 fibres) are presented as means ± SD.

Discussion

The important original findings from this study were that NaNO₂ exposure delayed fatigue development in single mammalian skeletal muscle fibres at a near-physiological $P_{{\text{O}}_2}$, but expedited fatigue development at a supra-physiological $P_{{\text{O}}_2}$, during a repeated tetanic contraction protocol. Moreover, when single skeletal muscle fibres were incubated with sodium lactate to replicate the contraction-induced decline in pH_i that is manifest in vivo, the blunting of fatigue development with NaNO₂ compared to the control condition at a physiological $P_{{\text{O}}_2}$ was greater than the same comparison without sodium lactate coincubation. The delayed fatigue development with NaNO₂ administration did not impact on peak [Ca²⁺]_c during the fatiguing contraction protocol but blunted the progressive decline in SR Ca²⁺ pumping rate and the associated increase in basal[Ca²⁺]_c in the recovery period between contractions. Incubation with NaNO₂ also alleviated the decline in myofilament Ca²⁺ sensitivity during the fatiguing contraction protocol. There was no difference in pH_i between the NaNO₂ and control conditions over the first 300 s of the fatigue-inducing contraction protocol, but pH_i was lower at task failure in the NaNO₂ condition compared to the control condition. These results suggest that, at a $P_{{\text{O}}_2}$ comparable to that observed in human skeletal muscle during fatigue-inducing contractions (Richardson et al. 1995), and in the absence of any alterations in perfusion, NO₂⁻ treatment can delay the development of fatigue in single skeletal muscle fibres by improving SR Ca²⁺ pumping, maintaining Ca²⁺ sensitivity, and permitting the attainment of a lower pH_i. These findings extend our understanding of the mechanisms by which increased muscle NO₂⁻ exposure, as occurs following dietary NO₃⁻ supplementation (Gillard et al. 2018;Wylie et al. 2019), can blunt skeletal muscle fatigue and suggest that the potential for NO₂⁻ administration to attenuate fatigue development is $P_{{\text{O}}_2}$ and pH dependent.

Influence of NaNO₂ administration on time to task failure at a near-physiological $P_{{\text{O}}_2}$

Incubation with NaNO₂ at a near-physiological $P_{{\text{O}}_2}$ increased time to task failure by 19% compared to the control condition with no fluorophore loading. Although NaNO₂ administration at a near-physiological $P_{{\text{O}}_2}$ did not increase time to task failure in fibres microinjected with FURA-2 to assess [Ca²⁺]_c dynamics, when these data were combined with data from fibres with no fluorophore loading, time to task failure was extended by 15%. The greatest increase (31% compared to the respective control condition) in time to task failure with NaNO₂ incubation in the current study was observed when skeletal muscle fibres were co-incubated with a near-physiological $P_{{\text{O}}_2}$ and sodium lactate to facilitate a decline in pH_i (Westerblad & Allen, 1992) and thus better reflect the pH_i dynamics in human skeletal muscle in vivo (Vanhatalo et al. 2011). The mean improvement in time to task failure with NaNO₂ incubation compared to the respective control conditions in the experiments completed with (FURA-2 and BCECF-AM) and without fluorophores was 21% at a near-physiological $P_{{\text{O}}_2}$ in the current study.

It has been reported that intracellular $P_{{\text{O}}_2}$ in both rodent and human skeletal muscle fibres drops from 30 Torr at restto3–4Torrduringintenseexercise (Richardson et al. 1995; Hirai et al. 2018). The extracellular $P_{{\text{O}}_2}$ was set at 15 Torr (2% O₂) in the current study as preliminary experiments revealed this to be the smallest extracellular $P_{{\text{O}}_2}$ that did not lower time to fatigue in single skeletal muscle fibres compared to experiments conducted at 20% O₂, which suggest that at 15 Torr fibres were not under hypoxic conditions during contractions. Furthermore, the extracellular $P_{{\text{O}}_2}$ used in the present work closely reflects the $P_{{\text{O}}_2}$ in the interstitial space between the capillaries and muscle fibres (Hirai et al. 2018), and was intended to replicate the intracellular $P_{{\text{O}}_2}$ during contractions in humans in vivo (Richardson et al. 1995). Although the control fatigue-inducing protocol always preceded the NaNO₂ fatigue-inducing protocol, there was no difference in evoked isometric force in the first tetanic contraction of the control and NaNO₂ fatigue-inducing protocols, and there was no difference in time to task failure between two fatigue-inducing protocols at 2% O₂ without NaNO₂ administration. Therefore, our results suggest that, at a near physiological $P_{{\text{O}}_2}$, the blunted rate of fatigue development in the second fatigue-inducing protocol completed with NaNO₂ administration was not confounded by differences in initial force production or fatigue development between the first and second fatigue-inducing contraction protocols.

Influence of NaNO₂ administration on time to fatigue at a supra-physiological $P_{{\text{O}}_2}$

In contrast to the delayed rate of fatigue development with NaNO₂ administration at a near-physiological $P_{{\text{O}}_2}$, fatigue development was expedited when NaNO₂ was administered at a supra-physiological $P_{{\text{O}}_2}$ of 20% O₂ (~150 Torr) in the present study. It has been reported that sarcomere shortening and [Ca²⁺]_c are greater in collagenase-digested single FDB muscle fibres excised from wild-type mice and stimulated by single twitches at 1% O₂ compared to 20% O₂, and that these effects are abolished in fibres excised from nNOS knock-out mice (Eu et al. 2003). These findings suggest that, at least in the unfatigued state, SR Ca²⁺ handling and skeletal muscle contractility are improved by NO production at a physiological $P_{{\text{O}}_2}$ compared to a supra-physiological $P_{{\text{O}}_2}$. It has been suggested that the one-electron reduction of NO₂⁻ to NO is inversely related to $P_{{\text{O}}_2}$ and that the oxidation of NO₂⁻ and NO to NO₃⁻ is increased at a higher $P_{{\text{O}}_2}$ (Lundberg & Weitzberg, 2009, 2010). The greater production of superoxide in hyperoxia (Clanton, 2007) will also act to scavenge NO generated from NO₂⁻ reduction (Sjöberg & Singer, 2013) leading to the formation of the potent oxidising agent, peroxynitrite (Radi, 2013). This potential for increased peroxynitrite synthesis with NO₂⁻ administration at a supra-physiological $P_{{\text{O}}_2}$ might have contributed to the earlier attainment of task failure compared to the control condition(Supinski et al.1999;Dutka et al. 2012). There is also evidence that hydrogen peroxide, which is generated through the dismutation of superoxide, can oxidise NO₂⁻ to NO₃⁻ through the enzyme catalase (Heppel & Porterfield, 1949). Therefore, the $P_{{\text{O}}_2}$-dependent effects of NO₂⁻ on fatigue development might be linked to the proportion of NO₂⁻ that is reduced to NO and its interaction with reactive oxygen species. Collectively, our results suggest that the effect of NaNO₂ administration on fatigue development in single skeletal muscle fibres is $P_{{\text{O}}_2}$ and pH dependent, with the greatest positive effect manifest when $P_{{\text{O}}_2}$ and pH_i dynamics most closely resemble those that are exhibited in human skeletal muscle in vivo.

Influence of NaNO₂ administration on skeletal muscle Ca²⁺ handling

In addition to measuring fatigue development, [Ca²⁺]_c transients were assessed during a series of single non-fatiguing contractions and during subsequent repeated fatigue-inducing tetanic contractions at a near-physiological $P_{{\text{O}}_2}$ in the absence and presence of NaNO₂. There were no differences in isometric force during a series of single contractions evoked prior to and 60 min following the completion of a repeated contraction fatigue-inducing protocol, suggesting that 60 min of recovery was sufficient to restore isometric force to baseline. Accordingly, any effect of NO₂⁻ on contractility during the second series of single non-fatiguing contractions would not be expected to be confounded by the preceding fatigue-inducing contraction protocol. During the series of single non-fatiguing contractions completed 60 min after the end of the first bout of fatiguing contractions and with NaNO₂⁻ incubation, isometric force during single submaximal (20–50 Hz) contractions was depressed compared to the initial control condition, but not at near-maximal or maximal (>50 Hz) contractions. [Ca²⁺]_c was lower with NO₂⁻ treatment during single 30–150 Hz contractions and during a single 120 Hz contraction in the presence of 10 mM caffeine to evoke maximum SR Ca²⁺ release, but due to the sigmoidal nature of the force-Ca²⁺ relationship, the decreased in peak [Ca²⁺]_c at high pulse frequencies with NaNO₂ did not alter the maximum force developed. Therefore, the suppression of isometric force development during sub-maximal contractions with NO₂⁻ exposure was a function of lower SR Ca²⁺ release with no change in Ca²⁺ sensitivity. In contrast, and despite administering the same NO₂⁻ dose as the current study, [Ca²⁺]_c was increased, and isometric force was attenuated in single mouse skeletal muscle fibres with NO₂⁻ treatment at 95% O₂ in a previous study by Andrade et al. (1998b). Since this reduction in myofibrillar Ca²⁺ sensitivity with NO₂⁻ treatment was also observed following the administration of different NO donors (Andrade et al. 1998b), these findings suggest that theeffectsofNO₂⁻ onSRCa²⁺ handling are NO-mediated or that NO₂⁻ and NO impact SR Ca²⁺ handling through common signalling mechanisms. The disparate effect of NO₂⁻ on SR Ca²⁺ handling and isometric force in the current study and the study by Andrade et al. (1998b) is likely to be a function of the inter-study difference in the experimental $P_{{\text{O}}_2}$ (Eu et al. 2003) and its influence on the crosstalk between redox and nitroso signalling (Spencer&Posterino, 2009). WhileitisunclearwhyNO₂⁻ administration suppressed isometric force production and peak [Ca²⁺]_c during 20–50 Hz submaximal contractions, and did not change isometric force production during maximal/near-maximal contractions at 100 Hz, these observations are in line with previous findings that NO donors are more likely to impair force production during unfused tetanus compared to fused tetanus (Murrant et al. 1997; Maréchal & Gailly, 1999).

During the fatigue-inducing repeated tetanic contraction protocol, peak [Ca²⁺]_c during contractions declined, basal [Ca²⁺]_c following contractions increased and myofibrillar Ca²⁺ sensitivity was lowered as the fatigue protocol progressed. These perturbations to SR Ca²⁺ handling are consistent with previous reports and are important contributors to the development of skeletal muscle fatigue (Westerblad & Allen, 1994; Allen et al. 2008). There was no difference in peak [Ca²⁺]_c during contractions between the NaNO₂ and control conditions throughout the fatigue run. However, after 80 s of the fatigue run, the slowing in SR Ca²⁺ pumping and the resultant increase in basal [Ca²⁺]_c following contractions were attenuated with NO₂⁻ treatment. Therefore, the blunted fatigue development in the NO₂⁻ trial compared to the control trial appears to be linked to a better maintenance of SR Ca²⁺ pumping. The pumping of Ca²⁺ by the SR is an active process that is coupled to the function (Ca²⁺ affinity and the coupling between ATP hydrolysis and Ca²⁺ transport) of the SR Ca²⁺-ATPase (SERCA). SERCA function is regulated by the phosphorylation status of two proteins with similar structures, phospholamban, which is mostly expressed in cardiac myocytes and in slow-twitch muscle fibres, and sarcolipin, which is mostly expressed in skeletal muscle fibres (Pant et al. 2016). Specifically, phosphorylated phospholamban and sarcolipin can improve SERCA function, whereas dephosphorylated phospholamban and sarcolipin can compromise SERCA function (Tupling, 2009). It has been reported that NO₂⁻ administration can increase the amount of phosphorylated phospholamban in striated muscle through nitrite reductase-dependent NO production (Huang et al. 2013). Therefore, it is possible that phosphorylation of phospholamban and/or sarcolipin might have contributed to the improved maintenance of SR Ca²⁺ pumping during the fatigue-inducing contraction protocol completed with NaNO₂ treatment at a low $P_{{\text{O}}_2}$ compared to the control trial in the current study. Moreover, since the rate of SR Ca²⁺ pumping is inversely related to basal [Ca²⁺]_c accumulation during repetitive contractions (Nogueira et al. 2013, 2018), the lower basal [Ca²⁺]_c accumulation during the fatigue-inducing contraction protocol would also probably have contributed to the better maintenance of SR Ca²⁺ pumping during the NO₂⁻ trial. Lowering the energy cost of actomyosin ATPase, through inhibiting cross-bridge cycling, has also been reported to abate the progressive slowing in SR Ca²⁺ pumping rate during fatiguing contractions (Nogueira et al. 2013). It has been reported that increasing NO₂⁻ via short-term dietary NO₃⁻ supplementation can lower the high-energy phosphate cost of sub-maximal (Bailey et al. 2010) and maximal (Fulford et al. 2013) skeletal muscle force production, which is compatible with findings from single intact skeletal muscle fibres that the energy cost of skeletal muscle contraction impacts SR Ca²⁺ pumping rate during fatiguing contractions (Nogueira et al. 2013).

Compared to the single non-fatiguing contractions completed during the construction of the force-frequency curve, myofilament Ca²⁺ sensitivity declined across the fatigue-inducing contraction protocol in the control trial, butnottheNaNO₂ trial. Therefore, improved preservation of myofilament Ca²⁺ sensitivity might have contributed to the slower rate of fatigue development in the NaNO₂ trial. It has been reported that treatment with NO donors can S-nitrosylate cysteine residues in the myosin heavy chain leading to slowed cross-bridge cycling (Nogueira et al. 2009), but increased force per power stroke (Evangelista et al. 2010). Since NO₂⁻ administration has also been reported to promote S-nitrosylation in striated muscle (Kovács et al. 2015), the improved maintenance of Ca²⁺ sensitivity with NaNO₂ administration in the current study might therefore be a function of direct NO₂⁻ action on the myofilaments. Alternatively, NO₂⁻ administration might have improved myofilament Ca²⁺ sensitivity indirectly through attenuating ROS production (Yang et al. 2015), or thwarting ROS-mediated oxidation of the myofilaments (Moonpanar & Allen, 2006).

Influence of NaNO₂ administration on skeletal muscle pH

Since a decline in muscle pH has been reported to compromise myofilament Ca²⁺ sensitivity (Fabiato & Fabiato, 1978) and SERCA function(Wolosker et al. 1997), and since NO has been reported to inhibit glycolysis via S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase (Mohr et al. 1996), pH_i was assessed to provide insight into the potential mechanisms for improved Ca²⁺ handling in the NaNO₂ trial. Since pH_i was not different between the NaNO₂ and control trials over the first 300 s of the fatigue run, the results presented in this study suggest that the improved maintenance of myofilament Ca²⁺ sensitivity and SR Ca²⁺ pumping rate in the NaNO₂ trial extended time to task failure and permitted the attainment of a lower pH_i at task failure compared to the control condition.

Limitations and areas for further research

We combined the time to task failure data from FURA-2 injected fibres and non-injected fibres as time to task failure was not significantly different with NaNO₂ compared to the control condition in FURA-2 microinjected fibres. Therefore, it should be acknowledged that the experiments in FURA-2 injected fibres to assess the effects of NaNO₂ administration on Ca²⁺ handling and fatigue at a near-physiological $P_{{\text{O}}_2}$ in the current study were underpowered. When the data from FURA-2 injected fibres and non-injected fibres were combined, time to task failure was significantly extended in the NaNO₂ condition, consistent with the experiments where NaNO₂ was co-incubated with sodium lactate to facilitate a decline in pH_i. Therefore, this approach does not undermine the conclusion that acute NaNO₂ administration can delay fatigue development at a near-physiological $P_{{\text{O}}_2}$ and we have based our conclusions on the effect of NaNO₂ administration on fatigue development when all fibres are pooled. Since acute NO₂⁻ exposure lowered [Ca²⁺]_c and force during 20–50 Hz isometric contractions in the current study, but elevating plasma [NO₂⁻] via chronic NO₃⁻ supplementation increased these variables in the same experimental model (Hernández et al. 2012), we cannot exclude the possibility that skeletal muscle contractility, fatigue and Ca²⁺ handling might be enhanced to a greater extent after chronic NO₃⁻ supplementation. It should be noted that since mammalian plasma nitrite is significantly lower (i.e. at high nanomolar range) after NO₃⁻ ingestion (e.g. Wylie et al. 2013) compared to the 100 μM NaNO₂⁻ that was used to perfuse the skeletal muscle fibres in the present investigation, the dose of NO₂⁻ administered in the current study was supraphysiological. Furthermore, since human skeletal muscles do not manifest the same changes in Ca²⁺-handling proteins following a 7-day NO₃⁻ supplementation (Whitfield et al. 2017) previously detected in rodent skeletal muscle(Hernández et al.2012), the extent to which our findings translate to humans is unclear and in need of further research.

While fatigue development was attenuated concomitant with improved Ca²⁺ handling following NaNO₂ administration at a physiological $P_{{\text{O}}_2}$, Ca²⁺ handling was not assessed during the supraphysiological $P_{{\text{O}}_2}$ experiments in the current study. Therefore, the mechanisms for the more rapid fatigue development with NaNO₂ administration at a supraphysiological $P_{{\text{O}}_2}$ remains to be determined. Moreover, although the contraction-induced pH_i changes observed in the present study were qualitatively similar to the contraction-induced pH_i changes in human skeletal muscle in vivo, it should be acknowledged that the solution that all fibres were superfused with was bubbled with 5% CO₂ for an extracellular final pH 7.4. Therefore, pH_i was higher in the present study compared to human skeletal muscle completing fatiguing contractions in vivo. It is also acknowledged that a limitation of our study is the lack of measurement of NO/ROS markers. Since the NO fluorescent probes, DAF-FM and DAF-2, do not detect NO at low oxygen tensions (Namin et al. 2013), these probes could not be employed to assess myofibre NO production from NO₂⁻ reduction in our study. Further research is required to assess the effects of increased skeletal muscle NO₂⁻ exposure on NO and ROS signalling in skeletal muscle.

Translational perspective

Over the past decade there has been significant interest in supplementing the diet with NO₃⁻ to enhance exercise economy and performance (Jones, 2014), but the underpinning mechanisms for these effects were unclear. The present study indicates that NaNO₂ administration can delay fatigue development, improve the maintenance of myofilament Ca²⁺ sensitivity and attenuate the fatigue-induced slowing in SR Ca²⁺ reuptake during repeated tetanic contractions in ex vivo skeletal muscle fibres at a near-physiological $P_{{\text{O}}_2}$ of 2% O₂. These findings using a single skeletal muscle fibre model also demonstrate that increased NO₂⁻ exposure can blunt skeletal muscle fatigue development independently of any alterations in skeletal muscle perfusion by improving skeletal muscle Ca²⁺ handling. Therefore, our findings offer novel insights into some of the mechanisms that might underpin the ergogenic effects of increased skeletal muscle NO₂⁻ exposure, which might have implications for improving understanding of how dietary NO₃⁻ supplementation is ergogenic in humans during high-intensity endurance exercise.

In addition to evoking positive effects in skeletal muscle, NO₃⁻ supplementation or administration of NO donors can improve Ca²⁺ handling and contractile function in cardiomyocytes (Pironti et al. 2016; Tocchetti et al. 2007). There is also evidence to suggest that NO₃⁻ supplementation can improve skeletal muscle (Coggan et al. 2015b) and cardiac(Zamani et al. 2015) function, and exercise capacity(Zamani et al. 2015;Coggan et al. 2018) in heart failure patients. Therefore, increasedNO₂⁻ exposure following NO₃⁻ supplementation might have important therapeutic application in patients with diseases of the cardiovascular system.

In conclusion, acute treatment with NaNO₂ delayed time to task failure in intact skeletal muscle fibres at a physiological $P_{{\text{O}}_2}$ during a repeated tetanic isometric contraction protocol. The greatest attenuation of fatigue development was observed when sodium lactate was co-administered with NaNO₂ at 2% O₂ to elicit similar pH_i response dynamics to those manifest in vivo. Conversely, task failure was attained earlier following NaNO₂ administration when assessed at a supra-physiological $P_{{\text{O}}_2}$ equivalent to 20% O₂. The delay in fatigue development following NaNO₂ administration at 2% O₂ was accompanied by improved maintenance of myofilamentCa²⁺ sensitivity, increased SR Ca²⁺ pumping and a lower basal [Ca²⁺]_c in the recovery period between contractions, and a greater decline of pH_i. Therefore, NaNO₂ administration can delay fatigue development in intact, ex vivo skeletal muscle fibres at a near-physiological $P_{{\text{O}}_2}$, with this effect linked to improved myocyte Ca²⁺ handling. These results provide new insight into the mechanisms by which increased NO₂⁻ exposure can mitigate skeletal muscle fatigue development and the ergogenic potential of dietary NO₃⁻ ingestion.

Keypoints.

• Dietary nitrate supplementation increases plasma nitrite concentration, which provides an oxygen-independent source of nitric oxide and can delay skeletal muscle fatigue.

• Nitrate supplementation has been shown to increase myofibre calcium release and force production in mouse skeletal muscle during contractions at a supra-physiological oxygen tension, but it is unclear whether nitrite exposure can delay fatigue development and improve myofibre calcium handling at a near-physiological oxygen tension.

• Single mouse muscle fibres acutely treated with nitrite had a lower force and cytosolic calcium concentration during single non-fatiguing contractions at a near-physiological oxygen tension.

• Nitrite treatment delayed fatigue development during repeated fatiguing isometric contractions at near-physiological, but not at supra-physiological, oxygen tension in combination with better maintenance of myofilament calcium sensitivity and sarcoplasmic reticulum calcium pumping.

• These findings improve understanding of the mechanisms by which increased skeletal muscle nitrite exposure might be ergogenic and imply that this is related to improved calcium handling

Biography

Dr Stephen Bailey is a lecturer in sport and exercise nutrition in the School of Sport, Exercise and Health Sciences at Loughborough University. Dr Bailey’s research interests surround exercise-induced fatigue, oxidative metabolism, cardiovascular function, nitric oxide and redox biology, and how these responses can be positively impacted by nutritional and exercise interventions.