Nitric oxide (NO) is a potent signaling molecule formed via the canonical oxygen-dependent L-arginine/NO synthase pathway (29). However, it is now known that NO can also be formed via the sequential reduction of inorganic nitrate (NO3−) and nitrite (NO2−) taken in through the diet in a process dependent on commensal anaerobic bacteria in the oral cavity (14,36). Therefore, supplementation with inorganic NO3− has become a practical method for increasing circulating plasma NO2−, and therefore NO, and has been shown to reduce blood pressure (4,10,39), improve vascular control (11), decrease oxygen (O2) consumption (V˙O2) during exercise (3,4,8,23,26,38,40), increase muscle contractile function (13,16), and improve exercise performance (7,8,23,32).
Despite the growing body of work researching the diverse biological effects of supplementing with dietary NO3−, the underlying mechanism of action remains to be elucidated. Of particular interest is the finding that supplementation decreases V˙O2 during exercise, as this challenges the classic physiological principle that the oxygen cost for an individual at a given submaximal power output is fixed, regardless of age or training status (34). TO reduce the energy cost of exercise, NO3− would need to improve mitochondrial coupling and therefore reduce the O2 cost of ATP production and/or reduce the ATP cost of force production. There is now evidence demonstrating that supplementation with NO3− in the form of beetroot juice (BRJ) does not alter mitochondrial bioenergetics (19,40), whereas early findings using sodium NO3− (24) have not been subsequently reproduced (19). Taken altogether, this suggests that improvements in mitochondrial efficiency and a reduction in ATP production costs are unlikely to explain the aforementioned decrease in whole-body V˙O2. By contrast, there is now a growing body of evidence that NO3− may improve the coupling of ATP hydrolysis to force production. Bailey et al. (3) have demonstrated decreased ATP turnover after BRJ supplementation during both low- and high-intensity exercise, as well as decreased PCr cost per unit force during maximum voluntary contractions (MVC) (13). These findings therefore suggest that NO3− decreases the ATP cost of skeletal muscle force production and does not increase reliance on anaerobic metabolism.
During exercise, one of the largest sources of energy consumption is the sarcoplasmic reticulum Ca2+-ATPase (SERCA) (5), suggesting that modifications of this ATPase could be a potential candidate to explain the decrease in ATP cost of contraction. Work using murine muscle has shown improvements in contractile function after NO3− supplementation, with fast-twitch muscle displaying greater peak force production in response to low-frequency (≤50 Hz) stimulation (18,19). This was due to an increase in cytosolic free calcium concentrations during tetanic stimulation (18), and increased expression of calsequestrin 1 and the dihydropyridine receptor, two key proteins involved in calcium uptake and release during skeletal muscle contraction (18,19). Although similar improvements in excitation–contraction coupling to low-frequency stimulation have been demonstrated in humans (16), the possibility that NO3− results in similar adaptations in calcium handling proteins in human skeletal muscle has not been investigated.
Although it is tempting to speculate that improving the efficiency of SERCA contributes to the observed improvement in contractile efficiency, paradoxically, incubation of resting murine skeletal muscle fibers with the NO donor S-nitro-N-acetyl cysteine inhibits SERCA activity in the absence of changes in force production (1), raising further questions as to how NO3− supplementation is altering skeletal muscle metabolism. However, we previously demonstrated that 7 d of BRJ supplementation resulted in increased mitochondrial emission of H2O2, a reactive oxygen species (ROS) (40). In contrast to NO donors, small increases in H2O2 have been shown to increase force production by directly interacting with the contractile apparatus (2) and can also directly increase the mechanical efficiency of skeletal muscle (2). Furthermore, the effects of acute increases in ROS on force production are more pronounced at submaximal frequencies because of their location on the steep portion of the force–[Ca2+] curve (21). Thus, an increase in calcium sensitivity mediated by intracellular redox signaling could result in a large effect on force generation and may represent a possible mechanism for the ergogenic effects of BRJ.
Therefore, the primary aim of this study was to examine the effects of 7 d of BRJ supplementation on in vivo skeletal muscle contractile characteristics and function and to evaluate whether changes in force production could be explained by structural and/or redox-mediated changes. We hypothesized that in human skeletal muscle, BRJ would improve force production at low-stimulation frequencies. However, contrary to the effects of NO3− in rodent muscle, we hypothesized this would occur in the absence of changes in protein expression for calcium handling targets, as acute (~2.5 h) BRJ supplementation in humans has been demonstrated to elicit similar reductions in whole-body V˙O2 when compared with supplementation for 5 and 15 d (38), a timeline that would preclude changes in protein content. By contrast, we hypothesized that BRJ supplementation would result in a shift to a more oxidized cellular environment, as determined by the ratio of reduced to oxidized glutathione (GSH:GSSG ratio), suggesting that H2O2 emission may be the underlying mechanism of action for the ergogenic effects of BRJ.
Eight healthy, recreationally active males (26.6 ± 1.2 yr, 77.1 ± 1.2 kg) were initially recruited for this study and underwent transcutaneous electrical muscle stimulation (TEMS) for the assessment of skeletal muscle contractile characteristics before and after supplementation. An additional eight male subjects (22.3 ± 1.2 yr, 77.9 ± 2.1 kg) then underwent skeletal muscle biopsies for the determination of mechanistic features related to calcium handling. Before participation, written informed consent was obtained from all subjects. All procedures were approved by the Research Ethics Boards of the University of Guelph (Guelph, Ontario), the University of Waterloo (Waterloo, Canada), and the McMaster University (Hamilton, Ontario) and conformed to the Declaration of Helsinki.
After the completion of a familiarization trial and an overnight fast, eight subjects performed TEMS and eight subjects had two biopsies taken from the vastus lateralis of one leg as previously described (6). The biopsies were flash-frozen for the determination of protein content via Western blotting and determination of cellular redox status (GSH:GSSG ratio) using HPLC. After the baseline TEMS or biopsy trial, subjects began supplementing with concentrated BRJ (2 × 6.5-mmol NO3−/70 mL Beet It Sport, taken twice daily for a total daily intake of ~26 mmol NO3−; James White Drinks, Ipswich, United Kingdom) for 7 d in an effort to maintain elevated blood plasma NO3− and NO2− levels. This protocol has been used by us previously and resulted in elevated plasma [NO3−] and [NO2−] concentrations throughout the supplementation period (40). On the morning of day 7 after an overnight fast, subjects consumed 2× Beet It Sport shots 90 min before repeating either the TEMS protocol or having two biopsies taken from the vastus lateralis of the opposite leg for postsupplementation measurements. Subjects were asked to refrain from consuming foods rich in nitrates for the duration of the study and to abstain from the use of antibacterial mouthwash, as this has been shown to attenuate the conversion of NO3− and NO2− by commensal bacteria (14).
Muscle contractile characteristics were assessed using TEMS as described previously (15). Briefly, for all force measurements, the participant sat upright in a straight-backed chair with the lower leg at 90° to the thigh and with the arms folded across the chest. A strap was used to secure the hips and thigh to minimize any extraneous movement that could affect force production. Twitches and tetani were delivered to the quadriceps from a Grass model S48 stimulator. A 5-cm-wide plastic cuff placed around the right leg just proximal to the malleoli was tightly attached to a linear variable differential transducer (LVDT). The LVDT output was amplified by a Daytronic carrier preamplifier and recorded on a two-channel Hewlett Packard 7402A recorder. Positioning of the LVDT was such that an angle of pull at 180° was achieved for each participant. Calibration was performed before each test session with weights of known amounts. Two brass electrodes coated with warm electrode gel were used to deliver the electrical impulse to the quadriceps muscle. The ground electrode was placed centrally on the anterior aspect of the thigh just above the patella, whereas the active electrode was placed toward the hip on the belly of the vastus lateralis. Each electrode was secured firmly with rubber straps wrapped around the leg and over the top of the electrode to ensure good contact between the skin and the electrode, and the position of the electrodes was recorded for each participant to ensure consistent placement for subsequent trials. Twitches were evoked using a single supramaximal (150 V) impulse of 50-μs duration. Tetani at low (10, 20, and 30 Hz) and high (50 and 100 Hz) frequencies were induced using a voltage that elicited 40% of presupplementation MVC separated by 30 s with a pulse duration of 50 μs and a train rate and duration of 1 s−1 and 1 s, as this was determined to be the maximum tolerable stimulus intensity. Tetanic force, regardless of frequency of stimulation, was taken as the peak force recorded. At each measurement point, a standardized protocol was used (Fig. 1) that consisted of measuring the MVC, two twitches, the tetani (in order of increasing frequency), and two further twitches. To assess MVC, 5-s contractions were performed. The average of two trials was used to represent MVC. One week before starting the experiment, participants were familiarized with all the electrical stimulation procedures. This session was also used to determine the voltage that elicited 40% of the participant’s MVC at 100 Hz.
Western blotting was performed on whole muscle homogenate using methods described previously (17). Samples were loaded equally for α-tubulin (ab7291; Abcam, Cambridge, United Kingdom), SERCA2a (MA3-919; Thermo Fisher Scientific, Waltham, MA), SERCA1a (A52, gift from Dr. David MacLennan, University of Toronto), ryanodine receptor (RyR, MA3-925, Thermo Fisher Scientific), dihydropyridine receptor (DHPR, MA3-920, Thermo Fisher Scientific), global calsequestrin (CSQ, MA3-913, Thermo Fisher Scientific), calsequestrin 1 (CSQ1, ab191564, Abcam), and calsequestrin 2 (CSQ2, ab108289, Abcam). Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane, and were incubated in a 5% nonfat skim milk blocking solution for 1 h, followed by primary antibody, and the corresponding secondary antibody (goat antimouse, sc-2005; Santa Cruz Biotechnology, Dallas, TX) diluted as specified by the supplier. All samples for a given target were detected from the same Western blot by cutting gels and transferring onto a single membrane to limit variability. To determine the amount of protein required for each target, linearity of signal for each antibody was confirmed by loading 1–15 μg of protein. SERCA1a, SERCA2a, global CSQ, and DHPR were loaded with 3 μg of protein; CSQ1 and CSQ2 were loaded with 5 μg; and RyR was loaded with 7 μg of protein. Membrane proteins were detected by enhanced chemiluminescence (Perkin Elmer, Woodbridge, ON) and quantified by densitometry (Alpha Innotech Fluorchem HD2; Fisher Scientific, Ottawa, ON).
Approximately 30 mg of wet muscle was removed from each biopsy for the determination of muscle GSH and GSSG contents. Tissue was homogenized in 7% perchloric acid/phenanthroline homogenization medium in a 1:10 wt/vol ratio and incubated for 10 min before centrifugation at 1000g at 4°C. Supernatant (250 μL) was then added to 10 μL of 0.4 M iodoacetic acid (Sigma Aldrich, Zwijndrecht, the Netherlands) and neutralized by the addition of excess NaHCO3. An internal 25 μL standard (G3640-25 mg, Sigma Aldrich) was added, and samples were incubated at room temperature for 1 h in a darkened room. Samples were then vortexed after the addition of 2 μL of dinitrofluorobenzene and incubated at room temperature in the dark for a further 8 h. Finally, samples (25 μL) were injected in an HPLC (LC-20AD; Shimadzu, Kyoto, Japan) coupled with a Microsorb 100-5 NH2 S250x4.6 mm HPLC column (Agilent Technologies, Santa Clara, CA) and measured with a flow of 0.5 mL·min−1 at 350 nM. Retention times were 18.5, 21, and 23 min for the internal standard, GSH, and GSSG, respectively.
A Student’s paired t-test (two-tailed) was used to detect differences between pre- and postsupplementation data in muscle contractile characteristics, protein content after Western blotting, and GSH:GSSG ratio. The force–frequency relationship was assessed by a two-way (supplementation–frequency) repeated-measures ANOVA. If significance was detected, a Bonferonni post hoc was applied. Significance was determined at P < 0.05 with confidence intervals ≥95%.
Muscle contractile properties
We demonstrate that after 7 d of BRJ supplementation, both MVC and peak force produced during electrically induced contractions at 100 Hz are unaltered (Fig. 2A and B). By contrast, the peak force measured during evoked twitches was increased after BRJ supplementation when performed both before and after the force–frequency curve (Fig. 2C and D). Specifically, peak force was increased by ~20% (P < 0.01) in the first set of twitches (Fig. 2C) and was increased ~11% (P < 0.05) in the second set (Fig. 2D). The rates of force development (+dF/dTmax) and relaxation (−dF/dTmax) were also increased for both sets of twitches after supplementation (P < 0.05); however, there was no difference in half-relaxation time (RT1/2) (Table 1).
As peak force was not different after supplementation, we therefore normalized the force produced by each individual subject at the different stimulation frequencies to their respective peak force at 100 Hz (Fig. 3A). We have demonstrated that there was a main effect of frequency on force production, with a significant interaction between frequency and BRJ supplementation (P < 0.05). A Bonferonni post hoc was therefore used to elucidate differences between pre- and post-BRJ measures at different stimulation frequencies. Although there was no difference in force production from 20 to 100 Hz, the peak force at 10 Hz was significantly greater after BRJ supplementation (pre-BRJ, 37.5% ± 2.4% vs post-BRJ, 41.1% ± 2.3% of peak force, P < 0.05). Traces of force production of a representative subject at 10 and 100 Hz are presented in Figure 3B and C.
Expression of calcium handling proteins
As low-frequency force is thought to be influenced by calcium, and previous work in rodents has shown that oral NO3− supplementation increases protein expression of CSQ and DHPR (18,19), we next used immunoblotting to measure the expression of key target proteins related to skeletal muscle calcium regulation. There was no difference in expression of SERCA1a and SERCA2a after 7 d of BRJ supplementation (Fig. 4A), and in contrast to results seen in rodent muscle (18,19), there was no difference in the expression of DHPR or RyR (Fig. 4B) or global CSQ (Fig. 4C). Because of the mixed fiber-type composition of human muscle (27,37), we also determined protein expression of both CSQ1 and CSQ2, and consistent with the global CSQ antibody, there was no difference in either isoform (Fig. C). Altogether, these data show that alterations in the human skeletal muscle force production to low-frequency stimulation cannot be explained by changes in protein expression.
Cellular redox status
Given the unaltered protein expression of proteins involved in calcium homeostasis, we next examined a potential signal/mechanism that regulates calcium release. Specifically, ROS has been shown to induce calcium release (2,9), whereas conversely antioxidants attenuate low-frequency force (35). Because we have previously shown that BRJ increases the capacity for mitochondrial ROS emission (40), we examined the reduced (GSH) and oxidized (GSSG) state of glutathione, a sensitive barometer of cellular redox stress. However, contrary to our hypothesis, there was no change in reduced (Fig. 5A), oxidized (Fig. 5B), or total glutathione (Fig. 5C) and, therefore, no difference in the ratio of GSH:GSSG (Fig. 5D, P = 0.241), suggesting the cellular redox state is unaltered after BRJ supplementation.
In this study, we used the combined techniques of TEMS and skeletal muscle biopsies to address modifications in contractile properties of human skeletal muscle after 7-d supplementation with BRJ. We have shown that consistent with previous reports in both humans (16) and rodents (18,19), peak force production is increased at low frequencies of stimulation after NO3− supplementation. However, in contrast to rodent data, this improvement cannot be explained by changes in key proteins associated with intracellular calcium handling in human skeletal muscle. These data therefore contribute to a growing body of literature, suggesting that the improvements in exercise efficiency seen after NO3− supplementation with either BRJ or sodium NO3− are likely due to modifications in myofibrillar force production.
Supplementation with NO3− improves muscle contractile function
In the current study, we have shown that the intrinsic contractile properties of skeletal muscle are increased after 7-d supplementation with BRJ. Specifically, we have demonstrated greater peak force during evoked isometric twitches after supplementation, and this corresponded to an increase in both the rate of force production and relaxation. The magnitude of the changes in peak twitch force seen in the current study (~11%–20%) is higher than previously reported in humans (~7% ) and may reflect the significantly higher NO3− dose (~26 vs 9.7 mmol·d−1) used in the current study. However, both studies show similar effects of BRJ on the force–frequency relationship of contraction, with increased contractile force production at low frequencies (10 Hz in the current study, 1–20 Hz ). This leftward shift of the force–frequency curve is also in agreement with the original findings in rodent muscle (18) and reflects enhanced excitation–contraction coupling at these frequencies.
Calcium handling protein expression is unaltered in humans
In contrast to our contractile characteristics, our finding that key calcium handling proteins were not altered after 7 d of BRJ supplementation does not support what has been previously reported in rodents (18,19). Hernández et al. (18) demonstrated increased protein expression of CSQ and DHPR in fast-twitch, but not slow-twitch, muscles taken from C57bl/6 male mice given 1 mM sodium NO3− for 7 d, and this finding has subsequently been replicated (19). Interestingly, work performed in rats has also shown that BRJ results in elevated blood flow (11) and microvascular PO2 (12) in type II, but not type I, muscles. Thus, it has been suggested that NO3− may preferentially target type II fibers (reviewed in ). In the current study, muscle was taken from human vastus lateralis, which contains displays considerable variability between subjects, but is estimated to be composed of approximately 50% type I and 50% type II fibers (27,37). It is therefore possible that because of the heterogeneous nature of the muscle used for assessment of protein content, any effects on type II fibers within the sample may be diluted by the presence of type I fibers. Given that the subjects recruited for biopsy experiments represent a different cohort compared with our contractile properties experiments, it is also possible that subjects with increased muscle contractile characteristics may have improved skeletal muscle calcium handling. However, given the consistency in which we have demonstrated a decrease in whole-body V˙O2 consumption (40) and force–production (current study) using this dosing protocol, this seems unlikely.
Furthermore, this fiber-type hypothesis is confounded by recent work demonstrating that contractility in murine cardiac tissue is also improved after NO3− supplementation (33), despite being highly oxidative. This study demonstrated an improvement in rates of pressure development and relaxation, and larger Ca2+ transients as a result of increased DHPR, but not CSQ expression. Interestingly, all of the aforementioned studies in murine skeletal (18,19) and cardiac (33) muscle have relied on changes in protein expression after chronic supplementation to explain the underlying changes in function. By contrast, acute (~2.5 h) supplementation in humans elicits similar reductions in whole-body V˙O2 when compared with supplementation for 5 and 15 d (38), a timeline that would preclude changes in protein content and supports the current findings. In addition, there are differences in NO3− metabolism (30), calcium handling, and SERCA function between species (22), suggesting that the regulation of this ATPase, and the role it plays in different fiber types after NO3− supplementation, may differ appreciably in humans compared with earlier work in rodents. Taken altogether, our findings suggest that the improvement in force observed in human muscle is unlikely to be explained by an increase in the content of proteins involved in maintaining calcium homeostasis during muscle contraction.
BRJ supplementation and redox signaling
The redox state of skeletal muscle is a highly regulated variable and is dictated by the rate of ROS production relative to intracellular antioxidant buffering capacity. In terms of force production, ROS elicits a hormetic response, as low levels of H2O2 can directly interact with the contractile apparatus to increase force production in the absence of changes in cytosolic calcium concentrations, whereas higher levels have also been shown to decrease myofibrillar calcium sensitivity and muscle force production (31). We have previously demonstrated that there is an increase in mitochondrial H2O2 emission in resting muscle after BRJ supplementation (40), and thus this represented a possible mechanism to connect the improvements in excitation–contraction coupling seen both previously (16,18,19) and in the current study. However, we have not been able to detect any changes in cellular redox state via protein carbonylation, lipid peroxidation, global nitrosylation (40), or changes in the GSH:GSSG ratio (current study), which is consistent with findings in human skeletal muscle after supplementation with sodium NO3− (25). However, these are markers of oxidative stress, providing a snapshot of intramuscular redox status and, therefore, cannot conclusively rule out a change in redox signaling (28). In this regard, a more sophisticated approach may be required to determine whether indeed there is a NO3−-mediated redox signaling cascade and what the downstream targets are. Furthermore, thus far, the majority of muscle analysis has been performed at rest, whereas the functional changes have been measured during exercise and/or skeletal muscle contraction. Future studies should therefore consider using more advanced techniques (i.e., proteomics and lipidomics, as reviewed in ) to examine alterations in the cellular redox balance after NO3− supplementation during exercise.
Taken altogether, we have demonstrated that after 7-d supplementation with NO3− in the form of BRJ, human skeletal muscle force production is increased at low-frequency stimulation, as are the rates of force development and relaxation during evoked twitches. This alteration in the force–frequency relationship is consistent with previous reports in both rodents (18,19) and humans (16) and suggests that human muscle can be activated at a lower frequency to achieve a given force output. This may reduce the number of motor units required to complete a given task and may explain the decrease in ATP turnover during exercise previously seen after BRJ supplementation (3). However, in contrast to results in rodent muscle, these improvements cannot be explained by increased expression of proteins associated with intracellular calcium handling. Furthermore, it remains to be determined if NO3− supplementation results in alterations in sarcoplasmic reticulum calcium uptake or release in humans or improvements in calcium sensitivity of the contractile machinery. In this regard, future studies should also include an assessment of changes in function after acute ingestion (i.e., <3 h) of BRJ. Mechanistic studies performed thus far have focused on the effects of chronic supplementation, with explanations relying on changes in structural protein expression skeletal muscle. However, similar reductions in whole-body V˙O2 have been demonstrated 2.5 h post-BRJ ingestion (38). This timeline would therefore likely rule out alterations in protein expression, and thus post-translational modifications, whether by redox signals or other mechanisms, remain a likely candidate to explain the effects of NO3− supplementation.
This work was funded by the Natural Sciences and Engineering Research Council of Canada (L. L. S. and G. P. H.), and infrastructure was purchased with the assistance of the Canadian Foundation for Innovation (G. P. H.) as well as the Ontario Research Fund (G. P. H.). The authors do not have any conflicts to disclose. The results of the present study have been presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation and do not constitute endorsement by the American College of Sports Medicine.
J. W. and G. P. H. designed experiments, interpreted the data, and wrote the manuscript. L. L. S. and A. R. T. designed experiments and interpreted the data. J. W., D. G., G. J. F. H., and L. J. C. v. L. performed experiments. All authors edited and approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.
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