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Nitrate Supplementation Enhances the Contractile Properties of Human Skeletal Muscle

HAIDER, GEORG; FOLLAND, JONATHAN P.

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Medicine & Science in Sports & Exercise: December 2014 - Volume 46 - Issue 12 - p 2234-2243
doi: 10.1249/MSS.0000000000000351
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Abstract

Dietary nitrate supplementation has been found to have a variety of effects on human physiology, with recent evidence that short-term supplementation (1–7 d) with either beetroot juice or sodium nitrate lowers resting blood pressure (22,41), reduces the energetic cost of exercise (1,19,22), and may improve endurance exercise performance (5,18,44).

Nitrate is an inorganic anion that is ingested via diet and is particularly abundant in green leafy vegetables such as spinach, beetroot, rocket, or lettuce (8,23). Nitrate itself is inert but after ingestion approximately 5%–7% is converted into nitrite (8) mainly by facultative bacteria in the oral cavity (13). The nitrate–nitrite conversion seems to be necessary for nitrate supplementation to exert the documented physiological effects (15,41). Nitrite then enters the systemic circulation and is further reduced to nitric oxide (NO) and other (bioactive) nitrogen oxides in several tissues (24). Because of the wide-ranging bioactive properties of NO as a vital signaling molecule (32,33), it seems to mediate the physiological effects of dietary nitrate supplementation (1). Parallel to the nitrate–nitrite–NO pathway, NO is also generated endogenously by NO synthases that catalyze the oxidation of L-arginine (24). Endogenous NO can subsequently be converted into nitrate and thereby complements nitrate exposure from dietary sources (24).

Much of the research attention regarding the physiological effects of nitrate supplementation has focused on changes in cardiovascular function (22,41) or energy metabolism (1,19,22). A recent study, however, found dietary nitrate supplementation in mice to have profound effects on the contractile properties of skeletal muscle (14). Intact fast-twitch, but not slow-twitch muscle, of nitrate-fed mice had 50%–100% greater peak force response to low frequencies (≤50 Hz) of electrical stimulation compared with age-matched controls, and this effect seemed to be due to improved intracellular calcium handling and increased crossbridge sensitivity to calcium. Whereas peak force at high stimulation frequencies (>50 Hz) was similar between nitrate and control mice, rapid (explosive) force production of fast-twitch muscle was markedly enhanced in the early rising phase of 100-Hz tetanic contraction (35% reduction in the time to reach 50% peak force).

Hernández et al. (14) suggested that the dose of the nitrate supplementation they provided to mice was equivalent to that in recent human studies. However, the possibility that nitrate supplementation might change the contractile properties of the human skeletal muscle and, potentially, voluntary muscle performance, particularly explosive force production, has not been investigated. Therefore, the aim of this study was to investigate the effects of 7 d of dietary nitrate supplementation on the intrinsic contractile properties of the human skeletal muscle in vivo and voluntary muscle function. The intrinsic contractile properties were assessed with the force–frequency relationship in response to submaximal muscle stimulation and the evoked twitch and octet (drives the muscle at its maximum for explosive force production (7)) response to supramaximal nerve stimulation. We hypothesized that nitrate supplementation would enhance the intrinsic contractile properties, specifically the peak force response to low-frequency stimulation and the explosive force response to an evoked octet and, thus, also explosive voluntary force production in man.

METHODS

Participants

Nineteen healthy young men (mean ± SD: age, 21 ± 3 yr; body mass, 73 ± 10 kg; height, 1.82 ± 0.06 m) volunteered to participate, provided written informed consent, and completed this study that was approved by the Loughborough University ethical advisory committee. Participants were injury free, not involved in any systematic physical training, and had a low-to-moderate level of physical activity (1341 ± 698 MET·min·wk−1, International Physical Activity Questionnaire, short format (6)). In addition, none of the participants were taking any nutritional supplements, nor were they smokers. One participant had a very high habitual nitrate intake during the baseline period (34 mmol·wk−1, greater than fourfold group mean) and was excluded from the study.

Overview

This study used a randomized, crossover, double-blind design. Participants completed four measurement sessions over a 4-wk period: a familiarization trial, a baseline trial (no dietary intervention), and two main trials each after 7 d of nitrate (NIT, approximately 9.7 mmol·d−1) or placebo (PLA) supplementation in a randomized order. There were 7 d between each of the first three measurement sessions and 9 d between the main trials. During the supplementation periods, participants also attended the laboratory every morning (8:30–10:00) to consume the supplement in a double-blind manner and to record their 24-h vegetable and fluid intake to monitor habitual dietary nitrate intake. Participants were instructed to abstain from caffeine, alcohol, and strenuous exercise for 6, 24, and 36 h, respectively, before measurement trials and not to use antibacterial mouthwash (known to diminish nitrate–nitrite conversion (13,39)) throughout the study period.

Measurement sessions were conducted at a consistent time of the day, with recordings of force and surface EMG during a series of voluntary and involuntary (electrically evoked) isometric contractions of the knee extensors of the dominant leg as well as blood pressure measurements. The familiarization trial involved all the voluntary and evoked contractions but without EMG recordings. The baseline and main trials involved an identical protocol according to a strict schedule. During voluntary contractions, maximum and explosive strength as well as neuromuscular activation (via surface EMG) of the agonist knee extensors and the antagonist knee flexors were assessed. Supramaximal femoral nerve stimulation was used to evoke maximal twitch and octet contractions (eight electrical impulses at 300 Hz, evokes the maximum capacity for explosive force production (7)). Maximal twitch contractions also elicited maximal compound muscle action potentials (M-waves) that were used for normalization of volitional EMG. Finally, the force response to submaximal muscle stimulation of the knee extensors (electrodes over superficial quadriceps) at a range of frequencies (1–100 Hz) was recorded to establish the force–frequency relationship. All the analysis was done blind for supplement, and exclusions were made before unblinding.

Dietary Intervention

Supplements were administered under supervision in a double-blind manner. An independent technician, not otherwise involved in this study, prepared the supplements. The supplements were provided in an opaque drinks bottle containing 300 g of fluid and were administered every morning (8:30–10:00) for 6 d before and 2.5 h before the start of each main trial (7th day of supplementation). In the NIT condition, participants consumed 120 g (1.5 × 70 mL) of concentrated beetroot juice (98% beetroot, 2% lemon juice, Beet It Sport; James White Drinks Ltd., Ipswich, United Kingdom) containing 0.6g nitrate (approximately 9.7 mmol) diluted in 180g low-nitrate mineral water (Buxton; Nestlé Waters UK Ltd., Rickmansworth, United Kingdom). The PLA consisted of 27g low-calorie blackcurrant juice cordial (negligible nitrate content, Sainsbury’s squash double concentrate; Sainsbury’s Supermarkets Ltd., London, United Kingdom), 3g lemon juice (Sainsbury’s lemon juice; Sainsbury’s Supermarkets Ltd., London, United Kingdom), and 270g low-nitrate mineral water. Participants were deliberately misinformed that the effects of two different nitrate supplements would be tested and compared to the baseline trial. They were instructed not to comment on the taste of the drinks or to report known side effects (e.g., pink urine/beeturia) to the investigators. Feedback forms completed after the study confirmed that participants were unaware of the actual research hypotheses during the study.

Habitual dietary nitrate intake was estimated via 24-h recall of vegetable and fluid intake administered daily during both supplementation periods. Vegetable, tap water, and beer intake accounts for, on average, approximately 85% of total dietary nitrate intake in the United Kingdom (8). Thus, before consuming each supplement, participants reported the number of 80-g vegetable portions that they ingested during the preceding 24 h from a list provided together with their tap water and beer consumption. Average 80-g portion sizes were defined using the United Kingdom National Health Service’s “5-a-Day” campaign portion size guide (28). Nitrate consumption was estimated from vegetable, beer, and tap water intake using published reference values for the nitrate content of vegetables (8,23), beer (25), and water (local tap water supplier) (31). A 30% reduction in nitrate intake was assumed for vegetables consumed after cooking, steaming, or boiling (8). The resulting 24-h nitrate intake values were summated to give a weekly habitual nitrate intake during both the NIT and PLA periods.

Measurements

Knee extension force

During all the contractions, participants sat in a custom-built isometric strength testing chair (29) with hip and knee joint angles of 110° and 120°, respectively (180°, full extension). Chair configuration was established during the familiarization trial and replicated thereafter. Waist and shoulder straps were tightly fastened to prevent extraneous movements. An ankle strap was placed approximately 4 cm proximal to the medial malleolus in series with a calibrated linear response S-beam load cell (FSBS; Force Logic, Swallowfield, United Kingdom) that was positioned perpendicular to the tibia. The analog force signal was amplified (×370) and sampled at 2 kHz with an external analog-to-digital converter (Micro 1401; CED Ltd., Cambridge, United Kingdom). A personal computer (PC) recorded and displayed the data with the Spike2 software (CED Ltd., Cambridge, United Kingdom). In offline analysis, the force data were low-pass filtered at 500 Hz using a fourth-order zero-lag Butterworth filter. Baseline resting force was subtracted from all force recordings to correct for the effects of gravity.

EMG

Surface EMG was recorded from the superficial quadriceps (vastus medialis (VM), vastus lateralis (VL), rectus femoris (RF)) and hamstrings (biceps femoris long head (BF) and medial hamstrings (MH)) using a wireless EMG system (Trigno; Delsys, Inc., Boston, MA). After preparation of the skin (shaving, abrading, and cleansing with 70% ethanol), single differential electrodes (1-cm interelectrode distance; Delsys Inc., Boston, MA) were attached over the muscles using adhesive interfaces. Two electrodes were placed over each of the superficial quadriceps muscles (located 1 cm laterally and medially on the longitudinal midline of the muscle belly), and one electrode, over each hamstrings site. Electrodes were positioned parallel to the presumed orientation of the muscle fibers at specific percentages of thigh length (distance of the lateral knee joint center to the greater trochanter) proximal to the superior border of the patella (VM, 25% and 35%; VL, 45% and 55%; RF, 55% and 65%) or to the popliteal fossa (BF, 45%; MH, 45%). EMG signals were amplified at source (×300; 20- to 450-Hz bandwidth) before further amplification (overall effective gain, ×909) and subsequently sampled at 2 kHz using the same analog-to-digital converter and PC software to enable synchronization with the force data. In offline analysis, the EMG data were time-aligned with the force signal (inherent 48-ms delay of EMG signal) and band-pass filtered (6–500 Hz) using a fourth-order zero-lag Butterworth filter.

Electrical stimulation

A constant current variable voltage stimulator (DS7AH; Digitimer Ltd., Welwyn Garden City, United Kingdom) was used to assess the contractile properties of the knee extensors while the participant was voluntarily passive. Electrical impulses (square wave pulses of 0.2-ms duration) were delivered via the following: (i) supramaximal femoral nerve stimulation to evoke maximal twitch and octet contractions and (ii) transcutaneous submaximal muscle stimulation to evoke contractions at a range of frequencies (1–100 Hz) to assess the force–frequency relationship. Although supramaximal stimulation is preferable to ensure maximal force responses, the force–frequency relationship involves sustained tetanic contractions that are not tolerable with supramaximal stimulation, and therefore, submaximal stimulation was necessary. However, submaximal femoral nerve stimulation does not give consistent responses because of the influence of contraction/movement on the current delivered to the nerve; therefore, submaximal transcutaneous muscle stimulation was used.

Protocol

Main trial measurements started 2.5 h after the final supplement intake and were completed in the following order, according to a consistent schedule.

Blood pressure

Participants rested in a seated position for 5 min before their blood pressure was measured using an automatic sphygmomanometer placed over the brachial artery of the left arm (Omron M5-I; Omron Healthcare Ltd., Milton Keynes, United Kingdom). Three measurements were taken at 1-min intervals, with an average of the last two used for further analysis. One participant was excluded from blood pressure analysis because of highly variable measurements.

Maximum voluntary contractions

After EMG preparation and positioning of the participant in the isometric apparatus, a brief warm-up of six submaximal knee extension contractions at 50% (×3), 75% (×2), and 90% of the participants’ perceived maximal effort was performed (contractions lasted 3 s each and were separated by approximately 20 s). Thereafter, participants completed four maximum voluntary contractions (MVC) of the knee extensors (≥30 s apart). They were instructed to contract “as hard as possible” for approximately 3 s. Strong verbal encouragement and biofeedback (online force signal and a marker of their maximum force during that session, which served as a target) were provided on a PC screen directly in front of the participant.

The greatest instantaneous force during either the knee extensor MVC (29 of 38 main trials) or explosive voluntary contractions (see later portion) of that trial was defined as maximum voluntary force (MVF). After filtering, correcting for EMG signal time offset (48-ms delay) and any baseline offset, the root mean square of the EMG signal (RMSEMG) at MVF (500-ms period surrounding MVF for an MVC, 200-ms for an explosive contraction) was calculated for each recording site (2 × VM, 2 × VL, 2 × RF, 1 × BF, 1 × MH). RMSEMG for each quadriceps recording site was first normalized to the corresponding maximal M-wave area (Mmax) (see below) and then averaged across all six sites to calculate the mean quadriceps value. The RMSEMG of each hamstring electrode (BF, MH) was normalized to the highest RMSEMG (500-ms period) measured during knee flexion MVC (HEMGmax) (see below) before calculating a mean hamstrings value.

Explosive voluntary contractions

After a 5-min rest, 15 isometric explosive voluntary knee extensions were performed (each separated by ≥15 s). Participants attempted to extend their leg “as fast and hard as possible” for approximately 1 s from a completely relaxed state in reaction to an auditory signal. Specific biofeedback, verbal feedback, and encouragement were provided throughout these contractions. The force–time curve was displayed together with a marker indicating 80% of peak force achieved in the preceding MVC, which participants were expected to exceed during each explosive contraction. The baseline force was displayed on a sensitive scale, and any pretension or countermovement was highlighted. Finally, the rate of force development (RFD, 10-ms time epoch) was displayed with peak RFD indicated by a marker, which provided a target and an index of their explosive performance.

During offline analysis, all force and EMG onsets were identified manually by visual identification by a trained investigator using a systematic approach (35,36) which is considered to be more valid than automated methods (36). Contractions were excluded if baseline force changed by >0.5 N during the 250 ms before force onset (i.e., pretension or countermovement) or if peak force was lower than 80% MVF. The three valid contractions with the greatest force at 100 ms after force onset were analyzed, and measurements were averaged across these contractions. Force was measured at 25-ms intervals up to 150 ms after force onset. The RMSEMG of each EMG site was measured over three consecutive 50-ms periods from EMG onset of the first agonist muscle to be activated. Thereafter, RMSEMG at each site was normalized (to Mmax and HEMGmax) (see below) and averaged for the quadriceps and hamstrings, respectively. The time difference between EMG onset and force onset was used to measure voluntary electromechanical delay (EMD).

Twitch and octet contractions (via supramaximal femoral nerve stimulation)

Femoral nerve stimulation involved a cathode stimulation probe (1-cm diameter; Electro-Medical Supplies Ltd., Wantage, United Kingdom) firmly pressed into the skin over the femoral nerve in the femoral triangle and an anode (7 × 10 cm carbon rubber electrode; Electro-Medical Supplies Ltd., Wantage, United Kingdom) coated with electrode gel and taped to the skin over the greater trochanter. A series of single electrical impulses (≥12 s apart) were delivered to determine the precise cathode location (greatest twitch response to a submaximal current). The current intensity was increased until a plateau in the amplitudes of peak twitch force and peak-to-peak M-wave occurred. The current was then increased by 40% to ensure supramaximal stimulation, and 7–8 min after the last voluntary contraction, three single impulses were delivered (15 s apart) to elicit three maximal twitch and M-wave responses. Subsequently, octet contractions (eight impulses at 300 Hz) were evoked at progressive currents (≥15 s apart) until a plateau in the amplitudes of peak force and peak RFD were achieved. Then, three discrete pulse trains (≥15 s apart) were delivered with a higher current (>15% to ensure supramaximal stimulation) to evoke maximal octet contractions.

The force response to the maximal twitch and octet contractions was measured at 10-ms intervals from force onset up to 50 ms, and analyzed for peak force, time to peak force, and half-relaxation time (for the twitch only). The M-wave response at each quadriceps EMG site during the twitch contractions was analyzed as the cumulative area (Mmax) from EMG onset after the stimulation artifact to the point at which the signal returned to baseline. Volitional quadriceps EMG was normalized to Mmax. The time difference between M-wave onset (first electrode site to be activated) and twitch force onset was used to measure evoked EMD. All twitch, octet, and M-wave measurements were averaged across the three analyzed contractions. One participant could not tolerate the octet contractions, and thus, eighteen participants completed this measurement.

Force–frequency relationship (via submaximal transcutaneous muscle stimulation)

The surfaces of two carbon rubber electrodes (14 × 10 cm; Electro-Medical Supplies Ltd., Wantage, United Kingdom) were coated with electrode gel and taped over the superficial quadriceps (approximately 10 cm apart). For calibration purposes, 100-Hz contractions were evoked at increasing current intensities to determine the current that elicited 50% of baseline MVF. This current (typically 80–140 mA) was then used for the range of force–frequency measurements. The last calibration contraction and the measured contractions were separated by 8 min while knee flexion MVC were performed. The force–frequency relationship contractions consisted of two sets of five twitch contractions (1 Hz), followed by two contractions of 1-s duration at each of the five different frequencies (10, 20, 30, 50, and 100 Hz) performed in an ascending order, with 20 s between contractions. Peak force was defined as the greatest instantaneous force (1 and 10 Hz) or the mean force over the last 300 ms of the force plateau (20–100 Hz). Thereafter, the force values at each stimulation frequency were averaged and normalized to 100-Hz force (F100Hz). The 20:50 Hz force ratio was also calculated. Four participants were not able to avoid voluntary contractions during force–frequency measurements, and their data were excluded.

Knee flexion MVC

After a series of warm-up contractions (identical to that for the knee extensors), participants performed three knee flexion MVC (≥30 s apart) to establish reference values for hamstrings (BF and MH) EMG normalization. Participants were instructed to contract their hamstrings “as hard as possible” for approximately 3 s and received strong verbal encouragement throughout. For each hamstring muscle (BF, MH), the highest RMSEMG (500-ms period) during these MVC was defined as HEMGmax and used for normalization of the antagonist hamstring muscle activation during voluntary knee extensions.

Statistical Analysis

Shapiro–Wilk tests confirmed normal distribution of the data. Dependent variables measured over several time points/periods (force and EMG during explosive voluntary contractions, evoked twitch and octet force) were analyzed using a two-way (condition–contraction time) repeated-measures ANOVA. Similarly, the force–frequency relationship was assessed by a two-way (condition–frequency) repeated-measures ANOVA. Significant interaction effects were followed up by Bonferroni-corrected paired t-tests, and a Greenhouse–Geisser correction was applied to the degrees of freedom when the Mauchly test of sphericity indicated heterogeneous variances. Paired t-tests (paired t) were used to compare all other dependent variables. The change in group mean values between conditions was used to calculate the percentage change values presented. Bivariate relationships were assessed using the Pearson product-moment correlation coefficient. Statistical analyses were completed using SPSS version 20 (SPSS Inc., Chicago, Ill), and statistical significance was accepted at P < 0.05. Data are presented as mean ± SD. Effect sizes (ES) were calculated as Pearson r, which varies from 0 (no effect) to a maximum of 1 (9).

RESULTS

Habitual Nitrate Intake and Dietary Intervention

Mean nitrate intake was greater than eightfold higher during NIT compared with PLA (77.4 ± 5.3 vs 8.4 ± 4.5 mmol·wk−1; paired t, P < 0.001; ES > 0.99), although nitrate intake via the diet alone was also higher during NIT than that during PLA (9.6 ± 5.3 vs 8.4 ± 4.5 mmol·wk−1; paired t, P = 0.026; ES = 0.50). Averaged over both supplementation weeks, the habitual dietary nitrate intake was 9.0 ± 4.8 mmol·wk−1 (range, 0.6–18.9 mmol·wk−1).

Voluntary Force Production

MVF

MVF was very similar in both conditions (paired t, P = 0.539; ES = 0.15) (Fig. 1A). Agonist EMG at MVF (paired t, P = 0.750; ES = 0.08) (Fig. 1B) and antagonist EMG (paired t, P = 0.433; ES = 0.19) were also similar between NIT and PLA.

FIGURE 1
FIGURE 1:
MVF (A) and agonist EMG at MVF (B) during isometric knee extensions after NIT and PLA. Data are mean ± SD (n = 19). RMSEMG, 500-ms period at MVF.

Explosive voluntary force

Explosive voluntary force measured every 25 ms throughout the first 150 ms of contraction was similar for NIT and PLA (ANOVA: condition, P = 0.510, ES = 0.16; condition–time, P = 0.467) (Fig. 2A). There were also no significant differences between conditions for agonist EMG (ANOVA: condition, P = 0.824, ES = 0.05; condition–time, P = 0.164) (Fig. 2B) or antagonist EMG (ANOVA: condition, P = 0.445, ES = 0.18; condition–time, P = 0.394) during the first 150 ms of explosive voluntary efforts. Furthermore, voluntary EMD was very similar for the two conditions (NIT, 25 ± 4 ms, vs PLA, 25 ± 5 ms; paired t, P = 0.985). Peak force achieved during the explosive voluntary contractions was 90% ± 5% MVF.

FIGURE 2
FIGURE 2:
Explosive voluntary force (A) and agonist EMG (B) during explosive isometric knee extensions after NIT and PLA. Data are mean ± SD (n = 19). RMSEMG, 50-ms periods.

Intrinsic Contractile Properties

Maximal twitch contractions (via femoral nerve stimulation)

Evoked force measured every 10 ms during the first 50 ms of maximal twitch contractions was 8%–14% greater after NIT compared with after PLA (ANOVA: condition, P = 0.009, ES = 0.57) (Fig. 3), and this effect was influenced by contraction time (ANOVA: condition–time, P = 0.018). Bonferroni-corrected post hoc tests revealed that force was significantly greater after NIT compared with that after PLA at 20 ms (24 ± 7 vs 21 ± 5 N; Bonferroni, P = 0.029, ES = 0.59) and at 50 ms (118 ± 34 vs 110 ± 28 N; Bonferroni, P = 0.048, ES = 0.56) after force onset.

FIGURE 3
FIGURE 3:
Evoked isometric twitch force (initial 50 ms and peak) of the knee extensors in response to supramaximal nerve stimulation after NIT and PLA. Data are mean ± SD (n = 19). Post hoc comparisons at specific time points during the explosive (rising) phase of contraction: r = 0–1; *Bonferroni NIT > PLA, P < 0.05. **Paired t-test NIT > PLA, P < 0.01.

Twitch peak force was also 7% greater after NIT than that after PLA (149 ± 41 vs 138 ± 37 N; paired t, P = 0.008, ES = 0.56). However, evoked EMD (both 9 ± 1 ms; paired t, P = 0.199) and the time course of the twitch contractions remained similar (time to peak force, both 80 ± 7 ms; paired t, P = 0.702; half-relaxation time, NIT, 67 ± 8 ms, vs PLA, 68 ± 7 ms; paired t, P = 0.865, ES = 0.04).

Maximal octet contractions (via femoral nerve stimulation)

Evoked force measured every 10 ms during the initial 50 ms of maximal octet contractions was 3%–15% greater after NIT compared with that after PLA (ANOVA: condition, P = 0.023; ES = 0.52) (Fig. 4), but this effect was not significantly influenced by contraction time (ANOVA: condition–time, P = 0.101). There were no significant differences in octet peak force (NIT, 618 ± 96 N, vs PLA, 614 ± 101 N; paired t, P = 0.430, ES = 0.19) or time to reach octet peak force (NIT, 121 ± 8 ms, vs PLA, 122 ± 7 ms; paired t, P = 0.167, ES = 0.33) between conditions.

FIGURE 4
FIGURE 4:
Evoked isometric octet force (initial 50 ms) of the knee extensors in response to supramaximal nerve stimulation (eight impulses at 300 Hz) after NIT and PLA. Data are mean ± SD (n = 18). *Main effect of condition, ANOVA NIT > PLA, P < 0.05; ES, r = 0–1.

Force–frequency relationship (via submaximal transcutaneous muscle stimulation)

Peak force during the 100-Hz contractions (F100Hz) was very similar between conditions (NIT, 428 ± 79 N, vs PLA, 431 ± 67 N; paired t, P = 0.664, ES = 0.12), indicating appropriate calibration of the stimulation current used for the force–frequency relationship. For the range of frequencies, peak force was higher after NIT compared with that after PLA (ANOVA: condition, P = 0.039, ES = 0.52) (Fig. 5A), and this main effect was influenced by stimulation frequency (ANOVA: condition–frequency, P = 0.018). Follow-up uncorrected paired t-tests indicated that peak force during low-frequency stimulation (1, 10, and 20 Hz) was 5%–10% higher after NIT than that after PLA (P = 0.010–0.034, ES = 0.53–0.63), and for 10-Hz contractions, this force difference remained significant after Bonferroni correction (NIT, 21.8% ± 4.2%, vs PLA, 19.8% ± 4.1% F100Hz; Bonferroni, P = 0.048, ES = 0.63). The 20:50 Hz ratio also showed higher values after NIT (0.678 ± 0.059 vs 0.648 ± 0.052; paired t, P = 0.018, ES = 0.58). The percentage change in the peak force response to low-frequency electrical stimulation at 1, 10, and 20 Hz after NIT (vs PLA) was negatively associated with habitual dietary nitrate intake (−0.56 ≤ r ≤ −0.62, P = 0.013–0.029, ES = 0.56–0.62) (Fig. 5B), i.e., individuals with low habitual nitrate intake had a greater force increase after NIT.

FIGURE 5
FIGURE 5:
A. Force–frequency relationship of the knee extensors; isometric peak force evoked at a range of frequencies in response to submaximal muscle stimulation after NIT and PLA. All forces are normalized toF100Hz. Data are mean ± SD (n = 15). Post hoc comparisons at specific frequencies, ES, r = 0–1; *Bonferroni NIT > PLA, P < 0.05. B. Relationship between habitual nitrate intake via the diet (averaged over the two supplementation periods) and change (Δ) in isometric peak force after NIT (vs PLA) of the knee extensors in response to 10-Hz submaximal muscle stimulation. Data are individual values for each participant (n = 15).

Blood Pressure

Neither systolic blood pressure (SBP) (NIT, 116 ± 6 mm Hg, vs PLA, 115 ± 9 mm Hg; paired t, P = 0.346, ES = 0.23) nor diastolic blood pressure (DBP) (both 69 ± 5 mm Hg; paired t, P = 0.665) was significantly different between the two conditions. However, the individual change in SBP (NIT vs PLA) was negatively correlated with SBP measured at baseline (r = −0.53, P = 0.024, ES = 0.53), i.e., individuals with high baseline values exhibited a greater decrease after NIT. The change in DBP, however, was not significantly related to DBP at baseline (r = −0.01, P = 0.955, ES = 0.01).

DISCUSSION

In the nitrate condition, mean dietary nitrate intake was greater than eightfold higher than that in the PLA condition and was associated with increased peak force responses to low-frequency stimulation (supramaximal twitch and submaximal 1- to 20-Hz contractions) as well as enhanced explosive force, but not peak force, responses to a supramaximal evoked octet. These positive changes in excitation–contraction coupling were not, however, accompanied by changes in voluntary force production.

The Dietary Nitrate Intervention

Participants were unaware of the study hypothesis, and all measurements were collected in a double-blind manner with a randomized crossover design. The habitual nitrate intake from vegetables, beer, and tap water captured by the 24-h dietary records used throughout both supplementation periods is thought to account for approximately 85% of total dietary nitrate intake (8). Considering this likely underestimation, the mean habitual nitrate intake of 9 mmol·wk−1 in the current experiment was comparable with that in previous reports (11–12 mmol·wk−1) (26,45). Besides uncertainties in the accuracy of dietary recall, it is also worth noting that variations in the nitrate content of food (8), the amount of endogenous nitrate production (40), the storage of nitrate in bodily tissues (e.g., in skin (27)), and the degree of nitrate–nitrite conversion (8) could all influence the biological effect of dietary nitrate intake. Nonetheless, the nitrate supplementation used during the current study seemed to provide a substantial (eightfold) increase in mean nitrate intake compared with the PLA period. For nitrate supplementation to exert any physiological effects, the conversion of nitrate into nitrite seems to be necessary (15,41). Although plasma nitrite concentration was not assessed in the current study, numerous previous reports have unanimously found nitrate supplementation via beetroot juice to be effective in increasing plasma nitrite concentration (2,38,43). In the present study, the nitrate supplementation was provided as beetroot juice and compared with a placebo of blackcurrant juice cordial (1,2,38). Therefore, it cannot be excluded that other components of beetroot juice (e.g., antioxidants, betaines) contributed to the effects we have observed. Use of a nitrate-depleted beetroot juice (5,18,19) in future work may represent a neater placebo that differs only in nitrate content.

Resting SBP for the whole cohort was not significantly affected by nitrate supplementation (NIT), but the change in SBP after NIT (vs PLA) was negatively correlated with SBP measured at baseline (r = −0.53), indicating that individuals with the highest SBP showed a greater decrease. The absence of a cohort level effect of NIT on SBP is in agreement with some recent studies (5,20,42) but is in contrast with others (19,22,41). This contrast could be due to the variability in the baseline SBP of different cohorts, as Kapil et al. (16) also found participants with the highest SBP to show the greatest reductions. Thus, the relatively low SBP in the current study may have limited the potential for reductions in SBP with NIT.

Intrinsic Contractile Properties of the Knee Extensors

Nitrate supplementation enhanced force production during twitch contractions evoked by supramaximal nerve stimulation, with increases of 8%–14% during the rising phase (10–50 ms) and 7% at peak force compared with PLA, but there was no change in the time course of the contraction. High-frequency octet contractions (eight impulses at 300 Hz) evoked by supramaximal stimulation were used to examine the maximum capacity of the knee extensors for explosive force production after NIT and showed increased force during the initial 50 ms of contraction by 3%–15%, without any changes in peak octet force or the time to reach this peak. These results are consistent with, although less dramatic than, those of Hernández et al. (14). Using a comparable dose of nitrate supplementation, relative to body mass, as the current study, they found enhanced peak twitch force (by approximately 100%) and early-phase explosive force production but not peak force of high-frequency (100 Hz) stimulation in mouse fast-twitch muscle. These authors reported no changes in slow-twitch muscle after nitrate feeding. Thus, it seems likely that in the present study, NIT affected the evoked contractile properties of only a proportion of knee extensor muscle fibers.

Submaximal muscle stimulation was used to assess the effect of NIT on the force–frequency relationship of the knee extensors. In the current study, F100Hz was calibrated to a prescribed level that was very similar for both conditions (NIT, 428 N, vs PLA, 431 N) and the contractile force response to lower frequencies of stimulation was compared with responses to F100Hz. NIT enhanced the contractile force response to low frequencies (1–20 Hz) of stimulation by 5%–10% (ES = 0.53–0.63) relative to those in F100Hz, and the 20:50 Hz ratio, but had no effect at higher frequencies (30 and 50 Hz) of stimulation (ES = 0.04–0.20). The nature of the current study, with in vivo measurements of contractile properties in humans, prevented the use of prolonged trains of supramaximal stimulation, and thus, only relative forces (normalized to F100Hz), and not absolute forces, could be assessed. Therefore, these results cannot be directly compared with those reported by Hernández et al. (14), who found large increases in absolute forces (approximately 50%–100%), with 1- to 50-Hz stimulation of mouse fast-twitch muscle after NIT. In the current study, however, the shape of the force–frequency relationship was influenced by NIT, with higher relative contractile force at low frequencies of stimulation (excitation) that reflects enhanced excitation–contraction coupling at these frequencies, and is broadly in agreement with the study of Hernández et al. (14).

The current study also found that individual habitual nitrate intake via diet explained approximately one-third (32%–39%) of the variation in the percentage change of peak forces at low stimulation frequencies (1–20 Hz) after NIT and indicates that NIT enhanced low-frequency excitation–contraction coupling the most in individuals with the lowest habitual nitrate intake. This association occurred despite the limitations in the measurement of dietary nitrate intake by dietary recall, which might indicate an even stronger influence of habitual nitrate intake on the efficacy of nitrate supplementation than that we have been able to capture. Furthermore, if habitual nitrate intake mediates some of the effects of NIT, this implies that studies, which restricted the intake of nitrate-rich food (1,21,22) or provided standardized, presumably low-nitrate meals (5,43), could have overestimated the physiological effects of NIT for populations with average or high levels of habitual nitrate intake.

Overall, this study found moderate effects (ES, r = 0.52–0.63) of NIT on the contractile properties, i.e., increased peak force during low-frequency stimulation and enhanced explosive force production in response to twitch and 300-Hz octet stimulation. These effects, although quantitatively smaller, were remarkably similar in pattern to those observed by Hernández et al. (14) who attributed the effects of NIT primarily to improved intracellular calcium handling with increases in the myoplasmic free calcium concentration. Increases in intracellular calcium concentrations are most likely to be beneficial in situations where calcium saturation is incomplete, i.e., during the explosive/rising phase of contraction or at low frequencies of stimulation. In these situations, increased intracellular calcium concentration in response to the same excitation would be expected to enhance force production. In nitrate-fed mice, a greater expression of two calcium handling proteins (calsequestrin 1 and dihydropyridine receptor) has been found in fast-twitch muscles, compared with controls (14), and would be expected to lead to improved calcium handling, greater calcium transients in the cytoplasm, and, thus, enhanced force production (14). Alternatively, some of the data from Hernández et al. (14) also point to altered crossbridge sensitivity to calcium, as follows: greater force at low-frequency stimulation (15 Hz) in nitrate-fed mice compared with that in controls despite similar myoplasmic free calcium concentration. Furthermore, altered crossbridge sensitivity to calcium alone has also been shown to increase peak force during low-frequency stimulation and explosive force production in response to low- and high-frequency stimulation (37). Moreover, it seems plausible that the results of the present study were due to similar adaptations to those proposed by Hernández et al. (14), but the exact mechanisms of how NIT might influence protein expression or crossbridge sensitivity to calcium in vivo remain to be determined.

Voluntary Force Production

Nitrate supplementation had no effect on maximum or explosive voluntary force production. The stable MVF found in this study is consistent with that found in another report after short-term NIT in humans (12) and the stable peak tetanic force of mouse muscle after NIT (14). However, small increases in MVF have been documented after acute NO administration (11).

Given the changes in the contractile properties that we have observed, including the explosive force response during the first 50 ms of an evoked octet, it might initially be considered surprising that explosive voluntary force was not affected by NIT. However, explosive voluntary force production during this period is known to be relatively unreliable (within-participant coefficient of variation for force at 50 ms, 13%–17%) (4,35) and also seems to be primarily determined by volitional neuromuscular activation rather than the contractile properties of the muscle (10). Considering these factors, it is understandable that the relatively subtle changes in the contractile properties we have observed did not influence explosive voluntary force production. It seems possible, however, that in a highly trained group with more effective neuromuscular activation, explosive voluntary force production may be both more consistent and more reliant upon the intrinsic contractile properties, in which case, NIT may be more beneficial. Alternatively, it has been suggested that trained endurance athletes have greater endogenous NO production than that in untrained individuals, which may reduce any beneficial effects of dietary nitrate supplementation (42).

Furthermore, during repeated submaximal activation of skeletal muscle fibers such as during endurance exercise, the benefits of enhanced excitation–contraction coupling at low frequencies of stimulation may become more apparent. Although data on the precise motor unit firing frequency during dynamic endurance exercise are not available, assuming neuromuscular activation of <50% of maximum then firing frequencies of ≤20 Hz would be expected within the knee extensors (17,30). For enhanced excitation–contraction coupling at these frequencies to be advantageous for endurance exercise, it would need to be accompanied by a reduced energy cost of contraction. The adenosine triphosphate (ATP) cost of calcium handling in human skeletal muscle fibers is approximately 30%–50% (depending on the fiber type) of the total ATP cost of contractile activity (3,34). If excitation–contraction coupling is enhanced because of improved calcium handling and/or increased crossbridge sensitivity to calcium, then the same contractile force output may be possible at a lower level of excitation and a reduced ATP cost of calcium handling after NIT. Consequently, the total ATP cost for a given level of contractile force output would be lower after NIT, which is consistent with a recent finding (1). Thus, the enhanced excitation–contraction coupling that we have found could contribute to the reported reductions in energy/oxygen cost of submaximal exercise after NIT (2,19,22,38). Future research is required to investigate the exact mechanisms underlying the changes in the contractile properties of the human skeletal muscle that we have observed and the implications for metabolic efficiency during exercise.

CONCLUSIONS

The results of the current study indicate that short-term dietary nitrate supplementation with concentrated beetroot juice (approximately 9.7 mmol nitrate·d−1) enhanced excitation–contraction coupling of human skeletal muscle in vivo. Specifically, nitrate supplementation increased peak force at low frequencies of electrical stimulation (1–20 Hz) and explosive force production at low (1 Hz) and high (300 Hz) frequencies of stimulation during unilateral isometric contractions of the knee extensors in untrained individuals. These moderate effects on the contractile properties did not translate into any significant changes in maximum or explosive voluntary force production but may be beneficial for other types of voluntary contractile activity such as endurance exercise.

Financial support for this study was provided by the Gatorade Sports Science Institute, a division of PepsiCo, Inc.

The authors’ contributions were as follows: G. H., lead experimenter and author; J. P. F., supervising experimenter and author

The authors declare no conflicts of interest. The views expressed in this article are those of the authors and do not necessarily reflect the position or policy of PepsiCo, Inc.

The results of the present study do not constitute endorsement by the American College of Sports Medicine.

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Keywords:

BEETROOT JUICE; FORCE–FREQUENCY RELATIONSHIP; EXCITATION–CONTRACTION COUPLING; MUSCLE STRENGTH; EXPLOSIVE FORCE PRODUCTION

© 2014 American College of Sports Medicine