Opposing effects of dietary nitrate on muscle contractile function in fast and slow human muscles

Abstract

Introduction/Purpose:

Increasing nitric oxide bioavailability via nitrate (NO₃⁻) ingestion enhances muscle contractility, which may be due to phosphorylation of the regulatory light chain (RLC) of myosin. If so, there should be an interaction between NO₃⁻ supplementation and post-activation twitch potentiation, which acts via the same pathway.

Methods:

A double-blind, placebo-controlled, randomized crossover study was conducted to determine the influence of dietary NO₃⁻ on the contractile properties of the triceps brachii (60–70% fast-twitch, or type II) and triceps surae (60–70% slow-twitch, or type I) muscles of healthy young men (n=14). Participants were studied after acute ingestion of 2.2 mL/kg of beetroot juice either containing or lacking 200 μmol/kg of NO₃⁻, with neuromuscular electrical stimulation used to determine muscle function.

Results:

NO₃⁻ supplementation did not alter unpotentiated or potentiated peak twitch torque or the maximal rates of torque development or relaxation in either muscle group. On the other hand, NO₃⁻ ingestion resulted in significant changes in the torque-frequency relationship of both the triceps brachii (P=0.019) and the triceps surae (P<0.001). This was due to a leftward shift of this sigmoidal relationship in the triceps brachii, but a rightward shift in the triceps surae.

Conclusions:

We conclude that dietary NO₃⁻ has opposing effects on the contractile properties of fast and slow human muscles, which could be due to differential changes in Ca²⁺ sensitivity. However, potentiation was unaltered, suggesting that this occurs independently of changes in RLC phosphorylation. Additional research will be required to determine the underlying biochemical mechanisms.

Introduction

The free radical gas nitric oxide (NO) regulates many aspects of muscle physiology, including blood flow, glucose uptake, mitochondrial respiration, and contractile function (1–3). NO production in muscle is primarily due to the activity of Ca²⁺-regulated NO synthases (NOS), especially NOS1 (nNOS), which generate NO via the O₂-dependent conversion of L-arginine to citrulline (1). However, NO can also be produced in muscle (and other tissues) via reduction of nitrate (NO₃⁻) and/or nitrite (NO₂⁻) to NO by endogenous nitroreductases, e.g., deoxyhemoglobin/deoxymyoglobin, xanthine oxidoreductase (4,5). Unlike NOS-mediated NO production, this alternative pathway is active at low pO₂ and is stimulated rather than inhibited by low pH, i.e., under the conditions that exist in intensely contracting muscle. Thus, although at rest approximately 95% of NO in the body is produced via the action of NOS (6,7), it has been hypothesized that the NO₃⁻/NO₂⁻ pool within muscle serves as an important source of NO during intense exercise (8).

Recognition of this non-canonical pathway for NO production has led to numerous studies over the last two decades of the effects of dietary NO₃⁻ supplementation, usually from beetroot juice (BRJ), on exercise capacity and/or associated physiological responses (cf. Ref. 9 for review). Although such research initially focused on aerobic exercise, more recent studies have demonstrated that dietary NO₃⁻ also has significant effects on the inherent contractile properties of muscle (cf. Ref. 10 for review). For example, we have found that acute NO₃⁻ supplementation significantly increases muscle speed and power in a variety of participant populations, i.e., healthy young adults (11,12), athletes (13), older individuals (14–16), and patients with heart failure (17). Others have reported that NO₃⁻ intake enhances the rate of torque development (RTD) during maximal voluntary isometric contractions (MVIC) (18–21) as well as peak twitch torque (PTT) and RTD during electrically evoked contractions (22,23). Muscle contractile function is a key determinant of both athletic performance and the ability to perform ordinary activities of daily living (24,25), making these findings of great practical and clinical significance. It should be noted, however, that not all studies have found improvements in muscle function in response to NO₃⁻ supplementation (e.g., Ref. 26), and that not all individuals seem to benefit (12).

Although it is now clear that NO₃⁻ ingestion often enhances muscle contractility, the precise mechanism(s) responsible is/are still uncertain. Hernández et. al. (27) originally reported that dietary NO₃⁻ improves the contractile function of fast-twitch, or type II, but not slow-twitch, or type I, mouse muscle by altering Ca²⁺ handling, via increased expression of calsequestrin and the dihydropyridine receptor. Whitfield et al. (23), however, found that NO₃⁻ supplementation does not alter the expression of these or other proteins of the sarcoplasmic reticulum in mixed human muscle (i.e., the quadriceps), even though like Hernández et. al. (27) they observed significant changes in the torque-frequency relationship during electrically stimulated contractions. At least in humans, changes in protein expression can also seemingly be ruled out by the fact that muscle function is increased by an acute dose of NO₃⁻, with no further improvement with repeated dosing (16,28) Thus, the effects of dietary NO₃⁻ on human muscle function presumably must be due to some other factor(s), e.g., post-translational modification of existing proteins. In fact, we have previously hypothesized that the effects of dietary NO₃⁻ may be due to phosphorylation of the regulatory light chain (RLC) of myosin (29), which enhances Ca²⁺ sensitivity by increasing the proportion of myosin heads available to bind with actin (1). This hypothesis was based on the fact that NOS activity has been shown to influence RLC phosphorylation in C2C12 myotubes (30) and that both dietary NO₃⁻ and RLC phosphorylation have been reported to increase PTT, RTD, Ca²⁺ sensitivity/torque during low frequency stimulation, and maximal muscle speed and power without altering maximal force (cf. Ref. 29 for review). Although Kumar et al. (31) have subsequently reported that dietary NO₃⁻ does not increase RLC phosphorylation in mouse diaphragm muscle, as acknowledged by these authors their method for assessing global changes in protein phosphorylation may not have been sensitive enough to detect any such changes. Regardless, if our original hypothesis is correct then NO₃⁻ supplementation should attenuate the extent of post-activation twitch potentiation, since the latter is also largely the result of RLC phosphorylation (32). To date, however, this hypothesis has not been tested, as previous neuromuscular electrical stimulation (NMES) studies of the effects of dietary NO₃⁻ in humans have measured twitch contractile properties only in the unpotentiated (22,28), potentiated (23; 33–36), or an unspecified/unclear (37) state, but not both.

In rodents, NOS-dependent NO signaling is more prominent in type II vs. type I muscle fibers (1). In particular, expression of NOS1 and increases in RLC phosphorylation during contractile activity are greatest in rodent type II fibers and minimal or absent in type I fibers (1). Based partially on such data, Jones et al. (38) have hypothesized that dietary NO₃⁻ specifically benefits physiological responses in type II fibers. Unlike in rodents, however, in humans both NOS1 activity and contraction-induced increases in RLC phosphorylation are comparable in type II and type I fibers (1). Furthermore, in a previous cross-sectional study of a group of highly heterogeneous individuals we found no relationship between maximal knee extensor speed or power (in vivo indicators of muscle fiber type distribution (39,40)) in the absence of NO₃⁻ intake and the magnitude of the increase following NO₃⁻ supplementation (12). Thus, whether dietary NO₃⁻ preferentially enhances the contractile function of human type II muscle fibers is still unclear.

Based on the above, the purpose of the present study was two-fold: 1) to determine, using NMES, whether there was an interaction between the effects of acute NO₃⁻ supplementation and post-activation potentiation on the contractile properties of human muscle, and 2) to determine whether there was a difference in the effects of such supplementation in predominantly type II (i.e., triceps brachii, 60–70% type II) versus predominantly type I (i.e., triceps surae, 60–70% type I) human muscles (41,42 – see Tables S1 and S2 of Supplemental Data for additional references). We hypothesized that dietary NO₃⁻ would increase PTT and/or RTD during unpotentiated twitches in both muscle groups, but that this effect would be diminished or lost in the potentiated state. We also hypothesized that NO₃⁻ ingestion would result in a leftward shift of the torque-frequency relationship of both muscles, commensurate with an increase in Ca²⁺ sensitivity due to myosin RLC phosphorylation.

Methods

Ethical approval:

The Human Subjects Office at Indiana University approved this study and written informed consent was obtained from each participant.

Participants:

Haider and Folland (22) have previously reported that dietary NO₃⁻ increases unpotentiated PTT, considered the primary outcome in this study, with an effect size of r = 0.53, equivalent to Cohen’s f = 0.625 (43). Based on G*Power 3.1.9.7 (44), a sample size of n = 12 would be required to detect this effect size with a power (1-β) of 0.95 at α = 0.05. We therefore studied 14 men with a mean (±SD) age, height, weight, and BMI of 26±7 y, 1.76±0.07 m, 78.1±19.3 kg, and 25.1±4.9 kg/m², respectively. Based on the short-form International Physical Activity Questionnaire (IPAQ), all were low to moderately active, expending 1197±785 MET·min/wk in physical activity. In particular, none engaged in endurance or resistance exercise on a regular basis. Only men were included due to difficulty reliably isolating the triceps brachii during NMES and because of recent data indicating that pre-menopausal women may not benefit from NO₃⁻ supplementation (18,45). Highly active individuals (i.e., those performing >3000 MET-min/wk of moderate physical activity or >1500 MET min/wk of vigorous physical activity) were excluded because exercise training may diminish the beneficial effects of NO₃⁻ supplementation on muscle contractile function (10). Other exclusion criteria were age <18 or >44 y; current use of antibiotics, phosphodiesterase inhibitors, tobacco, or any supplements intended to increase muscle mass or function; diagnosis of epilepsy or presence of pacemaker or other implantable cardiac device; resting blood pressure >140/90 mmHg; an answer of yes to any of the seven general health questions of the Physical Activity Readiness Questionnaire (PAR-Q); or the inability to provide informed consent.

Experimental design:

All experiments were performed in the temperature-controlled Exercise Physiology Laboratory at Indiana University Indianapolis. After completion of a screening visit, which included practicing the entire NMES protocol as described below, each participant was tested on two occasions using a double-blind, placebo-controlled, crossover design. During one visit, participants were studied following acute ingestion of 2.2 mL/kg body mass of a concentrated BRJ supplement (Beet It Sport, James White Drinks, Ipswich, UK) containing (based on direct measurement (46)) 200 μmol/kg of NO₃⁻. We have previously demonstrated that this dose is efficacious at increasing maximal knee extensor power, at least in older individuals, whereas a dose of 400 μmol/kg of NO₃⁻ is not (15). During the other visit, they were studied following ingestion of the same volume of NO₃⁻-free BRJ. The order of these visits was randomized, with a washout period of 12±7 (range 5–27) d in between. Participants were asked to avoid strenuous physical activity, alcohol, caffeine, chewing gum, and foods high in NO₃⁻ (e.g., spinach, arugula, beets) for 24 h and to not brush their teeth, use mouthwash, or eat or drink anything except water for 8 h before each study visit. All study visits for a given participant took place at the same time of day (±1 h) to control for any diurnal or circadian variation in muscle contractile function.

Experimental protocol:

Upon reporting to the laboratory, an intravenous catheter was placed and a baseline blood sample was obtained for subsequent analysis of plasma NO₃⁻ and NO₂⁻ concentrations (see below). The participant then ingested the BRJ supplement and rested quietly for 2 h, with blood sampling repeated at the 1 h and 2 h time points. During this time the skin over the triceps brachii and triceps surae was shaved, abraded, and cleaned with an alcohol wipe, after which two 5.08 cm x 8.89 cm self-adhesive electrodes (Dura-Stick Plus, Chattanooga Medical Supply, Chattanooga, TN) were applied to the specific muscles of interest. For the triceps brachii, the cathode was placed diagonally across the proximal posterolateral portion of the long and lateral heads, whereas the anode was placed transversely ~5 cm proximal to the olecranon process. For the triceps surae, the cathode was placed transversely over the gastrocnemius muscle ~5 cm distal to the popliteal fossa, whereas the anode was placed over the soleus muscle ~10 cm proximal to the calcaneus. For each participant, the exact positioning of the electrodes was adjusted as necessary during the screening visit to avoid co-contraction of other muscles, then photographed to permit replication of their location on subsequent visits.

After the 2 h blood sample was obtained, an isokinetic dynamometer (Biodex System 4 Pro, Biodex Medical Systems, Shirley, NY) interfaced with a computer via a 16-channel data acquisition system (Biopac MP160, Biopac, Goleta, CA) operating at 1000 Hz was used to assess the contractile properties of the triceps brachii and triceps surae. The order of testing of the two muscle groups was randomized between participants but kept constant within an individual to assure comparable timing across study visits. Participants were positioned on the dynamometer with 1) a chair back angle of 1.48 rad (85°) and with the elbow of the dominant arm at a joint angle of 1.57 rad (90°), with the shoulder adducted and at an elevation of 0 rad (0°), or 2) a chair back angle of 0.96 rad (55°), with the knee and ankle joints of the ipsilateral leg of the dominant arm at an angle of 0 rad (0°). After adjustment of the dynamometer and its attachments, straps were placed over the participant’s waist, torso, and forearm or thigh to restrict extraneous movement. During testing of the triceps brachii, a plastic and fabric brace (Roylan Ulnar Deviation Splint, Performance Health, Cedarburg, WI) and an elastic wrap were also used to prevent any movement of the wrist or fingers. During testing of the triceps surae, straps were placed across the instep and metatarsals to prevent any movement of the foot with respect to the footplate. The positioning of each participant during their screening visit was recorded and carefully reproduced during the subsequent experimental visits, as were the currents used in each muscle group during both phases of testing (see below).

Once the participant was positioned, a constant current stimulator (Digitimer DS-7AH, Hertfordshire, UK) was used to elicit isometric contractions of triceps brachii or surae. First, single stimuli (400 V, 200 μs) of increasing current were applied at 3–5 s intervals until a plateau in twitch torque was observed. After a brief rest, the amperage resulting in the highest torque was then used to elicit four unpotentiated twitches at approximately 1 s intervals. The participant then performed a 6 s MVIC, immediately after which four potentiated twitches were elicited, also at approximately 1 s intervals. Strong verbal encouragement was provided during the MVIC, during which participants were instructed to “chop downward” with their forearm or “floor the gas pedal” with their foot. This sequence was repeated twice, with at least 10 min in between to allow for reversal of the potentiation (verified via application of a test twitch) (45,47). After a further 10 min of rest, participants were stimulated at 100 Hz with 1 s trains of increasing current to determine the amperage eliciting approximately 33% of their maximal voluntary isometric torque. After a brief rest, this amperage was then applied at 3–5 s intervals as a single stimulus and in 1 s trains at (in order) 5, 10, 15, 20, 25, 30, 35, 40, 60, 80, and 100 Hz to determine the torque-frequency relationship. The participant was then repositioned on the dynamometer and the other muscle group tested using the same procedures. A final blood sample was then drawn and the participant was released.

Measurement of plasma NO₃⁻ and NO₂⁻ concentrations:

Blood samples were immediately centrifuged at 4° C for 10 min at 2000 g to obtain plasma, which was transferred to a microfuge tube and frozen at −20° C until subsequent analysis. Upon thawing, plasma was deproteinized via addition of an equal volume of methanol, centrifuged at 4° C for 10 min at 10,000 g, and an aliquot of the protein-poor supernatant used to determine NO₃⁻ and NO₂⁻ concentrations via HPLC as previously described (12, 14–17).

Data analysis:

Torque data were analyzed using Biopac AcqKnowledge version 5.08. The signal was first smoothed using a 10 ms rolling average filter, after which PTT, time to peak torque (TPT), and one-half relaxation time (HRT) were determined for each twitch. The maximal RTD and rate of relaxation (RR) were also calculated based on the 1^st derivative of the smoothed torque over a 20 ms time interval. The averages of these values for the eight twitches under each condition (i.e., unpotentiated and potentiated) were then used in subsequent calculations. The torque-frequency data were analyzed by smoothing the data as above, extracting the peak torque from the single twitch and each of the 1 s trains at 5–100 Hz, and expressing the data relative to the peak torque measured at 100 Hz.

Statistical analysis:

Statistical analyses were performed using GraphPad Prism version 10.6.1 (GraphPad Software, La Jolla, CA). Normality of data distribution was assessed using the D'Agostino and Pearson omnibus test. Two-way (i.e., time x treatment) analysis of variance (ANOVA) was used to determine the effects of NO₃⁻ supplementation on plasma NO₃⁻ and NO₂⁻ concentrations. Two-way (i.e., condition x treatment) ANOVA was also used to determine the effects of dietary NO₃⁻ on twitch contractile properties within each muscle group. The torque-frequency results from each muscle group were analyzed by fitting the data to a four-parameter sigmoidal function (45,49–51):

$$\begin{gathered} Torque\left(\mathrm{\%}of100Hz\right) \\ =minimumtorque\left(\mathrm{\%}of100Hz\right) \\ +\frac{maximumtorque\left(\mathrm{\%}of100Hz\right)-minimumtorque\left(\mathrm{\%}of100Hz\right)}{{\left(\frac{F_{50}\left(Hz\right)}{frequency\left(Hz\right)}\right)}^{Hilln}+1} \end{gathered}$$

where F₅₀ is the midpoint of this relationship and Hill n is the steepness at this point. Within each muscle group, an extra-sum-of-squares F test was used to compare the effects of fitting data from the NO₃⁻ and placebo conditions separately to a global fit using data from both treatments. The above equation was also fit to the data from each trial in each participant, and individual changes due to treatment within each muscle compared using paired t tests. All data are reported as mean ± standard deviation (SD). Two-tailed P values <0.05 were considered statistically significant.

Results

Plasma nitrate and nitrite concentrations:

No significant changes in either plasma NO₃⁻ or NO₂⁻ were observed after ingestion of the NO₃⁻-free placebo BRJ (Figure 1). However, plasma NO₃⁻ was increased >15 fold at all time points following ingestion of NO₃⁻-containing BRJ. This increase in plasma NO₃⁻ was accompanied by a much smaller (i.e., ~45%), but still statistically significant, increase in plasma NO₂⁻ at 2 h and ~3.5 h post-ingestion. The intervention was therefore successful in elevating the availability of these NO precursors in the circulation.

Figure 1.

Effect of acute dietary NO₃⁻ supplementation on plasma NO₃⁻ (top panel) and NO₂⁻ concentrations (bottom panel).

Twitch contractile properties:

Twitch contractile properties of the triceps brachii are shown in Table 1, whereas those of the triceps surae are shown in Table 2. Both muscle groups exhibited distinct post-activation potentiation, with PTT, RTD, and RR all being significantly greater, and TPT and HRT being significantly shorter, immediately after a 6 s MVIC, except for the relative RR and HRT in the triceps surae. However, dietary NO₃⁻ did not alter the twitch contractile properties of either muscle group in either the unpotentiated or potentiated states, with all treatment and interaction effects being non-significant.

Table 1.

Effect of acute dietary NO₃⁻ supplementation on the twitch contractile properties of the triceps brachii.

		Condition		P value
Property	Treatment	Unpotentiated	Potentiated	Condition	Treatment	Interaction
PTT (Nm)	Placebo	3.4 ± 1.4	8.0 ± 2.6	<0.001	0.993	0.575
	Nitrate	3.5 ± 1.7	7.9 ± 2.3
RTD (Nm/s)	Placebo	74.6 ± 37.5	220.4 ± 83.4	<0.001	0.979	0.572
	Nitrate	77.7 ± 45.5	216.8 ± 81.5
RTD (%/s)	Placebo	2120 ± 213	2717 ± 368	<0.001	0.725	0.659
	Nitrate	2155 ± 253	2715 ± 378
TPT (ms)	Placebo	83 ± 6	78 ± 7	0.018	0.467	0.678
	Nitrate	82 ± 7	77 ± 11
RR (Nm/S)	Placebo	−37.0 ± 15.3	−96.6 ± 39.4	<0.001	0.416	0.220
	Nitrate	−38.1 ± 23.5	−89.3 ± 27.8
RR (%/s)	Placebo	−1082 ± 158	−1202 ± 216	<0.001	0.151	0.695
	Nitrate	−1048 ± 190	−1155 ± 167
HRT (ms)	Placebo	64 ± 8	59 ± 9	0.043	0.732	0.729
	Nitrate	65 ± 11	60 ± 9

Values are mean ± SD for n=14. PTT, peak twitch torque. RTD, rate of torque development. TPT, time to peak torque. RR, rate of relaxation. HRT, half-relaxation time.

Table 2.

Effect of acute dietary NO₃⁻ supplementation on the twitch contractile properties of the triceps surae.

		Condition		P value
Property	Treatment	Unpotentiated	Potentiated	Condition	Treatment	Interaction
PTT (Nm)	Placebo	15.6 ± 5.4	19.0 ± 6.4	<0.001	0.996	0.843
	Nitrate	15.6 ± 6.2	19.0 ± 6.9
RTD (Nm/s)	Placebo	221.3 ±71.8	284.5 ± 89.1	<0.001	0.461	0.450
	Nitrate	223.0 ± 82.6	292.3 ± 97.7
RTD (%/s)	Placebo	1446 ± 155	1521 ± 172	<0.001	0.156	0.136
	Nitrate	1458 ± 176	1576 ± 238
TPT (ms)	Placebo	129 ± 14	123 ± 13	<0.001	0.423	0.211
	Nitrate	129 ± 15	120 ± 13
RR (Nm/S)	Placebo	−130.7 ± 50.4	159.1 ± 57.2	<0.001	0.306	0.616
	Nitrate	−128.7 ± 53.0	−161.9 ± 71.9
RR (%/s)	Placebo	−838 ± 108	−838 ± 115	0.625	0.908	0.566
	Nitrate	−830 ± 104	−852 ± 227
HRT (ms)	Placebo	95 ± 15	95 ± 15	0.463	0.947	0.386
	Nitrate	96 ± 15	94 ± 22

Values are means ± SD for n=14. PTT, peak twitch torque. RTD, rate of torque development. TPT, time to peak torque. RR, rate of relaxation. HRT, half-relaxation time.

Torque-frequency relationship:

In contrast to the lack of effect of NO₃⁻ supplementation on twitch contractile properties, acute NO₃⁻ ingestion significantly altered the torque-frequency relationship of both muscle groups. Specifically, NO₃⁻ intake resulted in a leftward shift of this relationship in the triceps brachii (Fig. 2, top panel), but a rightward shift in the triceps surae (Fig. 2, bottom panel).

Figure 2.

Effect of acute dietary NO₃⁻ supplementation on the torque-frequency relationship of the triceps brachii (top panel) and triceps surae (bottom panel) muscle groups. Thicker black solid and red dashed lines are the best-fit curves obtained via non-linear regression for the placebo and nitrate trials, respectively. Thinner solid black and red lines illustrate the corresponding 90% confidence intervals. Curves were compared using an extra-sum-of-squares F test, with the resultant P values shown. NO₃⁻ intake resulted in a significant leftward shift of the torque-frequency relationship in the triceps brachii, but a significant rightward shift in the triceps surae.

On an individual participant basis, dietary NO₃⁻ did not alter the fitted minimum torque, fitted maximum torque, or Hill n in either muscle group, but F₅₀ tended to be lower in the triceps brachii and was significantly higher in the triceps surae (see Table S3 of Supplemental Data). The change in F₅₀ due to NO₃⁻ intake was therefore also significantly different between the two muscle groups (see Fig. S1 of Supplemental Data).

Discussion

The purpose of the present study was to determine the effects of dietary NO₃⁻ on the contractile properties of the predominantly type II triceps brachii and predominantly type I triceps surae muscles of humans. We hypothesized that NO₃⁻ ingestion would enhance PTT and/or RTD in both muscles in the unpotentiated state, but that this effect would be reduced or absent in the potentiated state. We also hypothesized that NO₃⁻ supplementation would increase torque during low but not high frequency stimulation in both muscles. Contrary to our first hypothesis, however, we found no changes in the twitch characteristics of either muscle under either condition. Furthermore, although acute NO₃⁻ intake did result in significant changes in the torque-frequency relationship in both the triceps brachii and triceps surae, these changes were in opposite directions. This is the first study to specifically examine the possible interaction between dietary NO₃⁻ supplementation and post-activation potentiation, as well as the first to determine the effects of NO₃⁻ ingestion on the function of human muscles differing in their speed of contraction.

Numerous studies have now demonstrated that dietary NO₃⁻ can enhance muscle contractility in humans (cf. Ref. 10 for review), which we have previously hypothesized may be due to NO-stimulated phosphorylation of the myosin RLC (28). This hypothesis was based in part on the results of initial studies reporting increases in PTT and RTD following NO₃⁻ supplementation (22,23). Subsequent studies, however, have failed to replicate these findings (28,33–36). This was also true in the present investigation, as acute NO₃⁻ intake did not alter the twitch contractile properties of either the triceps brachii or the triceps surae in either the unpotentiated or potentiated states. These results therefore extend prior findings in a mixed muscle, i.e., the quadriceps (33–35,37) or just the predominantly type I triceps surae (28,36) to the predominantly type II triceps brachii. More importantly, however, our data apparently exclude increases in myosin RLC phosphorylation as a mechanism contributing to dietary NO₃⁻-induced improvements in muscle function. This is because there was no interaction between the effects of NO₃⁻ supplementation and post-activation potentiation, the latter of which is largely (albeit not entirely) due to myosin RLC phosphorylation (32). This conclusion is supported by the results of Kumar et al. (31), who as previously mentioned found that NO₃⁻ supplementation enhanced the maximal shortening velocity and peak power of the diaphragm muscle of aged mice but did so without apparently altering phosphorylation of the myosin RLC (or other proteins).

Although dietary NO₃⁻ had no effect on the twitch contractile properties of either the triceps brachii or triceps surae, it did alter the torque-frequency relationship in both muscle groups, shifting it leftward in the triceps brachii but rightward in the triceps surae. The former is in keeping with the results of some (22,23) but not all (37,51), previous studies that have used NMES to determine the effects of NO₃⁻ ingestion on the torque-frequency relationship of the quadriceps. On the other hand, this is the first study to find that dietary NO₃⁻ actually reduces torque during low frequency stimulation of the triceps surae, or for that matter, any muscle. In contrast, Wickham et al. (28) reported that high-dose NO₃⁻ supplementation (i.e., ~400 μmol/kg) for 1 or 8 d significantly increased torque during 10 Hz stimulation of this muscle in recreationally active young women on oral contraceptives. More recently, Rasica et al. (36) found that a lower dose of NO₃⁻ (i.e., ~150 μmol/kg) for 7 d had no influence on the torque-frequency relationship of the tricep surae in young men but increased torque during stimulation at 20–30 Hz in older men. However, it is difficult to compare these data to the present results due to the differences in NO₃⁻ dosing regimen and participant characteristics. Furthermore, the study of Wickham et al. (28) did not include a placebo trial. Regardless, the present findings are in keeping with the long-standing realization that NO can have both positive and negative effects on muscle contractile function (1,29,53). Indeed, as mentioned previously we have found that although acute ingestion of 200 μmol/kg of NO₃⁻ increases maximal knee extensor speed and power in healthy older men and women, these beneficial effects are diminished or even reversed at a dose of 400 μmol/kg (15). It is therefore clear that dietary NO₃⁻ supplementation can have divergent effects on muscle contractility in humans, which may vary as a function of dose (15,21), sex (18,45), age (36), potentially fiber type (present results), and possibly other factors as well.

The mechanism responsible for the above-described changes in the torque-frequency relationship are not clear. As with a twitch, however, the force produced during repetitive stimulation of a muscle depends upon numerous factors. These include the rate and magnitude of Ca²⁺ release and reuptake, the sensitivity to Ca²⁺, the amount of force generated per crossbridge, and the rates of crossbridge attachment and detachment, which influence the RTD and RR and hence the degree of summation. Thus, the observed shifts in the torque-frequency relationship could theoretically be due to a change in any (or several) of these factors. However, there were no changes in PTT or in tetanic torque (i.e., torque at 100 Hz), which would seem to rule out changes in Ca²⁺ release or force per crossbridge as possible explanations. Likewise, there were also no changes in RTD or RR during twitch contractions, which seemingly eliminates changes in the rates of crossbridge cycling or Ca²⁺ reuptake as mechanisms. It therefore appears that NO₃⁻ supplementation altered muscle contractility by changing Ca²⁺ sensitivity, enhancing it in the fast triceps brachii but reducing it in the slow triceps surae. As previously discussed (3), this could potentially be due to alterations in the phosphorylation and/or nitrosylation of one or more sarcomeric proteins other than the myosin RLC, e.g., myosin-binding protein C (54,55), titin (56). Future studies using proteomics to quantify dietary NO₃⁻-induced post-translational modification of these and other proteins will be required to test this hypothesis.

Regardless of the exact biochemical mechanism, it is interesting that in the triceps brachii this dietary NO₃⁻-induced shift in the torque-frequency relationship was greatest at 30–35 Hz, whereas in the triceps surae the difference in torque was greatest at 20–25 Hz. These values are comparable to the motor unit firing frequencies observed during intense voluntary contractions of these muscles in humans (57). This suggests that the divergent response of these two muscles to NO₃⁻ supplementation may reflect underlying differences in the role of NO in regulating the contractile function of human type II and type I muscle fibers. Specifically, it is tempting to speculate that by differentially altering Ca²⁺ sensitivity in the two fiber types, NO may increase whole-muscle maximal muscle speed and hence power in part by enhancing the contractility of faster fibers while limiting the “braking action” exerted by slower fibers. This is consistent with Maréchal and Gailly’s (53) suggestion that NO acts as a molecular “gear shift” within muscle, acutely altering contractile properties in a manner similar to that observed due to longer-term alterations in type II vs. type I myosin expression. This hypothesis could explain why dietary NO₃⁻ supplementation increases Vmax (11,12,14–17) despite no changes in RTD during electrically evoked twitch (28,34,36,37; Tables 1 and 2) or tetanic (52) contractions.

There are both strengths and weaknesses to the current investigation. The former include the robust assessment of muscle contractile properties via NMES and the minimization of intraindividual variability by adjusting the dose of NO₃⁻ relative to body mass. The most obvious limitation is that we did not verify the fiber type distribution of the triceps brachii and triceps surae muscles of our participants. However, it is well-established that these muscle groups are composed of predominantly type II and type I fibers, respectively, with essentially no overlap between them (41,42 – see Supplemental Data for additional references). This conclusion is reinforced by the observed differences in their in vivo contractile properties (Tables 1 and 2), which are highly correlated with differences in their myosin expression (42,58–60). Nonetheless, neither the triceps brachii nor the triceps surae are composed of purely type II or type I fibers, meaning that our results may in fact underestimate the effects of fiber type on the response to dietary NO₃⁻. It is also important to recognize that we only studied the effects of acute NO₃⁻ ingestion during electrically evoked contractions, which, while providing mechanistic insight, are not highly predictive of voluntary muscle function (51). In particular, it remains to be demonstrated that NO₃⁻ supplementation differentially alters the contractile properties of the triceps brachii and/or surae (or other muscles differing significantly in fiber type) during central nervous system-driven dynamic movements.

In summary, we have used NMES to determine the effects of acute ingestion of NO₃⁻-rich BRJ on the contractile properties of two human muscles differing markedly in fiber type distribution. No changes were observed in PTT, RTD, or RR in either the unpotentiated or potentiated state, seemingly ruling out changes in RLC phosphorylation as a mechanism by which NO₃⁻ supplementation alters muscle contractile function. NO₃⁻ supplementation did, however, result in changes in the torque-frequency relationship in both muscles, commensurate with an increase in Ca²⁺ sensitivity in the predominantly type II triceps brachii but a decrease in Ca²⁺ sensitivity in the predominantly type I triceps surae. These opposing effects likely contribute to heterogeneous findings in the literature regarding the effects of dietary NO₃⁻ on muscle contractile function. Additional research will be required to determine the underlying biochemical mechanisms.

Supplementary Material

Supplemental Digital Content

Supplemental Digital Content 1.docx