Fiber Type-Specific Effects of Dietary Nitrate

Abstract

Dietary nitrate supplementation increases circulating nitrite concentration, and the subsequent reduction of nitrite to nitric oxide is promoted in hypoxic environments. Given that PO₂ is lower in Type II compared with Type I muscle, this article examines the hypothesis that the ergogenicity of nitrate supplementation is linked to specific effects on vascular, metabolic, and contractile function in Type II muscle.

In Brief

The ergogenic effects of dietary nitrate supplementation may be related to specific physiological effects on Type II muscle fibers.

Key Points

• Nitric oxide (NO) is involved in an array of physiological processes that regulate skeletal muscle function. Dietary nitrate supplementation increases plasma nitrite concentration and therefore NO bioavailability. Nitrate supplementation may enhance exercise economy and performance, although its efficacy appears to be related both to the endurance training status of the participants and to the type of exercise being performed.
• We present evidence that nitrate supplementation may be particularly effective at augmenting physiological responses in type II (fast-twitch) muscle. The relatively low O2 tension surrounding type II fibers may promote the reduction of nitrite to NO, thereby improving: local perfusion, fatigue resistance, fiber contractility and, ultimately, whole body exercise performance.
• This targeted effect of nitrate supplementation on type II muscle may have important implications for improving performance during high-intensity (intermittent) exercise in healthy adults and for helping to restore functional capacity in senescent and patient populations where increased reliance on type II muscle fibers contributes to compromised exercise tolerance.

INTRODUCTION

Nitric oxide (NO) is essential to life. It is a gaseous signaling molecule that is produced continuously to enable or facilitate a wide range of physiological processes including neurotransmission, vasodilatation, angiogenesis, cellular respiration, mitochondrial biogenesis, and muscular contraction. A failure to produce or use sufficient NO occurs in senescence and is a characteristic of many cardiovascular and metabolic diseases. The production of NO often is attributed exclusively to the canonical pathway in which the semi-essential amino acid l-arginine is oxidized in a reaction catalyzed by the NO synthase (NOS) family of enzymes. The products of this reaction, nitrite (NO₂⁻) and nitrate (NO₃⁻), were thought originally to have no physiological relevance. However, it is now appreciated that, under certain physiological conditions (e.g., in low-O₂ environments that may exist in some tissues such as contracting skeletal muscle), NO₂⁻ may undergo a one-electron reduction to re-form NO.

Dietary supplementation with the NOS pathway substrate, l-arginine, seems to have a very limited effect on NO-related physiological processes (³⁷). In contrast, consuming foodstuffs such as green leafy vegetables (spinach, lettuce, rocket/arugula) and beetroot, which are rich in inorganic NO₃⁻, substantially increases the circulating plasma [NO₃⁻] and [NO₂⁻], thereby increasing the potential for O₂-independent NO synthesis (²⁴). A crucial step in this NO₃⁻-NO₂⁻-NO pathway is the conversion of NO₃⁻ to NO₂⁻ by facultative bacteria residing in the mouth. It has been shown repeatedly that dietary NO₃⁻ intake results in a significant reduction of resting blood pressure for several hours, consistent with an NO-mediated peripheral vasodilatation. Other physiological effects of NO₃⁻ ingestion also have been reported, including a reduction in the O₂ cost of submaximal exercise, at least in nonelite athletes ((^2,4,22,23) cf. (¹⁹)). This effect, which may arise as a consequence of a reduced adenosine triphosphate (ATP) cost of muscle force production (²) or to enhanced mitochondrial efficiency (²²), is surprising given that this variable has been considered to be essentially unaltered by factors such as subject health and age, as well as aerobic fitness and training status, at least for cycle exercise. Improved muscle efficiency and/or exercise economy would be anticipated to result in enhanced exercise performance, and this has led to many recreational and elite athletes consuming NO₃⁻-rich products, usually beetroot juice, precompetition and/or to support training (¹⁸). However, although NO₃⁻ supplementation has been reported to be performance enhancing in multiple studies involving recreationally active or moderately trained subjects (e.g., (^2,4,41,42)), it seems that elite endurance athletes do not benefit to the same extent (e.g.,^{6,9, see 18} for review).

Although there are several factors that might explain the difference in responsiveness to NO₃⁻ supplementation between elite and nonelite subjects, including dose and duration of NO₃⁻ supplementation as well as genetic or training effects on NOS activity, muscle oxygenation, and mitochondrial function, likely differences in muscle fiber type composition between these populations also might be important (¹⁸). Although there is considerable heterogeneity both between and within muscle fiber types, compared with Type I (slow-twitch) fibers, Type II (fast-twitch) fibers differ in terms of myofibrillar protein content and calcium handling, typically have lower mitochondrial and capillary density, and, therefore, rely relatively more on nonoxidative compared with oxidative pathways of ATP resynthesis (⁷). The lower O₂ availability in Type II muscle, reflected in O₂ tension (PO₂), in addition to possible differences in contractile function and efficiency characteristics, results in greater fatigability compared with Type I muscle. The predominant muscle fiber type in a given muscle reflects its function. For example, across muscles in the human leg, the soleus may be approximately 88% Type I fibers, whereas the rectus femoris may be only approximately 35% Type I fibers (¹⁷). Moreover, in elite athletes, endurance runners may have approximately 70% Type I fibers whereas sprinters may have approximately 40% Type I fibers in the quadriceps femoris (³³).

In this article, we explore the hypothesis (schematized in Fig. 1) that the efficacy of dietary NO₃⁻ supplementation may be linked to specific physiological effects on Type II muscle fibers.

Figure 1: Proposed mechanisms by which dietary NO₃ ⁻ supplementation improves exercise performance. 1. Fast-twitch Type II muscles have a substantially lower microvascular PO₂ (PO₂ mv) at rest and during contractions than their slow-twitch Type I counterparts (⁵). 2. This likely facilitates the reduction of NO₂ ⁻ to NO preferentially within fast-twitch Type II muscles, thus, increasing Q˙O₂ in these tissues (¹²). 3. Preferential increases in Q˙O₂ result in a greater Q˙O₂/V˙O₂ ratio in fast-twitch but not slow-twitch muscles (¹³). 4. This ultimately results in tighter metabolic control and thus a reduced accumulation of fatigue-associated metabolites such as blood lactate (¹²).

PHYSIOLOGICAL HYPOXIA AND THE EFFICACY OF NITRATE SUPPLEMENTATION

Because our hypothesis that Type II muscle fibers may be especially targeted by NO₃⁻ supplementation rests, at least in part, on differences in intramyocyte PO₂ between fiber types, we first consider the role of systemic and local O₂ availability on NO production and the efficacy of NO₃⁻ supplementation on physiological function in hypoxia.

Nitric oxide bioavailability and bioactivity are dictated by an intricate balance between NO and O₂. Dependent on the metabolic rate and the matching of tissue O₂ delivery (Q˙O₂), PO₂ fluctuates from approximately 40 mm Hg (5% O₂) at rest to as low as approximately 2 mm Hg (∼0.4% O₂) during strenuous exertion (³²). Nitric oxide production via NOS enzymes requires the presence of O₂. Given the role of NO as a master regulator of blood flow and cellular respiration, it is intuitive that a backup system is needed to guarantee NO bioavailability under the low-PO₂ conditions, which are native to skeletal muscle. Indeed, NO can be produced alternatively via the NOS-independent, single-electron reduction of NO₂⁻, which is obtained through dietary NO₂⁻ and NO₃⁻ as well as via oxidation of endogenous NO (²⁴). Importantly for maintenance of NO bioavailability, NO₂⁻ reduction to NO is stimulated by low PO₂ and low pH (²⁴). Therefore, the therapeutic potential of the NO₃⁻ reduction pathway to compensate for dysfunctional NOS is substantial.

There is convincing evidence from in vitro experiments that physiologically critical NO-mediated signaling pathways that regulate skeletal muscle Q˙O₂ and cellular bioenergetics are O₂ dependent (³⁵). Low physiological PO₂ in active muscle stimulates erythrocyte-derived NO-mediated vascular relaxation, which serves to match Q˙O₂ to local metabolic rate, whereas high PO₂ triggers vasoconstriction. NO regulates myocyte calcium flux via S-nitrosylation of thiols contained in ryanodine receptor (RyR1) Ca²⁺ channels. These are activated only only by NO at low physiological PO₂ (∼10 mm Hg), such that low PO₂ in active muscle increases contractility, and high physiological PO₂ has the opposite effect. NO also may regulate mitochondrial respiration by reversible inhibition of cytochrome c oxidase in competition with O₂, such that greater inhibition by NO would occur at low physiological PO₂. It has been proposed that, collectively, the actions of NO₂⁻ and/or NO on O₂ consumption and vasodilatation might enable a more precise local matching of blood flow to metabolic rate (³⁵). It is important to note here that although it is likely that the effects of dietary NO₃⁻ largely are mediated by NO, it is possible that NO₂⁻ and/or other bioactive NO₃⁻-derived molecules also play a role in the physiological effects observed after NO₃⁻ supplementation (¹).

Restricted Q˙O₂ to skeletal muscle reduces maximal oxidative metabolic rate (Q_max), which is reflected in a slowing of muscle phosphocreatine (PCr) recovery kinetics after exercise. For the same metabolic rate, there is a greater muscle metabolic perturbation (e.g., greater fall in muscle PCr concentration during exercise performed in hypoxia compared with normoxia). In vivo evidence suggests that the O₂ demand of exercising muscle may be reduced during normoxic exercise after dietary NO₃⁻ supplementation ((^2,4,22,23,36) cf. (¹⁹)), which might be consequent to an increased phosphate/oxygen ratio of mitochondrial respiration (²²) and/or a reduced ATP cost of force production (²). Given that NO (and/or NO₂⁻) has the potential to improve Q˙O₂ as well as reduce the O₂ demand for a fixed work rate, dietary NO₃⁻ supplementation might be particularly important for improving skeletal muscle functional capacity under conditions where O₂ availability is low, irrespective of whether this is considered in terms of environmental, systemic, or local tissue O₂ availability.

The influence of dietary NO₃⁻ on exercise tolerance in healthy humans under hypoxic conditions has been investigated using manipulation of the inspired O₂ fraction. It was first shown that 24 h of NO₃⁻ supplementation (9.3 mmol·NO₃⁻) reduced the extent of muscle metabolic perturbation during high-intensity knee-extension exercise and enhanced exercise tolerance in moderate hypoxia (14.5% O₂) ((³⁸) Fig. 2). Dietary NO₃⁻ supplementation also abolishes the reduction in the rate of pH-independent PCr recovery, which typically is observed in hypoxia, and essentially restores muscle Q_max to values similar to those observed in normoxia ((^38,39) Fig. 2). The speeding of Cr recovery was linked to improved muscle oxygenation during the immediate postexercise period in hypoxia but not in normoxia (assessed using effective transverse relaxation time weighted magnetic resonance signal) in healthy young adults (³⁹). These data, therefore, suggest that, in hypoxia, dietary NO₃⁻ supplementation may facilitate NO production and enable greater muscle oxygenation, offsetting the deleterious effects of hypoxia on muscle function.

Figure 2: Time to exhaustion during high-intensity knee-extension exercise (A) and the muscle (PCr) recovery time constant (τ, B) in normoxia (N), in hypoxia with a placebo supplement (H-PL), and in hypoxia with a dietary nitrate supplement (H-BR). *Different from N and H-BR, P < 0.05. Error bars indicate SE. The figure has been drawn based on data reported in Vanhatalo et al. (³⁸).

During whole body exercise in hypoxia, NO₃⁻ supplementation has been shown to reduce the O₂ cost of submaximal exercise by approximately 4% to 7% (^19,25,27). Moreover, muscle oxygenation, defined as the total oxygenation index derived from near-infrared spectroscopy (NIRS), was elevated by 4% to 5% at rest and during both submaximal and maximal-intensity exercise after NO₃⁻ supplementation in hypoxia (11% O₂) (²⁵). This may reflect the lower V˙O₂ observed (²⁵) or be indicative of NO-mediated enhanced bulk Q˙O₂ and/or microvascular O₂ distribution during hypoxic exercise. These same factors may underpin the faster pulmonary V˙O₂ kinetics after NO₃⁻ supplementation reported during exercise in hypoxia but not normoxia (¹⁹). Dietary NO₃⁻ ingestion also attenuated the hypoxia-induced reduction in exercise tolerance during both severe-intensity constant work rate exercise (by 9% (¹⁹)) and exhaustive incremental exercise (by 5% (²⁵)). It seems that, for the same NO₃⁻ dose, NO₃⁻ supplementation is more effective during whole body exercise in hypoxia than in normoxia, with the improvement in high-intensity exercise tolerance being related to the reduced O₂ cost of submaximal exercise (¹⁹) perhaps because this would permit a better preservation of arterial O₂ saturation. Importantly, the beneficial effects by NO₃⁻ ingestion on exercise tolerance in hypoxia may translate to improved exercise performance; in one study, a 16.1-km cycling time trial performance was reported to be improved by approximately 2% by NO₃⁻ supplementation compared with placebo under 15% inspired O₂ (²⁷).

The available evidence indicates that elevating NO bioavailability through a simple dietary intervention may particularly be beneficial in hypoxic conditions such as occurs during exposure to high altitude and in conditions where Q˙O₂ is impaired, including various cardiovascular, pulmonary, and sleep disorders. Although the potentially greater physiological effects of NO₃⁻ supplementation pertain to when an individual exercises at altitude or breathes a gas containing a low fraction of O₂ at sea level (¹⁹), it is important to note that the ratio between local muscle blood flow and metabolic rate is heterogeneous even within skeletal muscle, and that Type II muscles and muscle fibers operate at a lower PO₂ than do Type I muscles and muscle fibers (^5,26). It, therefore, is conceivable that the physiological effects of NO₂⁻ are more manifest in Type II than Type I muscle. In the next section, we consider the potential influence of local hypoxia on NO bioavailability and the efficacy of NO₃⁻ supplementation.

MUSCLE FIBER TYPE SELECTIVITY OF NITRATE SUPPLEMENTATION: ANIMAL STUDIES

Skeletal muscles are highly heterogeneous in terms of their structure and function. For instance, muscle activation patterns, fatigability, fiber type composition, oxidative/glycolytic enzyme activities, vascularity, and vascular control broadly range among and within muscles (²⁰). In humans, bipedalism has facilitated the additional flexibility of preferentially activating muscle groups in either the arms or legs and distributing the available (and potentially limited) cardiac output such that prodigious blood flows can be achieved when smaller muscle masses are activated. Through experimental evidence procured in humans, and especially animals, for the past few decades, our appreciation of heterogeneity with respect to blood flow among and within muscles has undergone a radical revision.

It is now recognized that both spatial and temporal heterogeneity of blood flow is of fundamental importance in the matching of increases in Q˙O₂ to local metabolic requirements (V˙O₂) (²⁰). The ability to drive O₂ from the capillary into the myocyte and, thus, to the mitochondria is dependent on the driving pressure (i.e., microvascular PO₂, PmvO₂) and the resultant pressure gradient. Because PmvO₂ principally is determined by the Q˙O₂/V˙O₂ ratio and because PmvO₂ determines blood-myocyte O₂ flux and also influences metabolic control via intracellular PO₂, it is intuitive that, unless Q˙O₂/V˙O₂ is regulated extremely tightly both spatially and temporally, V˙O₂ kinetics may be slowed and muscle function will be suboptimal, thereby reducing exercise tolerance (³⁰). By the same token, if the Q˙O₂/V˙O₂ ratio, and thus PmvO₂, can be increased at any given work rate, exercise performance may be enhanced.

Dietary Nitrate Supplementation, Type II Muscle, and Fatigue

Exercise tolerance is dictated by a plethora of physiological and physiochemical interactions that ultimately determine the fatigability of skeletal muscle. Of the potential mechanisms of fatigue, the recruitment of higher-order motor units that elicit contraction of fast-twitch Type II muscles is especially pertinent herein. Muscles predominantly composed of Type II fibers regulate their PmvO₂ very differently from their slow-twitch (Type I) counterparts. Specifically, significantly lower blood flow (relative to V˙O₂) results in a much lower contracting PmvO₂ (i.e., ∼10 vs 20 mm Hg; Fig. 1), ultimately forcing greater reliance on fractional O₂ extraction in Type II compared with Type I muscle (^5,26). The heterogeneous fiber type distribution evident in the rat and the ability to measure skeletal muscle vascular function across discrete muscle fiber types provide a powerful research tool to examine the impact of dietary NO₃⁻ on muscles of varying fiber type. It is important to note that human skeletal muscle also exhibits appreciable heterogeneity in fiber type (^17,33), and that murine models may provide important insight into fiber-specific vascular control (²⁰).

Given the propensity for a very low PO₂ environment and possibly greater acidity to reduce NO₂⁻ to NO, Ferguson et al. (¹²) tested the hypothesis that dietary NO₃⁻ preferentially would increase blood flow and thus PmvO₂ in exercising muscles composed of Type II fibers. In rats running at approximately 70% V˙O_2max, beetroot juice ingestion (1 mmol·NO₃⁻⁻¹·kg⁻¹ for 5 d) elevated blood flow to muscles and muscle portions composed of 66% or more Type II b+d/x muscle fibers (¹²) in concert with a lower blood lactate level. As expected, these selective vascular effects were found to raise PmvO₂ in Type II, but not Type I, muscles during contractions (¹³) providing the mechanistic basis for the reduced blood lactate level (Fig. 1).

Mechanistic Considerations

The preferential fiber type effects of NO₃⁻ supplementation is important for vascular control because NO synthesis via the NOS family of enzymes requires O₂ and consequently may become dysfunctional at the low PO₂ extant in contracting muscle. This may be especially true in Type II muscle given their inherently lower PO₂ compared with Type I muscle (^5,26). Thus, the reduction of NO₂⁻ to NO acts as a useful backup to maintain NO homeostasis and hyperemia in situations where NOS function is compromised. Indeed, it recently has been reported that bolus infusion of NO₂⁻ offsets acute pharmacological NOS inhibition and restores cardiovascular function to control values (¹¹), thus supporting the complementary role of the NO₃⁻-NO₂⁻-NO pathway.

Another potential means by which dietary NO₃⁻ may elicit its beneficial effects involves its impact on skeletal muscle calcium-handling proteins. For instance, Hernández et al. (¹⁶) demonstrated that 7 d of supplementation with NO₃⁻-enriched water resulted in an increased level of calsequestrin 1 and the dihydropyridine receptor, which ultimately increased the rate of muscle force development. Crucially, these effects were evident in the extensor digitorum longus (predominantly Type II) but not soleus (predominantly Type I) muscles, further supporting fiber type-specific effects of NO₃⁻ supplementation. Collectively, these results demonstrate that NO₃⁻ supplementation has the ability to impact skeletal muscle vascular and metabolic function during contractions, via both blood flow-dependent and -independent mechanisms.

From a performance standpoint, NO₃⁻-targeted effects likely take center stage during high-intensity exercise given that Type II fibers are recruited to a greater extent in the heavy-intensity to severe-intensity exercise domains (²¹). These higher-intensity domains result in an increased V˙O₂/Q˙O₂ ratio (decreased Q˙O₂/V˙O₂ and PmvO₂ (²⁶)), which further exacerbates the tissue hypoxia within Type II muscles, ultimately resulting in increased accumulation of ADP, [P_i], [K⁺], and [H⁺], all of which are associated with fatigue development. As previously mentioned, these low-PO₂/pH environments are ripe for reduction of NO₂⁻ to NO and thus likely facilitate the increase in Q˙O₂ (and potentially reduced V˙O₂) observed after NO₃⁻ supplementation. Moreover, the increased PmvO₂ and any reduced V˙O₂ may raise the intracellular PO₂, which will act to reduce the intracellular perturbation of the aforementioned metabolites, thereby resulting in a tighter metabolic control. This is especially important when considering that increased intracellular PO₂ reduces the ADP and nicotinamide adenine dinucleotide concentrations necessary to drive a given ATP production, consequently suppressing glycolysis and glycogen depletion. Finally, if Q˙O₂ is limiting V˙O₂ kinetics, it is quite possible that NO₃⁻ supplementation will help alleviate the O₂ deficit by speeding V˙O₂ kinetics in the first minutes of exercise (³⁰).

ROLE OF TYPE II MUSCLE FIBER RECRUITMENT IN EFFICACY OF NITRATE SUPPLEMENTATION IN HUMANS

Since the seminal findings of Larsen et al. (²³), most, although not all, studies have reported improvements in exercise economy and/or fatigue resistance in moderately trained participants (V˙O_2peak, ≤55 mL·kg⁻¹·min⁻¹) after NO₃⁻ supplementation ((^2,4,23,41) cf. (¹⁹)). Conversely, although some well-trained endurance athletes do exhibit improvements in exercise economy or performance, it seems that the ergogenicity of NO₃⁻ supplementation is attenuated in this population (e.g., (^6,9)). These observations are encapsulated in the recent study by Porcelli et al. (³¹), in which NO₃⁻ supplementation lowered V˙O₂ during moderate-intensity running and improved 3-km running performance in participants with low (V˙O_2peak, <45 mL·kg⁻¹·min⁻¹) and moderate (V˙O_2peak, <45–57 mL·kg⁻¹·min⁻¹), but not high (V˙O_2peak, >63 mL·kg⁻¹·min⁻¹), aerobic fitness, with the reductions in moderate-intensity V˙O₂ and 3-km completion time being positively correlated with V˙O_2peak. As previously reviewed (¹⁸), many NO₃⁻ supplementation studies may not have provided a sufficient NO₃⁻ dose and/or duration of supplementation to elicit physiological effects (⁴¹). It is plausible that elite athletes, in whom NOS activity, plasma [NO₂⁻], and muscle oxygenation will all be enhanced by intensive training, might benefit from greater and/or more protracted NO₃⁻ supplementation (¹⁸). However, given that high-level endurance athletes would, in general, be expected to have a lower percentage of Type II muscle fibers (³³) and that NO₃⁻ treatment seems to preferentially augment physiological responses in Type II muscle fibers in animal models (as described in the previous section), it is tempting to speculate that the efficacy of NO₃⁻ supplementation to improve physiological and functional responses in human participants might be linked to the population of muscle fibers recruited during exercise. Accordingly, recent research has sought to elucidate the potential for preferential effects of NO₃⁻ on human Type II skeletal muscle in vivo.

Influence of Muscle Fiber Type on the Effectiveness of Nitrate Supplementation in Humans

To examine the potential for a greater effectiveness of NO₃⁻ supplementation in human Type II muscle, recent studies have used experimental models to capitalize on differences in the power-velocity relationship between fiber types and the hierarchical recruitment of fiber types as force requirements are increased. Bailey et al. (³) observed greater muscle oxyhemoglobin concentration using NIRS, faster pulmonary V˙O₂ kinetics, and extended severe-intensity exercise tolerance (+22%) after NO₃⁻ supplementation when cycling at 115 rpm, but not 35 rpm (Fig. 3). These findings are consistent with other recent in vivo data that indicated that peak knee extensor torque was increased at an angular velocity of 360 degrees·s⁻¹ but not at angular velocities of 90, 180, and 270 degrees·s⁻¹ after NO₃⁻ supplementation (¹⁰). Assuming that the proportional contribution of Type II muscle fibers to force production is greater at higher contraction frequencies or velocities, these findings support the notion of enhanced Q˙O₂ and/or contractile function of Type II muscle in vivo after NO₃⁻ supplementation (^3,10), consistent with observations in murine models (^12,13,16).

Figure 3: Pulmonary oxygen uptake responses in a representative individual during a step increment from a low-intensity baseline to a moderate-intensity work rate (A), a step increment from a moderate-intensity work rate to a severe-intensity work rate (B), as well as step increments from a low-intensity baseline to a severe-intensity work rate when cycling at a low pedal cadence (35 rpm; C) or high pedal cadence (115 rpm; D). Responses after NO₃ ⁻-rich beetroot juice supplementation (BR) are shown in filled circles, with the responses after NO₃ ⁻-depleted beetroot juice (PLA) shown in open circles. Notice the faster adjustment of pulmonary oxygen uptake when the proportional contribution of Type II muscle to force production would be expected to be greater (B and D) compared with settings where the proportional contribution of Type II muscle to force production would be expected to be lower (A and C). Data redrawn from Bailey et al. (³) and Breese et al. (⁸).

Further support for a targeted effect of NO₃⁻ ingestion on human Type II muscle was provided in the study by Breese et al. (⁸). These authors administered an initial step work rate increment from a low-intensity baseline (20 W) to a moderate-intensity work rate (<gas exchange threshold; L→M), where predominantly Type I muscle fibers are recruited (²¹), and a subsequent step to a severe-intensity work rate (>critical power; M→S), where a greater recruitment of Type II muscle fibers has been reported (²¹). These double-step tests were completed after both NO₃⁻ and placebo supplementation. Compared with placebo, NO₃⁻ resulted in faster pulmonary V˙O₂ and muscle deoxyhemoglobin kinetics (reflective of faster muscle O₂ extraction kinetics) in M→S but not L→M (Fig. 3). In addition, exercise tolerance during M→S was extended by 22% after NO₃⁻ compared with placebo ingestion. Although necessarily speculative, taken together, these findings suggest that the physiological effects evoked by NO₃⁻ supplementation may be enhanced in Type II muscle fibers.

It is well known that short-duration high-intensity intermittent exercise mandates a significant recruitment of Type II muscle fibers. Accordingly, improved performance during such exercise would be hypothesized if NO₃⁻ supplementation was particularly effective at augmenting physiological processes in Type II muscle. However, the effect of NO₃⁻ supplementation on performance during short-duration high-intensity intermittent exercise is controversial (^{9,28,34,40,42}). From the existing studies, it seems that when participants received only an acute NO₃⁻ bolus (²⁸), or when elite endurance cyclists completed repeated cycle sprints after 6 d of NO₃⁻ supplementation (⁹), performance was not enhanced. The lack of an ergogenic effect with NO₃⁻ supplementation in these studies might be attributable to an insufficient supplementation dose and/or duration to elicit the skeletal muscle and mitochondrial remodeling that underpins some of the positive physiological responses reported elsewhere (^16,22) and to the lower percentage of Type II fibers in the locomotor muscles of elite endurance athletes (³³). An interesting exception in this regard is that NO₃⁻ ingestion resulted in significantly improved exercise economy in a laboratory exercise test and significantly improved on-water time trial performance in elite kayakers (²⁹). This is intriguing in relation to the hypothesis advanced in the current article because the upper body musculature typically has a higher fraction of Type II muscle fibers (¹⁷). However, other factors such as muscle volume, intramuscular tension, and perfusion pressure also may impact on the efficacy of NO₃⁻ supplementation in exercise of this type. On the other hand, when moderately trained team-sport athletes undergo short-term dietary NO₃⁻ supplementation, short-duration high-intensity intermittent exercise performance generally has been found to be enhanced (^34,40,42). Although further research is required, initial observations support an ergogenic effect after short-term NO₃⁻ supplementation in moderately trained team-sport athletes, but not necessarily after acute NO₃⁻ ingestion and/or when elite endurance athletes complete short-duration high-intensity intermittent exercise.

Functional and Clinical Implications and Future Directions

The capacity to complete repeated bouts of high-intensity exercise is linked to the metabolic homeostasis of Type II muscle fibers and is an important determinant of performance in team sports. Therefore, the apparent improvements in pulmonary V˙O₂ kinetics (^3,8), skeletal muscle metabolic homeostasis (^2,14), perfusion (¹²), oxygenation (^3,13), force production (^10,16), and fatigue resistance (^{2–4,8,34,42}) with NO₃⁻ when Type II muscle fiber recruitment is likely to be greater might be expected to improve performance in team sports or other exercise settings that mandate a significant recruitment of Type II muscle. It also is noteworthy that many patient groups including those with peripheral artery disease, chronic obstructive pulmonary disease, and heart failure have a higher proportion of Type II muscle fibers (e.g., (¹⁵)). Given the emerging evidence to support augmented physiological benefits in Type II compared with Type I skeletal muscle, NO₃⁻ supplementation might represent a simple dietary intervention to improve skeletal muscle perfusion, contractile function, and fatigue resistance in some patient populations.

To further our understanding of these specific effects, future investigations using a multidisciplinary approach should resolve the impact of dietary NO₃⁻ supplementation on the Q˙O₂/V˙O₂ relationship across the spectrum of fast- versus slow-twitch muscles. In particular, simultaneous measurement of Q˙O₂ and PO₂mv (potentially by combining the phosphorescence quenching with the radiolabeled microsphere technique) in murine muscles of varying fiber type would afford calculation of skeletal muscle V˙O₂ and shed further light on the impacts of NO₃⁻ supplementation on muscle vascular and metabolic control. In humans, important unresolved questions include whether 1) dietary NO₃⁻ supplementation increases skeletal muscle [NO₃⁻] and [NO₂⁻], 2) NO₂⁻ is “consumed” by contracting skeletal muscle (as determined by measurements of arterial-venous difference) and this correlates with changes in either Q˙O₂ or V˙O₂, 3) the energetics of isolated myocytes and/or mitochondria are altered on exposure to NO₃⁻ and NO₂⁻, and 4) NO₃⁻ supplementation impacts the heterogeneity of local blood flow to metabolic rate as assessed with time-resolved multichannel NIRS (²⁰). With specific regard to the potential fiber type sensitivity of NO₃⁻ supplementation highlighted in this article, future investigations might usefully compare the physiological responses to NO₃⁻ ingestion of athletes specializing in power and endurance sports (within whom fiber type distribution would be expected to be different) and/or characterize responders/nonresponders to NO₃⁻ supplementation on the basis of directly measured muscle fiber type.

SUMMARY

Nitric oxide is involved in an array of physiological processes that regulate cardiovascular and muscle function. Because the activity of NOS can decline in low-O₂ environments, various disease states, and in older age, the NO₃⁻-NO₂⁻-NO pathway represents an essential backup system for NO generation. Dietary NO₃⁻ supplementation increases plasma [NO₂⁻] and therefore NO bioavailability. Assuming a sufficient dose and duration of supplementation, the effectiveness of NO₃⁻ in positively altering exercise economy and performance seems to be related both to the aerobic fitness or endurance training status of the subject population and to the type of exercise being considered (¹⁸). In this article, we have presented evidence consistent with the hypothesis that NO₃⁻ may be particularly effective at augmenting physiological responses in Type II muscle (Fig. 1). The relatively low PmvO₂ therein may promote the reduction of NO₂⁻ to NO, thereby increasing local perfusion and Q˙O₂/V˙O₂, improving fatigue resistance, augmenting fiber contractility, and ultimately enhancing whole body exercise tolerance or performance. This targeted effect of NO₃⁻ supplementation on Type II muscle may have important implications for improving performance during intense exercise in healthy adults and for helping to restore functional capacity in senescent and patient populations, where increased reliance on Type II muscle fibers contributes to compromised aerobic capacity and exercise intolerance.

This study was supported by the National Heart, Lung, and Blood Institute grants HL-50306 and HL-108328.

A.M.J., A.V., and S.J.B. were supported, in part, by the Gatorade Sports Science Institute; D.C.P. was supported by a grant-in-aid from the American Heart Association.