Microvascular oxygen pressures in muscles comprised of different fiber types: Impact of dietary nitrate supplementation

Abstract

Nitrate (NO₃⁻) supplementation via beetroot juice (BR) preferentially improves vascular conductance and O₂ delivery to contracting skeletal muscles comprised predominantly of type IIb + d/x (i.e. highly glycolytic) fibers following its reduction to nitrite and nitric oxide (NO). To address the mechanistic basis for NO₃⁻ to improve metabolic control we tested the hypothesis that increased NO bioavailability via BR supplementation would elevate microvascular PO₂ (PO₂mv) in fast twitch but not slow twitch muscle. Twelve young adult male Sprague-Dawley rats were administered BR ([NO₃⁻] 1 mmol/kg/day, n=6) or water (control, n=6) for 5 days. PO₂mv (phosphorescence quenching) was measured at rest and during 180s of electrically induced 1-Hz twitch contractions (6–8 V) of the soleus (9% type IIb +d/x) and mixed portion of the gastrocnemius (MG, 91% type IIb + d/x) muscles. In the MG, but not the soleus, BR elevated contracting steady state PO₂mv by ~43% (control: 13.7 ± 0.5, BR: 19 ± 1.6 mmHg, (P<0.05). This higher PO₂mv represents a greater blood-myocyte O₂ driving force during muscle contractions thus providing a potential mechanism by which NO₃⁻ supplementation via BR improves metabolic control in fast twitch muscle. Recruitment of higher order type II muscle fibers is thought to play a role in the development of the $\stackrel{.}{V}{O}_2$ slow component which is inextricably linked to the fatigue process. These data therefore provide a putative mechanism for the BR-induced improvements in high-intensity exercise performance seen in humans.

1. Introduction

At exercise onset, the immediate increase in ATP turnover within contracting skeletal muscle mandates an elevated rate of O₂ delivery (QO₂), such that capillary blood flow is rapidly increased to meet the rising O₂ demand of contracting myocytes (i.e. O₂ uptake; $\stackrel{.}{V}{O}_2$) [1]. This augmented capillary flow is accomplished via elevated cardiac output and blood flow redistribution (neurohumoral activation) as well as local mechanical and vasomotor mechanisms [reviewed by 2]. Of the local controllers, the powerful signaling molecule nitric oxide (NO) promotes vasodilation of terminal arterioles within skeletal muscle, helping to facilitate this hyperemic response and better match QO₂ to the elevated $\stackrel{.}{V}{O}_2$ demands [3; 4; 5].

It is now understood that nitrate (NO₃⁻) and nitrite (NO₂⁻) can be converted to NO and other reactive nitrogen species in vivo following a stepwise reduction [reviewed by 6]. In humans, dietary NO₃⁻ supplementation has been shown to enhance muscle contractile [7] and mitochondrial [P/O ratio; 8] efficiency, both of which are associated with a reduction in the O₂ cost of submaximal exercise [7; 8; 9; 10; 11; 12; 13; 14] and improvements in tolerance to high intensity exercise [7; 9; 11; 13; 15; 16; 17; 18].

What is particularly interesting is that the improvements in performance have been seen predominantly during severe-intensity exercise [15; 19] rather than long term endurance exercise [20; 21]. Recent studies performed in murine models suggest that this phenomenon may be due to a fiber type selective enhancement in skeletal muscle vascular and metabolic control following NO₃⁻ ingestion [22; 23]. Specifically, our laboratory has demonstrated that rats supplemented with BR (NO₃⁻ concentration 1 mmol/kg/day) for five days had higher exercising blood flow and vascular conductance in muscles comprised principally of type II muscle fibers [22]. BR also raised the pressure head for capillary-myocyte O₂ flux during the crucial transition period from rest to muscle contractions (i.e., ~20–60 s) in the rat spinotrapezius muscle, which is composed of approximately 50% Type IIb+d/x muscle fibers [24; 25]. Moreover, Hernandez et al. [23] demonstrated improved calcium handling and rate of force development in type II but not type I muscles of NO₃⁻ supplemented mice.

Given the lower contracting PO₂mv reported in fast twitch muscles [26] and the evidence that NO₂⁻ reduction to NO is potentiated in environments with low PO₂ [27] the physiological effects of BR on the PO₂mv profile may be intensified in fast twitch muscles. Therefore the purpose of the present investigation was to examine the effects of 5 days of NO₃⁻ supplementation via BR (NO₃⁻ concentration 1 mmol/kg/day) on the PO₂mv profile of rat muscles comprised of predominantly type I (slow twitch) and type IIb+d/x (fast twitch) muscle fibers. We tested the hypothesis that BR would attenuate the fall in PO₂mv in the fast twitch mixed portion of the gastrocnemius (MG) across the rest-contraction transition with either a lesser or no effect in the slow twitch soleus muscle.

2. Methods

2.1 Animal selection and care

Twelve young adult male Sprague-Dawley rats (average body mass = 521±20 g, Charles River Laboratories, Wilmington, MA) were used in this investigation. Rats were maintained in accredited animal facilities at Kansas State University on a 12/12 hr light-dark cycle with food and water provided ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee of Kansas State University and conducted according to National Institutes of Health guidelines.

2.2 Supplementation protocol

Rats were randomly assigned to receive 5 days of BR supplementation with a NO₃⁻ dose of 1 mmol/kg/day (BR; n=6, Beet it™, James White Drinks, Ipswich UK, diluted with 100 ml of tap water) or NO₃⁻ depleted BR (control; n=6, Beet it™ placebo, diluted with 100 ml of tap water) with consumption monitored. This NO₃⁻ dose (1 mmol/kg/day) represents a NO₃⁻ concentration similar to that used in humans by Jones and colleagues [9; 11; 14; 18] after accounting for the resting metabolic rate of rats [~7× that of humans, 28]. In addition, this dose was used in our laboratory previously with significant vascular effects observed following supplementation [22; 25].

2.3 Surgical instrumentation

Rats were anaesthetized with a 5% isoflurane-O₂ mixture and maintained subsequently on 3% isoflurane-O₂ mixture. The carotid artery was cannulated and a catheter (PE-10 connected to PE-50, Intra-Medic polyethylene tubing, Clay Adams Brand, Becton, Dickinson and Company, Sparks, MD) inserted into carotid artery catheter for measurement of mean arterial pressure (MAP) and heart rate (HR), arterial blood sampling (Nova State Profile M, Waltham, MA, USA) and, infusion of the phosphorescent probe (see below). A second catheter was also placed in the caudal artery. Incisions were then closed and the rats were transitioned to pentobarbital sodium anesthesia (administered to effect and subsequently maintained via the caudal artery catheter) with level of anesthesia monitored continuously via the toe pinch and blink reflexes. If indicated, additional pentobarbital sodium was administered in supplemental dosage (5–10 mg/kg) as needed. Rats were then transferred onto a heating pad to maintain core body temperature at ~38°C (measured via rectal probe thermometer) and the carotid artery catheter was connected to a pressure transducer (Digi-Med BPA model 200, Louisville, KY, USA) for measurement of MAP and HR.

The muscles chosen for the present experiment (soleus and mixed portion of the gastrocnemius, MG) were selected based on their fiber type composition [24] and represent the spectrum of slow twitch (type I/IIa) and fast twitch (type IIb+d/x) muscle fiber types. The highly oxidative soleus [84% type I, 7% type IIa and 9% type IIb+d/x, 24] serves as a postural muscle whose primary functions are ankle stabilization and plantar flexion while the MG functions in plantar flexion and is comprised predominantly of highly glycolytic fast-twitch muscle fibers [3% type I, 6% type IIa, 91% type IIb+d/x, 24]. Each muscle was exposed for PO₂mv experiments in the following manner. Overlaying skin and fascia along the sagittal plane on the right hindlimb were reflected carefully to expose the muscles of the ‘calf’. For measurements made in the MG, silver wire electrodes were sutured (6–0 silk) to the proximal (cathode) and distal (anode) portions of the muscle. Following measurements of PO₂mv in the MG the soleus muscle was exposed by carefully reflecting overlaying tissue covering the peroneal muscle group (cornal plane) and silver wire electrodes were sutured (6–0 silk) in the same manner as for the MG. Measurement order was randomized to avoid the potential influences of an ordering effect caused by NO₃⁻ metabolism. The exposed muscles were continuously superfused with warmed (38°C) Krebs–Henseleit bicarbonate buffered solution equilibrated with 5% CO₂–95% N₂ and surrounding exposed tissue was covered with Saran wrap (Dow Brands, Indianapolis, IN). This method has been used previously in our laboratory and facilitates access to the MG and soleus muscles whilst minimizing perturbation caused by surgery [29].

2.4 Experimental protocol

The phosphorescent probe palladium meso-tetra (4 carboxyphenyl)tetrabenzoporphyrin-dendrimer (G2: 1–5 mg/kg dissolved in 0.4 ml saline) was infused via the carotid artery catheter. After a brief stabilization period (~10 min), the common end of the light guide of a frequency domain phosphorimeter (PMOD 5000, Oxygen Enterprises, Philadelphia, PA) was positioned ~2–4 mm superficial to the lateral surface of the exposed muscle (either MG or soleus) of the right hindlimb over a randomly selected muscle field absent of large vessels thus ensuring that the region contained principally capillary blood. PO₂mv was measured via phosphorescence quenching (see below) and reported at 2 s intervals throughout the duration of the 180 s contraction protocol (1 Hz, ~6 V, 2 ms pulse duration) elicited via a Grass stimulator (model S88, Quincy, MA). As an indicator of preserved vasomotor function, it was ensured that PO₂mv returned to baseline values following the contraction period. Rats were euthanized via pentobarbital sodium overdose (≥50 mg/kg administered into the carotid artery catheter). Power analysis based on a known sample variability of PO₂mv and anticipated supplementation effects [22; 25] indicate that six rats per group would be sufficient to demonstrate a statistical difference, if present.

2.5 PO₂mv measurement and curve-fitting

The Stern-Volmer relationship allows the calculation of PO₂mv through the direct measurement of a phosphorescence lifetime via the following equation [30]:

$${\mathrm{PO}}_{2\mathit{mv}}=[(\tau °/\tau )-1]/(k_{\mathrm{Q}}\times \tau °)$$

Where k_Q is the quenching constant and τ° and τ are the phosphorescence lifetimes in the absence of O₂ and the ambient O₂ concentration, respectively. For G2, k_Q is 273 mmHg/s and τ° is 251 μs at 38°C [31] and these characteristics do not change over the physiological range of pH and temperature in the rat in vivo and, therefore, the phosphorescence lifetime is determined directly by the O₂ pressure [30; 31].

Curve-fitting of the measured PO₂mv responses was performed with commercially available software (SigmaPlot 11.01, Systat Software, San Jose, CA) and the data were fit with either a one- or two-component model as described below:

$$\text{One component}:{\mathrm{PO}}_{2}m{v}_{(\mathrm{t})}={\mathrm{PO}}_{2}m{v}_{(\mathrm{BL})}-\mathrm{\Delta }{\mathrm{PO}}_{2}mv(1-{\mathrm{e}}^{-(t-\mathrm{TD})/\tau })$$

$$\text{Two component}:{\mathrm{PO}}_{2}m{v}_{(\mathrm{t})}={\mathrm{PO}}_{2}m{v}_{(\mathrm{BL})}-{\mathrm{\Delta }}_1{\mathrm{PO}}_{2}mv(1-{\mathrm{e}}^{-(t-\mathrm{TD}1)/\tau 1})+{\mathrm{\Delta }}_2{\mathrm{PO}}_{2}mv(1-{\mathrm{e}}^{-(t-\mathrm{TD}2)/\tau 2})$$

where PO₂mv_(t) represents the PO₂mv at any given time t PO₂mv _(BL) corresponds to the pre-contracting resting baseline PO₂mv, Δ₁ and Δ₂ are the amplitudes for the first and second component, respectively, TD1 and TD₂ are the time delays for each component, and τ₁ and τ₂ are the time constants (i.e., time to 63% of the final response value) for each component. The two component model was only used when the PO₂mv increased above its initial nadir during contractions. Goodness of fit was determined using the following criteria: 1) the coefficient of determination; 2) sum of the squared residuals; and 3) visual inspection and analysis of the model fits to the data and the residuals. The mean response time (MRT) of the kinetics response was calculated for the first component in order to provide an index of the overall principal kinetics response according to the following equation:

$${\mathrm{MRT}}_1={\mathrm{TD}}_1+{\tau }_1$$

where TD₁ and τ₁ are as described above. The delta of the initial PO₂mv fall following contractions onset was normalized to τ₁ (Δ₁ PO₂mv/τ₁) to provide an index of the relative rate of fall. Additionally, the time taken to reach 63% of the initial PO₂mv fall was determined independently from and prior to the modeling procedures (T₆₃) to ensure appropriateness of the model fits. Specifically, the raw PO₂mv data were interpolated, and the time coinciding with 63% of the total amplitude (Δtotal PO₂mv) was determined.

2.6 Blood sampling and measurement of plasma [NO₃⁻] and [NO₂⁻]

Post-supplementation blood samples were collected following the experiment via the caudal artery catheter to assess: 1) plasma [NO₃⁻] and [NO₂⁻]; and 2) pH; PO₂; and %O₂ saturation. For measurements of plasma [NO₃⁻] and [NO₂⁻], ~0.8 ml of blood was drawn into heparinized tubes and rapidly centrifuged at 6000 g at 4°C for 6 minutes. Plasma was then extracted and frozen immediately at −80°C for later analysis. A second ~0.3 ml blood sample was drawn and analyzed for blood [lactate], pH, PO₂, and %O₂ saturation (Nova Stat Profile M, Nova Biomedical, Waltham, MA, USA).

All measurements of plasma NO₃⁻ and NO₂⁻ were performed within 30 minutes of thawing via chemiluminescence with an Ionic/Sievers NO analyzer (NOA 280i, Sievers Instruments, Boulder, CO, USA). In order to obtain plasma NO₂⁻ levels and to avoid potential reduction of NO₃⁻, potassium iodide in acetic acid was used as a reductant. This reductant possesses the ability to reduce NO₂⁻ to NO but is incapable of reducing higher oxides of nitrogen (i.e. NO₃⁻) thus increasing the specificity for NO₂⁻. Plasma NO₃⁻ concentrations were then obtained using the same apparatus with the stronger reductant vanadium chloride in hydrochloric acid at a temperature of 95°C. This stronger reductant reduces the sum of all nitrogen oxides with an oxidation state of +2 or higher (predominantly NO₃⁻ [μM]) but also includes NO₂⁻ and nitrosothiols [nM]. Signals obtained using potassium iodide were then subtracted from those with vanadium chloride to provide a clearer representation of the NO₃⁻ concentrations.

2.7 Statistical analysis

Data are presented as mean ± SEM. Results were compared within and between groups using mixed 2-way ANOVAs (MAP and HR) with Student-Newman-Keuls post hoc tests where appropriate or unpaired student’s t-test (PO₂mv kinetics parameters, blood gasses, [NO₃⁻], [NO₂⁻]). Significance was accepted at P<0.05.

3. Results

3.1 Plasma [NO₃⁻] and [NO₂⁻]

BR rats had significantly higher plasma [NO₃⁻] compared to control (control: 29 ± 6, BR: 79 ± 17 μmol/l, P=0.02).). Relative to control, plasma [NO₂⁻] tended to be higher in BR rats, however these changes did not reach significance (control: 156 ± 66, BR: 216 ± 46 nmol/l, P=0.22).

3.2 MAP, HR, and arterial blood gases

There were no between group differences in arterial PO₂, PCO₂, or pH (data not shown, P>0.05 for all). Resting (control: 1.6 ± 0.1, BR: 1.1 ± 0.2 mmol/l, P>0.05) arterial blood [lactate] was not different between groups. There were no differences in resting or contracting steady-state MAP for the soleus (resting control: 122 ± 7, resting BR: 114 ± 6, contracting control: 124 ± 6, contracting BR: 117 ± 8 mmHg, P>0.05 for all) or MG (resting control: 114 ± 5, resting BR: 106 ± 6, contracting control: 116 ± 6, contracting BR: 110 ± 6 mmHg, P>0.05 for all). Furthermore, there were no differences in resting or contracting steady-state HR for the soleus (resting control: 364 ± 16, resting BR: 345 ± 25, contracting control: 366 ± 17, contracting BR 381 ± 45 mmHg, P>0.05 for all) or MG (resting control: 377 ± 16, resting BR: 372 ± 35, contracting control: 391 ± 19, contracting BR: 354 ± 52 mmHg, P<0.05 for all).

3.3 PO₂mv baseline, contracting steady-state and kinetics parameters

Baseline and contracting steady-state

As expected control PO₂mv_{(base line)} and contracting PO₂mv_{(steady-state)} (evident over the final ~30 s of contractions) were higher in the soleus compared to MG (Table 1, Figure 1). However, during contractions BR supplemented rats demonstrated an elevated PO₂mv_{(steady-state)} in the MG when compared to control (Table 1, P=0.01) with no significant difference evident in the soleus (Table 1, P=0.31). It was noteworthy that BR raised PO₂mv_{(steady-state)} in the MG to that found in the control soleus.

Table 1.

Microvascular O₂ partial pressure (PO₂mv) kinetics parameters during soleus and MG contractions in control and BR rats.

	Soleus		MG
	Control	BR	Control	BR
PO2mv(Base line) (mmHg)	32 ± 3	33 ± 3	24 ± 2†	25 ± 2†
Δ1PO2mv (mmHg)	11 ± 1	9 ± 2	11 ± 2	10 ± 1
Δ2 PO2mv (mmHg)			1 ± 0	4 ± 1
Δtotal PO2mv (mmHg)	11 ± 1	9 ± 2	9 ± 1	5 ± 1
PO2mv(steady-state) (mmHg)	20 ± 3	24 ± 2	14 ± 1†	20 ± 2*
Time delay 1 (s)	12 ± 1	8 ± 2	6 ± 2†	2 ± 1†
Time delay 2 (s)				51 ± 14
Time constant 1 (s)	25 ±5	26 ± 6	13 ± 2†	15 ± 4†
Time constant 2 (s)				58 ± 12
Mean response time (s)	37 ± 4	34 ± 6	19 ± 3†	17 ± 3†
T63 (s)	39 ± 5	34 ± 6	19 ± 3†	13 ± 2†

Values are mean ± SEM. Where second component model averages are shown the value reflects only those rats where a two-component model was applied to describe the PO₂mv data (control: n=1 of 6 BR: n=6 of 6 MG profiles).

^*P<0.05 vs. control.

^†P<0.05 vs. soleus.

Figure 1.

Mean percent delta PO₂mv profiles for the soleus (top panel) and MG (bottom panel) muscles of control and BR rats. Time “0” represents the onset of contractions. *P<0.05 versus control.

PO₂mv kinetics parameters

The kinetics following the onset of contractions for the soleus were adequately fit by a one-component model in all control and BR rats. In contrast, for the MG, the more complex two-component model was necessary for 1 of 6 control and all BR rats. Indeed, ~80% of the increased PO₂mv_{(steady-state)} for the BR group resulted from the secondary component increase of PO₂mv (Table 1). The coefficient of determination (r²) for soleus and MG (≥0.98 for control and BR) and low sum of squared residuals (RSS <20) for both groups suggested the appropriateness of the respective model fits.

Table 1 presents the mean PO₂mv kinetics parameters. The time delay and time constant for the first component as well as the mean response time and T₆₃ were greater in the soleus versus the MG (for both control and BR, P<0.05 for all, Table 1). There were no between-group differences in any kinetic parameter following the onset of contractions for the soleus or MG (Table 1, P>0.05 for all). Importantly, within the control and BR groups the model-dependent MRT₁ and model-independent T₆₃ were not different (Table 1) providing additional confidence in the model parameters.

4. Discussion

The present investigation provides the first demonstration that NO₃⁻ supplementation-induced (via BR) elevation of PO₂mv occurs preferentially in muscles comprised of fast twitch (MG) rather than slow twitch (soleus) fibers. This finding coheres with the presence of lower PO₂mv levels in fast twitch muscles [26; 29] and a physicochemical milieu (lower pH, higher lactate) that favors the reduction of NO₂⁻ to NO [27]. It is also true that the low PO₂ environment extant in these muscles which favors NO₂⁻ reduction will suppress the endogenous production of NO from the neuronal and endothelial nitric oxide synthase (nNOS, eNOS) pathways; of which nNOS is the most important in fast twitch muscles [32; 33; 34; 35]. NO₃⁻ supplementation (BR) raises PO₂mv (i.e., QO₂/ $\stackrel{.}{V}{O}_2$ ratio) by simultaneously increasing O₂ delivery [QO2, 22] and reducing the O₂ cost ( $\stackrel{.}{V}{O}_2$) of exercise via changes in mitochondrial and contractile function [6; 8; 12; 23]. Increases in PO₂mv in-and-of themselves have the capacity to improve blood-myocyte O₂ flux, increase intramyocyte PO₂ and consequently enhance mitochondrial oxidative phosphorylation whilst suppressing glycolysis. This behavior may underlie the reduced arterial [lactate] levels found during heavy intensity exercise in running rats [22].

4.1 Effects of BR on the PO₂mv Profile

As also shown in the present investigation, previous studies have reported pronounced fiber type differences in both kinetics parameters and the magnitude of the overall change in PO₂mv following the onset of contractions. Specifically, Behnke et al. [29] found a longer time delay, MRT, slower rate of PO₂mv fall, and a lower overall amplitude of PO₂mv fall (e.g. Δ PO₂mv) in the soleus (slow-twitch) vs. peroneal (fast-twitch) muscles during electrically induced contractions. Additionally, McDonough and colleagues [26] reported a higher PO₂mv_{(steady-state)} in soleus versus MG muscles. In the present investigation, despite BR raising the MG PO₂mv_{(steady-state)} to levels commensurate with those observed in the soleus muscle of control rats herein (PO₂mv_{(steady-state)} ↑43%, Table 1) the kinetics profile was unchanged. This situation does not mean that the kinetics profile is intransigent. Indeed, changes in the PO₂mv kinetics profile can be driven by both NOS-dependent and independent mechanisms invoked by 6–8 weeks of exercise training [33]. Interestingly, the training-induced adaptation of the PO₂mv kinetics profile occurred in the absence of an elevated PO₂mv_{(steady-state).} Thus, exercise training raises PO₂mv across the dynamic transition at a time when $\stackrel{.}{V}{O}_2$ is increasing most rapidly. In marked contrast, the elevation of PO₂mv in the MG after BR seen in the present investigation is delayed and appears to consist of a secondary effect that only becomes apparent after ~60 s of contractions (Figure 1). Such an augmented PO₂mv _{(steady-state)} is expected to facilitate fatigue resistance consequent to decreased metabolic perturbations during exercise [e.g.↓PCr breakdown, 36; 37].

4.2 Relationship to existing literature

BR supplementation elevates QO₂ preferentially in muscles comprised of ≥66% type IIb+d/x muscle fibers [22]. Those fiber type selective elevations in QO₂ observed during treadmill exercise, if present herein, would help explain the greater PO₂mv seen in the MG (Figure 1). As mentioned above, one potential explanation for the fiber type specific elevations in QO₂ (and thus PO₂mv) is that NO₂⁻ reduction is facilitated to a greater extent in fast twitch muscles as a result of lower contracting PO₂mv in type II vs. type I fibers [26; 29; 38]. This is supported by Cosby et al. [27] who demonstrated that NO₂⁻ reduction is potentiated in environments with low PO₂ and pH, such that may exist in skeletal muscle during exercise. Furthermore, activity of the nitric oxide synthase family of enzymes (nNOS and eNOS rather than iNOS being relevant here) may be reduced under such conditions [39] allowing the NO₃⁻ NO₂⁻-NO pathway to serve a complimentary role in the local regulation of NO bioavailability. In this respect also, there is a fiber type specificity: The Michaelis-Menten constant (Km) for nNOS (350 μ m) is over 15-fold greater than eNOS (23 μ m) [40]. Thus, the sensitivity of nNOS to the changes in PO₂ (and PO₂mv) in the MG evoked by BR may potentiate the overall NO bioavailability by allowing nNOS, the predominant NOS in fast twitch fibers, to function more effectively.

In addition to the beneficial vascular impacts of NO₃⁻ supplementation, there may be fiber type specific improvements in metabolic control brought about via improvements in contractile function. For example the improvements in rate of force development and tetanic contractile force reported by Hernandez et al. [23], consequent to improvements in intracellular calcium handling (↑ expression of calsequestrin 1 and the dihydropyridine receptor), may serve to reduce $\stackrel{.}{V}{O}_2$ in the face of elevated QO₂, further raising the QO₂/ $\stackrel{.}{V}{O}_2$ ratio. Furthermore, NO₃⁻ supplementation reportedly increases mitochondrial efficiency in the human vastus lateralis muscle [comprised of ~58% Type IIa/IIb+dx fibers, 41] in proportion to the reduced pulmonary $\stackrel{.}{V}{O}_2$ response to submaximal exercise [8]. Collectively, these studies support that simultaneous increases in muscle(s) QO₂ combined with reductions in $\stackrel{.}{V}{O}_2$ may account for the observed fiber type-specific elevations in contracting PO₂mv_{(steady-state)}.

With regards to performance, the fiber type-selective impacts of BR supplementation may delay the onset of fatigue given that phosphocreatine and glycogen degradation is greater in Type II vs. Type I muscles during maximal exercise [42]. Therefore, an elevated PO₂mv_{(steady-state)} has the means to contribute to BR-induced improvements in intense intermittent exercise [19] especially when considering that type II muscle fibers are heavily recruited during the transition from low to high metabolic rates [43; 44]. In addition, higher exercise intensities result in a greater muscle $\stackrel{.}{V}{O}_2$/QO₂ ratio exacerbating intramyocyte hypoxia and accumulation of [ADP], [Pi], [K⁺] and [H⁺] each of which may play a role in the fatigue process [45; 47]. That these perturbations may be ameliorated, at least in part, by BR-induced elevations in PO₂mv_{(steady-state)} carries significant implications for individuals suffering from diseases where derangements in skeletal muscle O₂ delivery/utilization balance (e.g. chronic heart failure, peripheral artery disease) expedite fatigue.

4.3 Experimental considerations

That the elevated PO₂mv_{(steady-state)} occurred in the absence of elevated plasma [NO₂⁻] suggests that alternate pools of NO₂⁻, perhaps within muscle tissue, contributed to this effect. None-the-less, an improved QO₂/ $\stackrel{.}{V}{O}_2$ ratio in the highly glycolytic muscles and muscle parts would presumably delay the onset of fatigue and thus, may be the mechanism responsible for the improvements in high intensity exercise seen in humans following BR supplementation [15; 19]. Contrary to our original hypothesis, BR did not impact PO₂mv during the immediate rest-contraction transition (i.e. rapid PO₂mv kinetics) as shown previously in the mixed fiber type spinotrapezius muscle [25]. This effect (or lack thereof) may be due to the length of the experimental procedure utilized herein which, given the relatively short half-life of plasma NO₂⁻ [~45 minutes in humans, 18; 48] may have allowed any significant elevation of circulating plasma [NO₂⁻] to subside reducing/abolishing any effect on PO₂mv kinetics. In this regard, it is important to note that muscle [NO₂⁻] can remain elevated after plasma [NO₂⁻] has returned to normal (see Calvert et al. [49]). The robust changes in PO₂mv_{(steady-state)} seen in BR supplemented rats herein combined with the results reported by Calvert et al. [49] suggests that high plasma [NO₂⁻] may not be obligatory to elicit beneficial physiological responses. Elevated tissue [NO₂⁻] may be the consequence of prolonged periods (i.e., several days to weeks) of high exposure for example with BR supplementation or exercise training.

It is important to note that PO₂mv_{(steady-state)} was numerically (~20%), but not significantly, higher in in the contracting soleus of BR supplemented rats (Table 1). This finding may be due to the very small proportion of fast twitch fibers in the soleus (~10%) and/or the reduced effect of BR on slow twitch fibers. Post-hoc power analysis revealed that 19 additional animals would be needed to achieve significance.

4.4 Potential limitations, future directions, and conclusions

Five days of BR-induced NO₃⁻ supplementation raised substantially the contracting PO₂mv_{(steady-state)} in the fast twitch MG with no significant changes evoked in the predominantly slow twitch soleus. That this effect occurred in the presence of unchanged plasma [NO₂⁻] seen in BR supplemented rats highlights the complex nature of NO₃⁻/NO₂⁻ bioactivation and suggest that potentially other storage pools of NO₂⁻ (i.e., within skeletal muscle) may impact skeletal muscle vascular and metabolic function. Importantly, the elevated PO₂mv_{(steady-state)} in the MG reflects an improved ability to maintain QO₂ relative to $\stackrel{.}{V}{O}_2$ and thus, is expected to ameliorate fatigue during high intensity exercise as demonstrated in humans [15; 19]. Future measurements of QO₂ and calculation of $\stackrel{.}{V}{O}_2$ will provide valuable insight into the relative contribution of vascular versus intramyocyte (mitochondrial, contractile machinery) mechanisms responsible for this effect.

In addition, while the lower PO₂mv in the gastrocnemius offers one putative mechanism for NO₂⁻ reduction to NO in vivo (i.e. ↓PO₂ ↑NO₂⁻ reduction) experiments in which tissue pH and/or mitochondrial function are manipulated may offer further insight into the precise mechanism(s) responsible for the fiber type preferential effect observed herein. Indeed, fiber type differences in tissue pH may afford enhanced bioactivation of NO₂⁻ particularly if tissue, rather than plasma, [NO₂⁻] is responsible for the effects on PO₂mv. In this regard, investigations into the impacts of tissue pH on NO₂⁻ bioactivation are warranted.

Considering the preponderance of fast twitch fibers in human locomotory muscles and the increased reliance on these fibers in diseased states (e.g. heart failure) NO₃⁻ supplementation via BR may constitute a novel and powerful “bench-to-bedside” therapeutic modality [47; 50]. Furthermore, considering that traditional organic NO₃⁻ therapies employing isosorbide mononitrate or nitroglycerine eventually lead to tachyphylaxis [51] it is plausible that utilizing the NO₃⁻-NO₂⁻-NO pathway will provide a viable NO-based treatment strategy for various disease conditions.

Highlights.

• Dietary nitrate (NO₃⁻) increases skeletal muscle function during exercise.

• Improvements were seen in muscles comprised predominantly of fast twitch fibers.

• Improved vascular function would be expected to raise microvascular PO₂ (PO₂mv).

• The impacts of NO₃⁻ on the PO₂mv profile of fast vs. slow twitch muscles is unknown.

• NO₃⁻ supplementation preferentially elevates PO₂mv in fast twitch muscle.