Effects of nitrate supplementation via beetroot juice on contracting rat skeletal muscle microvascular oxygen pressure dynamics

Abstract

NO₃⁻ supplementation via beetroot juice (BR) augments exercising skeletal muscle blood flow subsequent to its reduction to NO₂⁻ then NO. We tested the hypothesis that enhanced vascular control following BR would elevate the skeletal muscle O₂ delivery/O₂ utilization ratio (microvascular PO₂, PmvO₂) and raise the PmvO₂ during the rest-contractions transition. Rats were administered BR (~0.8 mmol/kg/day, n=10) or water (control, n=10) for 5 days. PmvO₂ was measured during 180 s of electrically-induced (1 Hz) twitch spinotrapezius muscle contractions. There were no changes in resting or contracting steady-state PmvO₂. However, BR slowed the PmvO₂ fall following contractions onset such that time to reach 63% of the initial PmvO₂ fall increased (MRT₁; control: 16.8±1.9, BR: 24.4±2.7 s, p<0.05) and there was a slower relative rate of PmvO₂ fall (Δ₁PmvO₂/τ₁; control: 1.9±0.3, BR: 1.2±0.2 mmHg/s, p<0.05). Despite no significant changes in contracting steady state PmvO₂, BR supplementation elevated the O₂ driving pressure during the crucial rest-contractions transients thereby providing a potential mechanism by which BR supplementation may improve metabolic control.

1. Introduction

At exercise onset, skeletal muscle blood flow (Qm) is elevated via a complex array of mechanical (muscle pump) as well as vasodilatory controllers (Joyner & Wilkins, 2007) in an effort to balance O₂ delivery (QO₂)-to-utilization (ṾO₂). Across species, for small and large muscle mass exercise QO₂ increases between 5 and 6 L per L ṾO₂ irrespective of muscle fiber-type composition (Whipp & Ward, 1982; Ferreira et al. 2006; Jones et al. 2012; reviewed by Poole & Jones, 2012) serving to meet the rising metabolic demands during the rest-contraction transient. However, owing to a lower intercept of the QO₂-to-ṾO₂ relation across the spectrum of muscle fiber-types, muscles or muscle portions comprised of fast-twitch (type II) fibers have a significantly lower QO₂ than their slow-twitch (type I) counterparts such that their fractional O₂ extraction at rest is higher and thus their microvascular PO₂ (PmvO₂) lower (Behnke et al. 2003; McDonough et al. 2005; Ferreira et al. 2006). Consequently, during contractions when ṾO₂ rises there is less room for further increases in fractional O₂ extraction giving rise to extremely low PmvO₂ values. This is particularly important given that low PmvO₂ values lead to reduced intramyocyte PmvO₂ (Hogan et al. 1992; Richardson et al. 1999; Kindig et al. 2003; Haseler et al. 2004; reviewed by McDonough et al. 2005) making it probable that the metabolic behavior of type II fibers (i.e. slowed ṾO₂ kinetics, elevated rate of glycolysis and lactic acid production) results, at least in part, from this phenomenon.

The ubiquitous signaling molecule nitric oxide (NO) plays a fundamental role in exercise induced vasodilation and thus QO₂ (Hirai et al. 2004; reviewed by Joyner & Tschakovsky, 2003) and also increases muscle mitochondrial oxidative (Larsen et al. 2012) and contractile (Andrade et al. 1998) efficiency. Emerging evidence suggests dietary nitrate (NO₃⁻), ingested for example via sodium NO₃⁻ salt or beetroot juice (BR), may impact skeletal muscle hemodynamic, metabolic and contractile function following its non-enzymatic reduction to nitrite (NO₂⁻) and NO in vivo (Larsen et al. 2007, Bailey et al. 2009, Bailey et al. 2010, Hernandez et al. 2012, Ferguson et al. 2013). In humans, acute NO₃⁻ supplementation via BR has been linked to improvements in muscle tissue oxygenation during exercise in a hypoxic environment (Vanhatalo et al 2011, Masschelein et al. 2012) and has been demonstrated to enhance local tissue oxygenation in peripheral artery disease patients in whom reduced local O₂ delivery is a defining characteristic responsible for exercise intolerance (Kenjale et al. 2011).

Recently, our laboratory demonstrated that BR supplementation in rats (NO₃⁻ dose 1 mmol/kg/day for 5 days) elevates QO₂ preferentially in muscles comprised of fast-twitch fibers during treadmill running (Ferguson et al. 2013). The resultant Qm increase would presumably elevate the QO₂/ṾO₂ ratio and thus PmvO₂, thereby improving metabolic control, which may help explain mechanistically the lowered arterial [lactate] seen in the running rat following BR supplementation (Ferguson et al. 2013).

To our knowledge there have been no reports on the effects of NO₃⁻ supplementation on the PmvO₂ profile of contracting skeletal muscle. We hypothesized that 5 days of BR supplementation (NO₃⁻ dose: ~0.8 mmol/kg/day) would increase PmvO₂ across the rest/contraction transient (i.e. slow PmvO₂ on kinetics) and raise the subsequent steady-state PmvO₂.

2. Methods

2.1 Animal selection and care

Twenty young adult male Sprague-Dawley rats (average body mass = 410±14 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 Institute of Health guidelines.

2.2 Supplementation protocol

Rats received 5 days of BR supplementation (BR; n=10) with a NO₃⁻ dose of 1 mmol/kg/day (Beet it™, James White Drinks, Ipswich UK, diluted with 100 ml of tap water) or untreated tap water (control; n=10) with consumption monitored daily (average NO₃⁻ consumption for BR rats = 0.79±0.04 (range = 0.66-0.90) mmol/kg/day). This dose is similar to the sodium NO₃⁻ dose administered to rats by Carlström et al. (2010) and accounts for the resting metabolic rate of the Sprague Dawley rat (~7× faster than humans, Musch et al. 1988). Moreover, we have reported recently that this identical BR dose and administration period elevates plasma [NO₃⁻] and [NO₂⁻] to levels similar to those seen in humans and improves skeletal muscle O₂ delivery in exercising rats (Ferguson et al. 2013).

2.3 Surgical preparation

Rats were anaesthetized with a 5% isoflurane-O₂ mixture and maintained subsequently on 3% isoflurane-O₂. 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 MAP and HR, infusion of the phosphorescent probe (see below), and arterial blood sampling. A second catheter was placed in the caudal artery. The incisions were then closed and rats were transitioned progressively to pentobarbital sodium anesthesia (administered into the caudal artery catheter to effect) with the level monitored continuously via the toe-pinch and blink reflexes and anesthesia supplemented as necessary. Rats were then placed on a heating pad to maintain core temperature at ~38 °C (measured via rectal probe). Overlying skin and fascia were reflected carefully from the mid-dorsal caudal region of each rat and the right spinotrapezius muscle was carefully exposed in a manner which ensured the integrity of the neural and vascular supply to the muscle (Bailey et al. 2000). Silver wire electrodes were sutured (6–0 silk) to the rostral (cathode) and caudal (anode) regions of the muscle. The exposed spinotrapezius muscle was continuously superfused with a 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). The spinotrapezius muscle was selected specifically based on its mixed muscle fiber-type composition and citrate synthase activity close to that found in human quadriceps muscle (Delp & Duan 1996; Leek et al. 2001).

2.4 Experimental protocol

The phosphorescent probe palladium meso-tetra (4 carboxyphenyl)porphyrin dendrimer (R2: 15–20 mg·kg⁻¹ 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 dorsal surface of the exposed right spinotrapezius muscle over a randomly selected muscle field absent of large vessels thus ensuring that the region contained principally capillary blood. PmvO₂ 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). Following the contraction period it was ensured that PmvO₂ returned to baseline values (indicative of preserved vasomotor function). Rats were euthanized via pentobarbital sodium overdose (≥50 mg/kg administered into the carotid artery catheter).

2.5 PmvO₂ measurement and curve-fitting

The Stern-Volmer relationship allows the calculation of PmvO₂ through the direct measurement of a phosphorescence lifetime via the following equation (Rumsey et al., 1988):

$$Pmv{O}_2=[{\tau }^{\circ }∕\tau -1]∕(k_Q\times {\tau }^{\circ })$$

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 R2, k_Q is 409 mmHg⁻¹·s⁻¹ and τ^○ is 601 μs (Lo et al., 1997) 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 (Rumsey et al., 1988; Lo et al., 1997).

The R2 phosphorescent probe binds to albumin, and consequently, is uniformly distributed throughout the plasma. A previous study from our laboratory investigated systematically the compartmentalization of R2 and confirmed that it remains within the microvasculature of exposed muscle over the duration considered in the present experiments, thereby ensuring a valid PmvO₂ measurement (Poole et al., 2004).

Curve-fitting of the measured PmvO₂ 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:

• One component: PmvO_{2 (t)} = PmvO_{2 (BL)} − Δ PmvO₂(1 − e^{−(t − TD)/τ)}

• Two component: PmvO_{2 (t)} = PmvO_{2 (BL)} − Δ₁ PmvO₂(1 − e^{−(t − TD₁)/τ₁}) + Δ₂PmvO₂(1 – e^{−(t − TD₂)/τ₂)}

where PmvO_2(t) represents the PmvO₂ at any given time t, PmvO_{2 (BL)} corresponds to the pre-contracting resting baseline PmvO₂, 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. 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 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 PmvO₂ fall following contractions onset was normalized to τ₁ (Δ₁PmvO₂/τ₁) to provide an index of the relative rate of fall. Additionally, the time taken to reach 63% of the initial PmvO₂ fall was determined independently from the modeling procedures (T₆₃) to ensure appropriateness of the model fits. Specifically, the raw PmvO₂ data were interpolated, and the time coinciding with 63% of the total amplitude (Δtotal PmvO₂) was determined.

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

A pre-supplementation period blood sample was taken from all rats to assess plasma [NO₃⁻]. In accordance with IACUC guidelines these pre-supplementation blood samples (i.e., when the rats were not instrumented with catheters) were taken from the sub-orbital plexus using a glass capillary pipette. Approximately ~0.8 ml of blood was sampled and centrifuged in heparnized tubes at 6000 g at 4°C for 6 minutes, plasma was extracted and frozen immediately at −80°C for later analysis. This required sampling strategy precluded accurate determination of pre-supplementation plasma [NO₂⁻] because of hemolysis in some samples.

Post-supplementation blood samples were collected following surgical instrumentation via the caudal artery catheter to assess 1) plasma [NO₃⁻] and [NO₂⁻] and 2) pH, PO₂, and %O₂ saturation. For plasma [NO₃⁻] and [NO₂⁻], ~0.8 ml of blood was drawn into heparinized tubes, rapidly centrifuged and frozen as described above. A second ~0.3 ml blood sample was drawn and analyzed for 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].

2.7 Statistical analysis

Data are presented as mean±SEM. Results were compared with mixed 2-way ANOVAs (plasma [NO₃⁻], MAP, and HR) with Student-Newman-Keuls post hoc tests where appropriate or unpaired Student’s t-tests ([NO₂⁻], blood gases, PmvO₂ kinetics parameters). Significance was accepted at p<0.05.

3. Results

3.1 Plasma [NO₃⁻] and [NO₂⁻]

Rats receiving BR had significantly higher plasma [NO₃⁻] and [NO₂⁻] when compared to control (Figure 1).

Figure 1.

Top panel: Pre- and post-supplemention plasma [NO₃⁻] for control and BR rats. Bottom panel: Post-supplemention plasma [NO₂⁻] for control and BR rats. *p<0.05 versus control.

3.2 MAP, HR, and arterial blood gasses

There were no differences in MAP or HR between control and BR rats prior to contractions or during the contracting steady-state (Table 1). Arterial blood pH (control: 7.50±0.01, BR: 7.51±0.01, p>0.05), PO₂ (control: 101±3, BR: 94±3 mmHg, p>0.05), and O₂ saturation (control: 98±1, BR: 98±1% mmHg, p>0.05) were not different between control and BR rats.

Table 1. MAP and HR prior to and during the steady-state of electrically-induced muscle contractions for control and BR rats.

	Control	BR
Rest (pre-contractions)
MAP (mmHg)	123±8	110±9
HR (bpm)	360±11	342±22
Contracting steady-state
MAP (mmHg)	123±8	111±7
HR (bpm)	360±11	349±17

Values are mean±SEM. There were no differences within or between groups (p>0.05).

3.3 PmvO₂ parameters

Representative raw PmvO₂ profiles, their model fits, and residuals for control and BR rats are presented in Figure 2. The responses were adequately fit by a one-component model in 2 of 10 control rats and 5 of 10 BR rats. The more complex two-component model was indicated in 8 of 10 control and 5 of 10 BR rats. The r² (control: 0.99 ± 0.01, BR: 0.98 ± 0.01) and sum of squared residuals (control: 20.2 ± 3.1, BR: 19.2 ± 3.7 mmHg) for both groups suggested the appropriateness of the model fits. Table 2 presents the average PmvO₂ kinetics parameters. There were no differences in the PmvO_2(BL). However, following the onset of contractions BR resulted in a longer TD₁, smaller first-component and overall (i.e., PmvO₂ baseline minus steady-state, Δ_totalPmvO₂) amplitudes, and slower PmvO₂ kinetics (i.e., longer MRT₁ and lower ΔPmvO₂/τ) following the onset of contractions. There were no significant differences in steady-state PmvO₂ during contractions. Importantly, within the control and BR groups the model-dependent MRT₁ and model-independent T₆₃ were not different (Table 2) increasing confidence in the model parameters. This conclusion was also supported by the very small and non-systematic pattern of the residuals of the model fits (Figure 2, middle panel).

Figure 2.

Top panel: Representative PmvO₂ profiles (black lines) and their model fits (gray lines) for one control and one BR rat. Middle panel: PmvO₂ residuals demonstrate excellent model fits. Bottom panel: Absolute PmvO₂ difference (BR-control) for responses shown in top panel. Time “0” represents the onset of contractions. Note greatest effect of BR across the initial transient (i.e. 0-60 s).

Table 2. Microvascular partial pressure of O₂ (PmvO₂) kinetics parameters during contractions for control and BR rats.

	Control	BR
PmvO2(BL), mmHg	30.2±2.0	28.6±2.3
Δ1PmvO2, mmHg	16.8±1.4	12.0±1.5*
Δ2PmvO2, mmHg	4.1±0.7	5.6±1.9
ΔtotalPmvO2, mmHg	13.5±1.4	9.2±1.3*
PmvO2(steady-state), mmHg	17.4±1.6	18.5±1.0
TD1, s	6.9±1.4	11.8±1.8*
TD2, s	46.7±10.2	26.0±3.5
τ1, s	9.9±1.1	12.6±1.9
τ2, s	88.0±16.2	76.6±12.9
MRT1, s	16.8±1.9	24.4±2.7*
T63, s	16.2±1.6	23.8±3.2*
Δ1PmvO2/τ1, mmHg/s	1.9±0.3	1.2±0.2*

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 PmvO₂ data (control: n=8 of 10; BR: n=5 of 10). PmvO₂(BL), pre-contracting PmvO₂; Δ₁PmvO₂, amplitude of the first component; Δ₂PmvO₂, amplitude of the second component; Δ_totalPmvO₂; overall amplitude regardless of one- or two-component model fit; PmvO_{2(steady-state)}, contracting steady-state PmvO₂; TD₁, time delay for the first component; TD₂, time delay for the second component; τ₁, time constant for the first component; τ₂, time constant for the second component; MRT, mean response time describing the overall kinetics response; T₆₃, time to reach 63% of the overall response determined independent of modeling procedures; Δ₁PmvO₂/τ₁, parameter describing the relative rate of PmvO₂ fall.

^*p<0.05 versus control.

4. Discussion

The primary novel finding of the present investigation was that, relative to control, 5 days of BR supplementation elevated plasma [NO₂⁻] and [NO₃⁻] and slowed significantly the PmvO₂ fall during the crucial rest-contraction transient despite no significant steady-state effects in the mixed fiber-type rat spinotrapezius muscle. The slowed PmvO₂ response (i.e. longer time delay, MRT, and slower relative rate of PmvO₂ fall) following the onset of contractions reflects an elevated QO₂/VO₂ ratio within the skeletal muscle microvasculature which serves to increase the pressure head vital for capillary-myocyte O₂ flux and potentially enhance oxidative function. Improvements in metabolic control at the onset of contractions would be expected to reduce glycolytic metabolism dependence, ultimately attenuating the accumulation of fatigue-associated metabolites. Therefore, these results suggest that acute BR supplementation may provide advantageous effects within skeletal muscle with important implications for exercise tolerance in health and disease.

4.1 Effects of BR on the PmvO₂ kinetics profile

The principal novel finding of this investigation was the slower PmvO₂ kinetics in BR supplemented rats. Behnke et al. (2003) found fiber type differences in the PmvO₂ response to electrical stimulation that included a longer time delay, MRT, and reduced rate of PmvO₂ fall at the onset of contractions in soleus (slow) versus peroneal (fast) muscles. Considering that the spinotrapezius muscle consists of predominantly fast-twitch type II muscles (59% type II; Delp & Duan, 1996) it appears that BR supplementation shifts the PmvO₂ profile, at least during the transient, to resemble the characteristic response seen in muscles composed of type I fibers. A recent study by Hirai et al. (2012) reported a similar slowing of the PmvO₂ kinetics in rats that completed an 8-week exercise training program compared to sedentary rats. The slower PmvO₂ fall in that report following exercise training was mediated by elevations in NO bioavailability (Hirai et al. 2012). In this regard it quite remarkable that a relatively short term (5 days) dose of a non-pharmacological aid mimics the effects of 8-weeks of exercise training with regards to effects on the microvascular oxygenation profile during muscle contractions.

Given our recent report that BR supplementation raises exercising muscle steady-state QO₂ substantially (Ferguson et al. 2013), the higher QO₂/ṾO₂ ratio seen herein may be due to a faster rate of QO₂ increase following the onset of contractions. In addition, BR supplementation may reduce ṾO₂ due to improvements in mitochondrial and/or muscle contractile efficiency (Larsen et al. 2007; Larsen et al. 2012; Bailey et al. 2009; Bailey et al. 2010; Vanhatalo et al. 2010; Hernandez et al. 2012). These mechanisms may contribute to the slower PmvO₂ kinetics found in the present investigation and could explain, at least in part, the reduced PCr breakdown and improved exercise tolerance following BR supplementation shown by Jones and colleagues (Bailey et al. 2010; Lansley et al. 2011; Vanhatalo et al. 2011). Moreover, elevations in O₂ pressures within the microvasculature reduce PCr breakdown (Haseler et al. 1998, Vanhatalo et al. 2011) and speed PCr recovery in hypoxic conditions (Haseler et al. 1999), which may also help explain the improvements in exercise tolerance seen in peripheral artery disease patients following acute BR supplementation (Kenjale et al. 2011). In this regard, BR supplementation may have vital implications for other diseases hallmarked by reduced QO₂ and elevated metabolic perturbation during exercise (e.g., chronic heart failure, reviewed by Poole et al. 2012) and could emerge as a non-pharmacological therapeutic modality used to increase adherence to, and efficacy of, cardiac rehabilitation programs in which exercise is a primary component.

In the present investigation we hypothesized initially that BR supplementation would elevate PmvO₂ not only across the rest-contraction transient but also during the contracting steady-state. However, no such steady-state PmvO₂ elevation was evident. This may be due, in part, to the apparent fiber-type specific effects of NO₃⁻ on skeletal muscle vascular control (Ferguson et al. 2013) and contractile function (Hernandez et al. 2012). Specifically, it has been demonstrated that BR supplementation elevates blood flow explicitly to exercising muscles and muscle parts composed predominantly of fast-twitch type IIb+d/x fibers (Ferguson et al. 2013). Moreover, a fiber-type selective effect also exists whereby NO₃⁻ supplementation augments Ca²⁺ handling and rate of force development in isolated mouse fast-twitch but not slow-twitch muscle (Hernandez et al. 2012). Considering that the spinotrapezius muscle utilized presently is composed of approximately 52% type IIb+d/x fibers (Delp & Duan, 1996) and that the elevations in blood flow demonstrated by Ferguson et al. (2013) were present only in muscles and muscle parts comprised of ≥66% type IIb+d/x muscle fibers it is possible that the spinotrapezius lacks the fiber-type composition necessary to illustrate the effects of BR supplementation on contracting steady-state PmvO₂. However, it is important not to understate the improved PmvO₂ kinetics seen herein given that humans and animals are rarely at a metabolic steady-state, but instead transition frequently among differing metabolic rates (reviewed by Jones et al. 2011; Poole & Jones, 2012). Therefore, the slowed PmvO₂ kinetics elicited by BR may underlie mechanistically the faster pulmonary ṾO₂ kinetics evident following BR consumption in certain circumstances (i.e. advanced age, Kelly et al. 2012).

4.2 Experimental considerations and future directions

The current experimental model differs from voluntary dynamic exercise in several respects, the most pertinent of these relates to the different skeletal muscle recruitment patterns evoked by electrical stimulation versus voluntary muscle contraction (Gollnick et al. 1974). This is particularly important when considering the fiber-type disparities discussed above. Namely, by using electrical stimulation it is presumed that all motor units are activated within a given stimulation field thereby contracting muscle fibers across the spectrum of both slow- and fast-twitch types. Therefore, the current experimental preparation maximized the opportunity to reveal any potential fiber-type specific effects of BR supplementation within the mixed fiber-type spinotrapezius muscle. In this regard, investigation into the effects of BR supplementation on the PmvO₂ profile in a muscle composed of primarily type IIb+d/x fibers are warranted and may unveil additional and/or greater effects both during the contracting steady state and across the rest-contraction transient. Moreover, blood flow measurements would allow calculation of muscle oxygen consumption using the Fick relationship (McDonough et al. 2001) thereby providing further insights into the potential metabolic effects of NO₃⁻ supplementation.

4.3 Conclusions

BR supplementation for 5 days slowed substantially (~45%) the PmvO₂ fall across the crucial rest-contraction transient with BR rats eliciting a longer time delay, MRT and blunted rate of PmvO₂ fall. However, in this mixed fiber-type muscle, steady-state PmvO₂ was not elevated significantly. This may be due to the fiber-type composition of the spinotrapezius muscle in that an effect may potentially be evident in muscles comprised of a greater proportion of fast-twitch fibers. The slowed kinetics seen following BR supplementation reflects an improved ability to increase O₂ delivery relative to metabolic demand (i.e. raise QO₂/ṾO₂ ratio) following the onset of muscle contractions. Muscle QO₂/ṾO₂ matching is compromised in multiple disease states (e.g. chronic heart failure, PAD) exacerbating metabolic perturbations and sowing the seeds for exercise intolerance. Consequently, these results provide compelling evidence that BR supplementation provides advantageous effects on skeletal muscle vascular and metabolic function during exercise.

Highlights.

• Dietary NO₃⁻ (via beetroot juice, BR) augments exercising muscle blood flow

• We examined the effects of BR on contracting rat muscle microvascular O₂ pressure (PmvO₂) dynamics

• BR slowed the PmvO₂ fall across the crucial rest-contractions transition

• There was no effect of BR on contracting steady-state PmvO₂

• This elevated O₂ driving pressure may improve metabolic control during exercise