Key points• Inorganic nitrate (NO 3 − ) supplementation with beetroot juice (BR) in humans lowers blood pressure and the O 2 cost of exercise and may improve exercise tolerance following its reduction to nitrite (NO 2 − ) and nitric oxide (NO).• The effect of inorganic NO 3 − supplementation with BR on skeletal muscle blood flow (BF) and vascular conductance (VC) within and among locomotory muscles during exercise is unknown.• Inorganic NO 3 − supplementation with BR in rats resulted in lower exercising mean arterial pressure, lower blood [lactate], and higher total skeletal muscle hindlimb BF and VC during submaximal treadmill running.• The greater BF and VC was found in muscles and muscle parts containing primarily type IIb + d/x muscle fibres.• These data demonstrate that inorganic NO 3 − supplementation improves vascular control and elevates skeletal muscle O 2 delivery during exercise predominantly in fast-twitch type II muscles, and provide a potential mechanism by which NO 3 − supplementation improves metabolic control. Abstract Dietary nitrate (NO 3− ) supplementation, via its reduction to nitrite (NO 2 − ) and subsequent conversion to nitric oxide (NO) and other reactive nitrogen intermediates, reduces blood pressure and the O 2 cost of submaximal exercise in humans. Despite these observations, the effects of dietary NO 3 − supplementation on skeletal muscle vascular control during locomotory exercise remain unknown. We tested the hypotheses that dietary NO 3 − supplementation via beetroot juice (BR) would reduce mean arterial pressure (MAP) and increase hindlimb muscle blood flow in the exercising rat. Male Sprague-Dawley rats (3-6 months) were administered either NO 3 − (via beetroot juice; 1 mmol kg −1 day −1 , BR n = 8) or untreated (control, n = 11) tap water for 5 days. MAP and hindlimb skeletal muscle blood flow and vascular conductance (radiolabelled microsphere infusions) were measured during submaximal treadmill running (20 m min −1 , 5% grade). BR resulted in significantly lower exercising MAP (control: 137 ± 3, BR: 127 ± 4 mmHg, P < 0.05) and blood [lactate] (control: 2.6 ± 0.3, BR: 1.9 ± 0.2 mM, P < 0.05) compared to control. Total exercising hindlimb skeletal muscle blood flow (control: 108 ± 8, BR: 150 ± 11 ml min −1 (100 g) −1 , P < 0.05) and vascular conductance (control: 0.78 ± 0.05, BR: 1.16 ± 0.10 ml min −1 (100 g) −1 mmHg −1 , P < 0.05) were greater in rats that received BR compared to control. The relative differences in blood flow and vascular conductance for the 28 individual hindlimb muscles and muscle parts correlated positively with their percentage type IIb + d/x muscle fibres (blood flow: r = 0.74, vascular conductance: r = 0.71, P < 0.01 for both). These data support the hypothesis that NO 3 − supplementation improves vascular control and elevates skeletal muscle O 2 delivery during exercise predominantly in fast-twitch type II muscles, and provide a potential mechanism by which NO 3 − supplementation improves metabolic control.

The capillary bed constitutes a vast surface facilitating exchange of O2, substrates and metabolites between blood and organs. In contracting skeletal muscle capillary blood flow and O2 diffusing capacity as well as O2 flux may increase two orders of magnitude above resting. Chronic diseases such as heart failure, diabetes and also sepsis impair these processes leading to compromised energetic, metabolic and ultimately contractile function. Among researchers seeking to understand blood-myocyte exchange in health and the bases for dysfunction in disease there is a fundamental disconnect between microcirculation specialists and many physiologists and physiologist clinicians. Whereas the former observe capillaries and capillary function directly (muscle intravital microscopy) the latter generally use indirect methodologies (e.g., post-mortem tissue analysis, 1-methyl xanthine, contrast enhanced ultrasound, permeability surface area product) and interpret their findings based upon August Krogh’s observations made nearly a century ago. “Kroghian” theory holds that only a small fraction of capillaries support red blood cell (RBC) flux in resting muscle leaving the vast majority to be “recruited” (i.e., initiate RBC flux) during contractions which would constitute the basis for increasing capillary exchange surface area and reducing capillary-mitochondrial diffusion distances. Experimental techniques each have their strengths and weaknesses and often the correct or complete answer to a problem emerges from integration across multiple technologies. Today Krogh’s entrenched “capillary recruitment” hypothesis is challenged by direct observations of capillaries in contracting muscle; something that he and his colleagues could not do. Moreover, in the peer-reviewed scientific literature, application of a range of contemporary physiological technologies including intravital microscopy of contracting muscle, magnetic resonance and near infrared spectroscopy and phosphorescence quenching combined with elegant in situ and in vivo models suggest that the role of the capillary bed, at least in contracting muscle, is subserved without the necessity for de novo capillary recruitment of previously non-flowing capillaries. When viewed within the context of the capillary recruitment hypothesis, this evidence casts serious doubt on the interpretation of those data that are based upon Kroghian theory and indirect methodologies. Thus today a wealth of evidence calls for a radical revision of blood-muscle exchange theory to one in which most capillaries support RBC flux at rest and, during contractions, capillary surface area is “recruited” along the length of previously flowing capillaries. This occurs, in part, by elevating capillary hematocrit and extending the length of the capillary available for blood-myocyte exchange (i.e., longitudinal recruitment). Our understanding of blood-myocyte O2 and substrate/metabolite exchange in health and the mechanistic bases for dysfunction in disease demands no less.

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.

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 trueV.O2 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.

Exercise training induces multiple adaptations within skeletal muscle that may improve local O(2) delivery-utilization matching (i.e., Po(2)mv). We tested the hypothesis that increased nitric oxide (NO) function is intrinsic to improved muscle Po(2)mv kinetics from rest to contractions after exercise training. Healthy young Sprague-Dawley rats were assigned to sedentary (n = 18) or progressive treadmill exercise training (n = 10; 5 days/wk, 6-8 wk, final workload of 60 min/day at 35 m/min, -14% grade) groups. Po(2)mv was measured via phosphorescence quenching in the spinotrapezius muscle at rest and during 1-Hz twitch contractions under control (Krebs-Henseleit solution), sodium nitroprusside (SNP, NO donor; 300 μM), and N(G)-nitro-L-arginine methyl ester (l-NAME, nonspecific NO synthase blockade; 1.5 mM) superfusion conditions. Exercise-trained rats had greater peak oxygen uptake (Vo(2 peak)) than their sedentary counterparts (81 ± 1 vs. 72 ± 2 ml · kg(-1) · min(-1), respectively; P < 0.05). Exercise-trained rats had significantly slower Po(2)mv fall throughout contractions (τ(1); time constant for the first component) during control (sedentary: 8.1 ± 0.6; trained: 15.2 ± 2.8 s). Compared with control, SNP slowed τ(1) to a greater extent in sedentary rats (sedentary: 38.7 ± 5.6; trained: 26.8 ± 4.1 s; P > 0.05) whereas l-NAME abolished the differences in τ(1) between sedentary and trained rats (sedentary: 12.0 ± 1.7; trained: 11.2 ± 1.4 s; P < 0.05). Our results indicate that endurance exercise training leads to greater muscle microvascular oxygenation across the metabolic transient following the onset of contractions (i.e., slower Po(2)mv kinetics) partly via increased NO-mediated function, which likely constitutes an important mechanism for training-induced metabolic adaptations.

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.

These data demonstrate important physiological roles for nNOS-derived NO during contractions in healthy rat skeletal muscle and implicate maladaptations in nNOS function in pathological conditions associated with reduced NO bioavailability.

Chronic heart failure (CHF) impairs nitric oxide (NO)-mediated regulation of skeletal muscle O2 delivery-utilization matching such that microvascular oxygenation falls faster (i.e., speeds PO2mv kinetics) during increases in metabolic demand. Conversely, exercise training improves (slows) muscle PO2mv kinetics following contractions onset in healthy young individuals via NO-dependent mechanisms. We tested the hypothesis that exercise training would improve contracting muscle microvascular oxygenation in CHF rats partly via improved NO-mediated function. CHF rats (left ventricular end-diastolic pressure = 17 ± 2 mmHg) were assigned to sedentary (n = 11) or progressive treadmill exercise training (n = 11; 5 days/wk, 6-8 wk, final workload of 60 min/day at 35 m/min; -14% grade downhill running) groups. PO2mv was measured via phosphorescence quenching in the spinotrapezius muscle at rest and during 1-Hz twitch contractions under control (Krebs-Henseleit solution), sodium nitroprusside (SNP; NO donor; 300 μM), and N(G)-nitro-l-arginine methyl ester (L-NAME, nonspecific NO synthase blockade; 1.5 mM) superfusion conditions. Exercise-trained CHF rats had greater peak oxygen uptake and spinotrapezius muscle citrate synthase activity than their sedentary counterparts (p < 0.05 for both). The overall speed of the PO2mv fall during contractions (mean response time; MRT) was slowed markedly in trained compared with sedentary CHF rats (sedentary: 20.8 ± 1.4, trained: 32.3 ± 3.0 s; p < 0.05), and the effect was not abolished by L-NAME (sedentary: 16.8 ± 1.5, trained: 31.0 ± 3.4 s; p > 0.05). Relative to control, SNP increased MRT in both groups such that trained CHF rats had slower kinetics (sedentary: 43.0 ± 6.8, trained: 55.5 ± 7.8 s; p < 0.05). Improved NO-mediated function is not obligatory for training-induced improvements in skeletal muscle microvascular oxygenation (slowed PO2mv kinetics) following contractions onset in rats with CHF.