Daniel M. Hirai

Impact of dietary nitrate supplementation via beetroot juice on exercising muscle vascular control in rats

•  Inorganic nitrate (NO3−) supplementation with beetroot juice (BR) in humans lowers blood pressure and the O2 cost of exercise and may improve exercise tolerance following its reduction to nitrite (NO2−) and nitric oxide (NO). •  The effect of inorganic NO3− supplementation with BR on skeletal muscle blood flow (BF) and vascular conductance (VC) within and among locomotory muscles during exercise is unknown. •  Inorganic NO3− 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 NO3− supplementation improves vascular control and elevates skeletal muscle O2 delivery during exercise predominantly in fast‐twitch type II muscles, and provide a potential mechanism by which NO3− supplementation improves metabolic control.

Critical speed in the rat: implications for hindlimb muscle blood flow distribution and fibre recruitment

Critical speed (CS) constitutes an important metabolic and performance demarcator. However, active skeletal muscle blood flow distribution specifically surrounding CS remains unknown. We tested the hypotheses that CS could be accurately determined in the running rat and that measurement of hindlimb inter‐ and intramuscular blood flow below and above CS would support that the greatest muscle fibre recruitment above, relative to below, CS occurs in the predominantly glycolytic muscles. Seven male Sprague–Dawley rats performed five constant‐speed tests to exhaustion at speeds between 95 and 115% of the speed that elicited to determine CS. Subsequent constant‐speed tests were performed at speeds incrementally surrounding CS to determine time to exhaustion, , and hindlimb muscle blood flow distribution. Speed and time to exhaustion conformed to a hyperbolic relationship (r2= 0.92 ± 0.03) which corresponded to a linear 1/time function (r2= 0.93 ± 0.02) with a CS of 48.6 ± 1.0 m min−1. Time to exhaustion below CS was ∼5× greater (P < 0.01) than that above. Below CS stabilized at a submaximal value (58.5 ± 2.5 ml kg−1 min−1) whereas above CS (81.7 ± 2.5 ml kg−1 min−1) increased to (84.0 ± 1.8 ml kg−1 min−1, P > 0.05 vs. above CS). The 11 individual muscles or muscle parts that evidenced the greatest blood flow increases above, relative to below, CS were composed of ≥69% Type IIb/d/x muscle fibres. Moreover, there was a significant correlation (P < 0.05, r= 0.42) between the increased blood flow above expressed relative to below CS and the percentage Type IIb/d/x fibres found in the individual muscles or muscle parts. These data validate the powerful CS construct in the rat and identify that running above CS, relative to below CS, incurs disproportionate blood flow increases (indicative of recruitment) in predominantly highly glycolytic muscle fibres.

Dynamics of Muscle Microcirculatory and Blood-myocyte O2 Flux During Contractions

The O2 requirements of contracting skeletal muscle may increase 100‐fold above rest. In 1919, August Krogh’s brilliant insights recognized the capillary as the principal site for this increased blood–myocyte O2 flux. Based on the premise that most capillaries did not sustain RBC flux at rest, Krogh proposed that capillary recruitment [i.e. initiation of red blood cell (RBC) flux in previously non‐flowing capillaries] increased the capillary surface area available for O2 flux and reduced mean capillary‐to‐mitochondrial diffusion distances. More modern experimental approaches reveal that most muscle capillaries may support RBC flux at rest. Thus, rather than contraction‐induced capillary recruitment per se, increased RBC flux and haematocrit within already‐flowing capillaries probably elevate perfusive and diffusive O2 conductances and hence blood–myocyte O2 flux. Additional surface area for O2 exchange is recruited but, crucially, this may occur along the length of already‐flowing capillaries (i.e. longitudinal recruitment). Today, the capillary is still considered the principal site for O2 and substrate delivery to contracting skeletal muscle. Indeed, the presence of very low intramyocyte O2 partial pressures (PO2s) and the absence of intramyocyte PO2 gradients, whilst refuting the relevance of diffusion distances, place an even greater importance on capillary hemodynamics. This emergent picture calls for a paradigm‐shift in our understanding of the function of capillaries by de‐emphasizing de novo‘capillary recruitment’. Diseases such as heart failure impair blood–myocyte O2 flux, in part, by decreasing the proportion of RBC‐flowing capillaries. Knowledge of capillary function in healthy muscle is requisite for identification of pathology and efficient design of therapeutic treatments.

Exercise training in chronic heart failure: improving skeletal muscle O2 transport and utilization

Chronic heart failure (CHF) impairs critical structural and functional components of the O2 transport pathway resulting in exercise intolerance and, consequently, reduced quality of life. In contrast, exercise training is capable of combating many of the CHF-induced impairments and enhancing the matching between skeletal muscle O2 delivery and utilization (Q̇mO2 and V̇mO2 , respectively). The Q̇mO2 /V̇mO2 ratio determines the microvascular O2 partial pressure (PmvO2 ), which represents the ultimate force driving blood-myocyte O2 flux (see Fig. 1). Improvements in perfusive and diffusive O2 conductances are essential to support faster rates of oxidative phosphorylation (reflected as faster V̇mO2 kinetics during transitions in metabolic demand) and reduce the reliance on anaerobic glycolysis and utilization of finite energy sources (thus lowering the magnitude of the O2 deficit) in trained CHF muscle. These adaptations contribute to attenuated muscle metabolic perturbations (e.g., changes in [PCr], [Cr], [ADP], and pH) and improved physical capacity (i.e., elevated critical power and maximal V̇mO2 ). Preservation of such plasticity in response to exercise training is crucial considering the dominant role of skeletal muscle dysfunction in the pathophysiology and increased morbidity/mortality of the CHF patient. This brief review focuses on the mechanistic bases for improved Q̇mO2 /V̇mO2 matching (and enhanced PmvO2 ) with exercise training in CHF with both preserved and reduced ejection fraction (HFpEF and HFrEF, respectively). Specifically, O2 convection within the skeletal muscle microcirculation, O2 diffusion from the red blood cell to the mitochondria, and muscle metabolic control are particularly susceptive to exercise training adaptations in CHF. Alternatives to traditional whole body endurance exercise training programs such as small muscle mass and inspiratory muscle training, pharmacological treatment (e.g., sildenafil and pentoxifylline), and dietary nitrate supplementation are also presented in light of their therapeutic potential. Adaptations within the skeletal muscle O2 transport and utilization system underlie improvements in physical capacity and quality of life in CHF and thus take center stage in the therapeutic management of these patients.

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

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.

Exercise training and muscle microvascular oxygenation: functional role of nitric oxide

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.

Aging alters the contribution of nitric oxide to regional muscle hemodynamic control at rest and during exercise in rats

Advanced age is associated with altered skeletal muscle hemodynamic control during the transition from rest to exercise. This study investigated the effects of aging on the functional role of nitric oxide (NO) in regulating total, inter-, and intramuscular hindlimb hemodynamic control at rest and during submaximal whole body exercise. We tested the hypothesis that NO synthase inhibition (N(G)-nitro-l-arginine methyl ester, l-NAME; 10 mg/kg) would result in attenuated reductions in vascular conductance (VC) primarily in oxidative muscles in old compared with young rats. Total and regional hindlimb muscle VCs were determined via radiolabeled microspheres at rest and during treadmill running (20 m/min, 5% grade) in nine young (6-8 mo) and seven old (27-29 mo) male Fisher 344 × Brown Norway rats. At rest, l-NAME increased mean arterial pressure (MAP) significantly by ∼17% and 21% in young and old rats, respectively. During exercise, l-NAME increased MAP significantly by ∼13% and 19% in young and old rats, respectively. Compared with young rats, l-NAME administration in old rats evoked attenuated reductions in 1) total hindlimb VC during exercise (i.e., down by ∼23% in old vs. 43% in young rats; P < 0.05), and 2) VC in predominantly oxidative muscles both at rest and during exercise (P < 0.05). Our results indicate that the dependency of highly oxidative muscles on NO-mediated vasodilation is markedly diminished, and therefore mechanisms other than NO-mediated vasodilation control the bulk of the increase in skeletal muscle VC during the transition from rest to exercise in old rats. Reduced NO contribution to vasomotor control with advanced age is associated with blood flow redistribution from highly oxidative to glycolytic muscles during exercise.

Effects of neuronal nitric oxide synthase inhibition on resting and exercising hindlimb muscle blood flow in the rat

Nitric oxide (NO) derived from endothelial NO synthase (eNOS) is an integral mediator of vascular control during muscle contractions. However, it is not known whether neuronal NOS (nNOS)‐derived NO regulates tissue hyperaemia in healthy subjects, particularly during exercise. We tested the hypothesis that selective nNOS inhibition would reduce blood flow and vascular conductance (VC) in rat hindlimb locomotor muscle(s), kidneys and splanchnic organs at rest and during dynamic treadmill exercise (20 m min−1, 10% grade). Nineteen male Sprague–Dawley rats (555 ± 23 g) were assigned to either rest (n= 9) or exercise (n= 10) groups. Blood flow and VC were determined via radiolabelled microspheres before and after the intra‐arterial administration of the selective nNOS inhibitor S‐methyl‐l‐thiocitrulline (SMTC, 2.1 ± 0.1 μmol kg−1). Total hindlimb muscle blood flow (control: 20 ± 2 ml min−1 100g−1, SMTC: 12 ± 2 ml min−1 100g−1, P < 0.05) and VC (control: 0.16 ± 0.02 ml min−1 100 g−1 mmHg−1, SMTC: 0.09 ± 0.01 ml min−1 100 g−1 mmHg−1, P < 0.05) were reduced substantially at rest. Moreover, the magnitude of the absolute reduction in blood flow and VC correlated (P < 0.05) with the proportion of oxidative muscle fibres found in the individual muscles or muscle parts of the hindlimb. During exercise, total hindlimb blood flow (control: 108 ± 7 ml min−1 100 g−1, SMTC: 105 ± 8 ml min−1 100 g−1) and VC (control: 0.77 ± 0.06 ml min−1 100g−1 mmHg−1; SMTC: 0.70 ± 0.05 ml min−1 100g−1 mmHg−1) were not different (P > 0.05) between control and SMTC conditions. SMTC reduced (P < 0.05) blood flow and VC at rest and during exercise in the kidneys, adrenals and liver. These results enhance our understanding of the role of NO‐mediated circulatory control by demonstrating that nNOS does not appear to subserve an obligatory role in the exercising muscle hyperaemic response in the rat.

The effects of antioxidants on microvascular oxygenation and blood flow in skeletal muscle of young rats

Alterations of skeletal muscle redox state via antioxidant supplementation have the potential to impact contractile function and vascular smooth muscle tone. The effects of antioxidants on the regulation of muscle O2 delivery–O2 utilization ( ) matching (which sets the microvascular partial pressure of O2; ) in young healthy muscle are not known. Therefore, the purpose of this study was to test the effects of acute antioxidant supplementation on rat spinotrapezius muscle force production, blood flow, and (phosphorescence quenching). Anaesthetized male Fischer 344 × Brown Norway rats (6–8 months old) had their right spinotrapezius muscles either exposed for measurement of blood flow and (n= 13) or exteriorized for measurement of muscle force production (n= 6). Electrically stimulated 1 Hz twitch contractions (∼7–9 V) were elicited for 180 s, and measurements were made before and after acute intra‐arterial antioxidant supplementation (76 mg kg−1 ascorbic acid, 52 mg kg−1 tempol) dissolved in saline and infused over 30 min. The principal effects of antioxidants were a ∼25% decrease (P < 0.05) in contracting spinotrapezius muscle force production concurrent with reductions in muscle blood flow and at rest and during contractions (P < 0.05 for both). Antioxidant supplementation reduced the resting baseline (before, 29.9 ± 1.2 mmHg; after, 25.6 ± 1.3 mmHg; P < 0.05), and this magnitude of depression was sustained throughout the rest‐to‐exercise transition (steady‐state value before, 16.4 ± 0.7 mmHg; after, 13.6 ± 0.9 mmHg; P < 0.05). In addition, the time constant of the decrease was reduced after antioxidant supplementation (before, 23.4 ± 4.3 s; after, 15.6 ± 2.7 s; P < 0.05). These results demonstrate that antioxidant supplementation significantly impacts the control of in young rats at rest and during contractions.

Muscle fibre‐type dependence of neuronal nitric oxide synthase‐mediated vascular control in the rat during high speed treadmill running

•  Neuronal nitric oxide (NO) synthase (nNOS) inhibition does not impact skeletal muscle blood flow or vascular conductance (VC) during low‐speed (20 m min−1) treadmill running. •  This may be due to the fact that low exercise intensities recruit primarily oxidative muscle and that nNOS‐derived NO contributes to vascular control primarily within glycolytic muscle. •  Rats ran in the severe‐intensity domain at 15% above critical speed (an important glycolytic fast‐twitch fibre recruitment boundary in the rat) before and after selective nNOS inhibition with S‐methyl‐l‐thiocitrulline (SMTC). •  SMTC reduced blood flow and VC during supra‐critical speed treadmill running (52.5 ± 1.3 m min−1) with the greatest proportional reductions observed in glycolytic fast‐twitch compared to oxidative slow‐ and fast‐twitch muscle. There were no effects of SMTC on muscle blood flow or VC during low‐speed running (20 m min−1). •  The present data reveal important fibre‐type‐ and exercise intensity‐dependent peripheral vascular effects of nNOS‐derived NO during whole‐body exercise.