Trenton D. Colburn

Skeletal Muscle Vascular Control During Exercise: Impact of Nitrite Infusion During Nitric Oxide Synthase Inhibition in Healthy Rats.

The nitric oxide synthase (NOS)-independent pathway of nitric oxide (NO) production in which nitrite (NO2−) is reduced to NO may have therapeutic applications for those with cardiovascular diseases in which the NOS pathway is downregulated. We tested the hypothesis that NO2− infusion would reduce mean arterial pressure (MAP) and increase skeletal muscle blood flow (BF) and vascular conductance (VC) during exercise in the face of NOS blockade via L-NAME. Following infusion of L-NAME (10 mg kg−1, L-NAME), male Sprague-Dawley rats (3-6 months, n = 8) exercised without NG-nitro-L arginine methyl ester (L-NAME) and after infusion of sodium NO2− (7 mg kg−1; L-NAME + NO2−). MAP and hindlimb skeletal muscle BF (radiolabeled microsphere infusions) were measured during submaximal treadmill running (20 m min−1, 5% grade). Across group comparisons were made with a published control data set (n = 11). Relative to L-NAME, NO2− infusion significantly reduced MAP (P < 0.03). The lower MAP in L-NAME+NO2− was not different from healthy control animals (control: 137 ± 3 L-NAME: 157 ± 7, L-NAME + NO2−: 136 ± 5 mm Hg). Also, NO2− infusion significantly increased VC when compared to L-NAME (P < 0.03), ultimately negating any significant differences from control animals (control: 0.78 ± 0.05, L-NAME: 0.57 ± 0.03, L-NAME + NO2−; 0.69 ± 0.04 mL min−1 100 g−1 mm Hg−1) with no apparent fiber-type preferential effect. Overall, hindlimb BF was decreased significantly by L-NAME; however, in L-NAME + NO2−, BF improved to a level not significantly different from healthy controls (control: 108 ± 8, L-NAME: 88 ± 3, L-NAME + NO2−: 94 ± 6 mL min−1 100 g−1, P = 0.38 L-NAME vs L-NAME + NO2−). Individuals with diseases that impair NOS activity, and thus vascular function, may benefit from a NO2−-based therapy in which NO bioavailability is elevated in an NOS-independent manner.

Dietary nitrate supplementation: impact on skeletal muscle vascular control in exercising rats with chronic heart failure

Chronic heart failure (CHF) results in central and peripheral derangements that ultimately reduce skeletal muscle O2 delivery and impair exercise tolerance. Dietary nitrate (NO3 (-)) supplementation improves skeletal muscle vascular function and tolerance to exercise. We tested the hypothesis that NO3 (-) supplementation would elevate exercising skeletal muscle blood flow (BF) and vascular conductance (VC) in CHF rats. Myocardial infarction (MI) was induced (coronary artery ligation) in young adult male rats. After 21 days of recovery, rats randomly received 5 days of NO3 (-)-rich beetroot juice (CHF + BR, n = 10) or a placebo (CHF, n = 10). Mean arterial pressure (carotid artery catheter) and skeletal muscle BF (radiolabeled microspheres) were measured during treadmill exercise (20 m/min, 5% grade). CHF-induced dysfunction, as determined by myocardial infarction size (29 ± 3% and 33 ± 4% in CHF and CHF + BR, respectively) and left ventricular end-diastolic pressure (18 ± 2 and 18 ± 2 mmHg in CHF and CHF + BR, respectively), and exercising mean arterial pressure (131 ± 3 and 128 ± 4 mmHg in CHF and CHF + BR, respectively) were not different (P > 0.05) between groups. Total exercising hindlimb skeletal muscle BF (95 ± 5 and 116 ± 9 ml·min(-1)·100 g(-1) in CHF and CHF + BR, respectively) and VC (0.75 ± 0.05 and 0.90 ± 0.05 ml·min(-1)·100 g(-1)·mmHg(-1) in CHF and CHF + BR, respectively) were 22% and 20% greater in BR-supplemented rats, respectively (P < 0.05). During exercise, BF in 9 and VC in 10 hindlimb muscles and muscle portions were significantly greater in the CHF + BR group. These results provide strong evidence that dietary NO3 (-) supplementation improves skeletal muscle vascular function during exercise in rats with CHF and, thus, support the use of BR as a novel therapeutic modality for the treatment of CHF.

Effect of sodium nitrite on local control of contracting skeletal muscle microvascular oxygen pressure in healthy rats.

Exercise intolerance characteristic of diseases such as chronic heart failure (CHF) and diabetes is associated with reduced nitric oxide (NO) bioavailability from nitric oxide synthase (NOS), resulting in an impaired microvascular O2 driving pressure (Po2mv; O2 delivery/O2 utilization) and metabolic control. Infusions of the potent NO donor sodium nitroprusside augment NO bioavailability yet decrease mean arterial pressure (MAP) thereby reducing its potential efficacy for patient populations. To eliminate or reduce hypotensive sequelae, [Formula: see text] was superfused onto the spinotrapezius muscle. It was hypothesized that local [Formula: see text] administration would elevate resting Po2mv and slow Po2mv kinetics [increased time constant (τ) and mean response time (MRT)] following the onset of muscle contractions without decreasing MAP. In 12 anesthetized male Sprague-Dawley rats, Po2mv of the circulation-intact spinotrapezius muscle was measured by phosphorescence quenching during 180 s of electrically induced twitch contractions (1 Hz) before and after superfusion of sodium nitrite (NaNO2 30 mM). [Formula: see text] superfusion elevated resting Po2mv (control: 28.4 ± 1.1 vs. [Formula: see text]: 31.6 ± 1.2 mmHg; P ≤ 0.05), τ (control: 12.3 ± 1.2 vs. [Formula: see text]: 19.7 ± 2.2 s; P ≤ 0.05), and MRT (control: 19.3 ± 1.9 vs. [Formula: see text]: 25.6 ± 3.3 s; P ≤ 0.05). Importantly, these effects occurred in the absence of any reduction in MAP (103 ± 4 vs. 105 ± 4 mmHg, pre- and postsuperfusion respectively; P > 0.05). These results indicate that [Formula: see text] supplementation delivered to the muscle directly through [Formula: see text] superfusion enhances the blood-myocyte oxygen driving pressure without compromising MAP at rest and following the onset of muscle contraction. This strategy has substantial clinical utility for a range of ischemic conditions. NEW & NOTEWORTHY Ischemic conditions as diverse as chronic heart failure (CHF) and frostbite inflict tissue damage via inadequate O2 delivery. Herein we demonstrate that direct application of sodium nitrite enhances the O2 supply-O2 demand relationship, raising microvascular O2 pressure in healthy skeletal muscle. This therapeutic action of nitrite-derived nitric oxide occurred without inducing systemic hypotension and has the potential to relieve focal ischemia and preserve tissue vitality by enhancing O2 delivery.

Skeletal muscle microvascular and interstitial from rest to contractions

Oxygen pressure gradients across the microvascular walls are essential for oxygen diffusion from blood to tissue cells. At any given flux, the magnitude of these transmural gradients is proportional to the local resistance. The greatest resistance to oxygen transport into skeletal muscle is considered to reside in the short distance between red blood cells and myocytes. Although crucial to oxygen transport, little is known about transmural pressure gradients within skeletal muscle during contractions. We evaluated oxygen pressures within both the skeletal muscle microvascular and interstitial spaces to determine transmural gradients during the rest–contraction transient in anaesthetized rats. The significant transmural gradient observed at rest was sustained during submaximal muscle contractions. Our findings support that the blood–myocyte interface provides substantial resistance to oxygen diffusion at rest and during contractions and suggest that modulations in microvascular haemodynamics and red blood cell distribution constitute primary mechanisms driving increased transmural oxygen flux with contractions.

Vascular KATP channels mitigate severe muscle O2 delivery-utilization mismatch during contractions in chronic heart failure rats.

The vascular ATP-sensitive K+ (KATP) channel is a mediator of skeletal muscle microvascular oxygenation (PO2mv) during contractions in health. We tested the hypothesis that KATP channel function is preserved in chronic heart failure (CHF) and therefore its inhibition would reduce PO2mv and exacerbate the time taken to reach the PO2mv steady-state during contractions of the spinotrapezius muscle. Moreover, we hypothesized that subsequent KATP channel activation would oppose the effects of this inhibition. Muscle PO2mv (phosphorescence quenching) was measured during 180s of 1-Hz twitch contractions (∼6V) under control, glibenclamide (GLI, KATP channel antagonist; 5mg/kg) and pinacidil (PIN, KATP channel agonist; 5mg/kg) conditions in 16 male Sprague-Dawley rats with CHF induced via myocardial infarction (coronary artery ligation, left ventricular end-diastolic pressure: 18±1mmHg). GLI reduced baseline PO2mv (control: 28.3±0.9, GLI: 24.8±1.0mmHg, p<0.05), lowered mean PO2mv (average PO2mv during the overall time taken to reach the steady-state; control: 20.6±0.6, GLI: 17.6±0.3mmHg, p<0.05), and slowed the attainment of steady-state PO2mv (overall mean response time; control: 66.1±10.2, GLI: 93.6±7.8s, p<0.05). PIN opposed these effects on the baseline PO2mv, mean PO2mv and time to reach the steady-state PO2mv (p<0.05 for all vs. GLI). Inhibition of KATP channels exacerbates the transient mismatch between muscle O2 delivery and utilization in CHF rats and this effect is opposed by PIN. These data reveal that the KATP channel constitutes one of the select few well-preserved mechanisms of skeletal muscle microvascular oxygenation control in CHF.

Sex and nitric oxide bioavailability interact to modulate interstitial PO2 in healthy rat skeletal muscle

Premenopausal women express reduced blood pressure and risk of cardiovascular disease relative to age-matched men. This purportedly relates to elevated estrogen levels increasing nitric oxide synthase (NOS) activity and NO-mediated vasorelaxation. We tested the hypotheses that female rat skeletal muscle would: 1) evince a higher O2 delivery-to-utilization ratio (Q̇o2/V̇o2) during contractions; and 2) express greater modulation of Q̇o2/V̇o2 with changes to NO bioavailability compared with male rats. The spinotrapezius muscle of Sprague-Dawley rats (females = 8, males = 8) was surgically exposed and electrically-stimulated (180 s, 1 Hz, 6 V). OxyphorG4 was injected into the muscle and phosphorescence quenching employed to determine the temporal profile of interstitial Po2 (Po2is, determined by Q̇o2/V̇o2). This was performed under three conditions: control (CON), 300 µM sodium nitroprusside (SNP; NO donor), and 1.5 mM Nω-nitro-l-arginine methyl ester (l-NAME; NOS blockade) superfusion. No sex differences were found for the Po2is kinetics parameters in CON or l-NAME ( P > 0.05), but females elicited a lower baseline following SNP (males 42 ± 3 vs. females 36 ± 2 mmHg, P < 0.05). Females had a lower ΔPo2is during contractions following SNP (males 22 ± 3 vs. females 17 ± 2 mmHg, P < 0.05), but there were no sex differences for the temporal response to contractions ( P > 0.05). The total NO effect (SNP minus l-NAME) on Po2is was not different between sexes. However, the spread across both conditions was shifted to a lower absolute range for females (reduced SNP baseline and greater reduction following l-NAME). These data support that females have a greater reliance on basal NO bioavailability and males have a greater responsiveness to exogenous NO and less responsiveness to reduced endogenous NO. NEW & NOTEWORTHY Interstitial Po2 (Po2is; determined by O2 delivery-to-utilization matching) plays an important role for O2 flux into skeletal muscle. We show that both sexes regulate Po2is at similar levels at rest and during skeletal muscle contractions. However, modulating NO bioavailability exposes sex differences in this regulation with females potentially having a greater reliance on basal NO bioavailability and males having a greater responsiveness to exogenous NO and less responsiveness to reduced endogenous NO.

Skeletal muscle interstitial O2 pressures: bridging the gap between the capillary and myocyte

The oxygen transport pathway from air to mitochondria involves a series of transfer steps within closely integrated systems (pulmonary, cardiovascular, and tissue metabolic). Small and finite O2 stores in most mammalian species require exquisitely controlled changes in O2 flux rates to support elevated ATP turnover. This is especially true for the contracting skeletal muscle where O2 requirements may increase two orders of magnitude above rest. This brief review focuses on the mechanistic bases for increased microvascular blood‐myocyte O2 flux (V̇O2) from rest to contractions. Ficks law dictates that V̇O2 elevations driven by muscle contractions are produced by commensurate changes in driving force (ie, O2 pressure gradients; ΔPO2) and/or effective diffusing capacity (DO2). While previous evidence indicates that increased DO2 helps modulate contracting muscle O2 flux, up until recently the role of the dynamic ΔPO2 across the capillary wall was unknown. Recent phosphorescence quenching investigations of both microvascular and novel interstitial PO2 kinetics in health have resolved an important step in the O2 cascade between the capillary and myocyte. Specifically, the significant transmural ΔPO2 at rest was sustained (but not increased) during submaximal contractions. This supports the contention that the blood‐myocyte interface provides a substantial effective resistance to O2 diffusion and underscores that modulations in erythrocyte hemodynamics and distribution (DO2) are crucial to preserve the driving force for O2 flux across the capillary wall (ΔPO2) during contractions. Investigation of the O2 transport pathway close to muscle mitochondria is key to identifying disease mechanisms and develop therapeutic approaches to ameliorate dysfunction and exercise intolerance.