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

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.

Abstract

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, $\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$ was superfused onto the spinotrapezius muscle. It was hypothesized that local ${\mathrm{N}\mathrm{O}}_2^{-}$ 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). $\mathrm{N}{\mathrm{O}}_2^{-}$ superfusion elevated resting Po₂mv (control: 28.4 ± 1.1 vs. $\mathrm{N}{\mathrm{O}}_2^{-}$: 31.6 ± 1.2 mmHg; P ≤ 0.05), τ (control: 12.3 ± 1.2 vs. $\mathrm{N}{\mathrm{O}}_2^{-}$: 19.7 ± 2.2 s; P ≤ 0.05), and MRT (control: 19.3 ± 1.9 vs. $\mathrm{N}{\mathrm{O}}_2^{-}$: 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 $\mathrm{N}{\mathrm{O}}_2^{-}$ supplementation delivered to the muscle directly through $\mathrm{N}{\mathrm{O}}_2^{-}$ 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.

sustained muscle contractions require a robust and appropriate hyperemic response that is contingent on arteriolar vasodilation increasing muscle O₂ delivery (Q̇o₂) in proportion to the elevated O₂ demands (V̇o₂). It is also important to recognize that the instantaneous Q̇o₂/V̇o₂ ratio sets microvascular O₂ pressures (Po₂mv) that are crucial for blood-myocyte O₂ flux and also the intracellular Po₂, which influences metabolic control (31, 57).

Within the spectrum of vasoactive mediators for the exercise hyperemia such as ADP, cAMP, and K⁺, nitric oxide (NO) has a key role. Thus blockade of the endogenous endothelial NO synthase (eNOS) and/or neuronal NOS (nNOS) systems reduces exercising muscle(s) blood flow and Q̇o₂ (10, 30), lowering Po₂mv (24) and impairing function. In diseases such as chronic heart failure (CHF), dysfunction of the NOS system reduces NO bioavailability as well as the capability to reduce vascular tone due to decreased arteriolar compliance. There is also a reduced efficacy of the muscle pump that is induced by venous congestion that reduces the muscle hyperemia following the onset of contractions (53). Thus in CHF, Po₂mv is compromised compared with healthy muscle especially in the transient phase immediately following the onset of contractions. This phenomenon slows V̇o₂ kinetics and contributes to the muscle dysfunction characteristic of CHF (17, 23, 28, 46).

Given the reduced capacity for endogenous NO production in disease, possibly as a consequence of tissue hypoxia and elevated reactive oxygen species, which constrain NOS function, there has been substantial interest in providing exogenous NO precursors such as sodium nitroprusside (SNP) and inorganic nitrate/nitrite ($\mathrm{N}{\mathrm{O}}_3^{-}$/$\mathrm{N}{\mathrm{O}}_2^{-}$) (39, 61). A particularly attractive feature of the $\mathrm{N}{\mathrm{O}}_3^{-}$/$\mathrm{N}{\mathrm{O}}_2^{-}$ pathway is that the pathological muscle hypoxia found in CHF and other patients promotes $\mathrm{N}{\mathrm{O}}_2^{-}$ reduction to NO and hence enhances Q̇o₂ locally with the potential to not compromise systemic blood pressure as might be expected for systemic vasodilators such as SNP or hydralazine, for example (58, 59). Recently, $\mathrm{N}{\mathrm{O}}_3^{-}$ supplementation (i.e., beetroot juice) has been advocated as a $\mathrm{N}{\mathrm{O}}_3^{-}$/$\mathrm{N}{\mathrm{O}}_2^{-}$ source that reduces mean arterial pressure, enhances muscle Q̇o₂ and Po₂mv, and improves muscle contractile function and efficiency (21, 22, 35). Unfortunately, after absorption in the gut the $\mathrm{N}{\mathrm{O}}_3^{-}$-$\mathrm{N}{\mathrm{O}}_2^{-}$ reduction requires salivary gland $\mathrm{N}{\mathrm{O}}_3^{-}$ secretion and the participation of commensal bacteria in the oral cavity before resorption into the blood stream as circulating $\mathrm{N}{\mathrm{O}}_2^{-}$. These steps typically take hours to raise plasma [$\mathrm{N}{\mathrm{O}}_2^{-}$] (60) and can be blocked by mouthwash-induced bactericide (41).

Given these limitations the utility of increasing vascular [$\mathrm{N}{\mathrm{O}}_2^{-}$] more directly has been considered (40). However, the results have been controversial with up to 36 µmol/min $\mathrm{N}{\mathrm{O}}_2^{-}$ infused intra-arterially in the brachial artery supplying the forearm of healthy subjects being unable to produce vasodilation (36). Arguing that hypoxia is crucial for the $\mathrm{N}{\mathrm{O}}_2^{-}$-NO reduction Maher et al. (40) subsequently demonstrated that direct arterial infusions into the resting forearm of subjects breathing 12% O₂ induced a robust arterial vasodilation. These low O₂ pressures are precisely the conditions extant in the microvasculature of contracting muscles. Specifically, in the rat during exercise muscle Po₂mv falls to ~20 mmHg in muscles comprised predominantly of slow-twitch fibers or ~10 mmHg in fast-twitch muscles (6, 42). Moreover, we have demonstrated that, when NO bioavailability is reduced by CHF (25) or NOS blockade (20), $\mathrm{N}{\mathrm{O}}_2^{-}$ infusions induce a robust increase in muscle Q̇o₂ that is especially pronounced in fast-twitch muscles. Yet, the effects of $\mathrm{N}{\mathrm{O}}_2^{-}$ on systemic blood pressure have been inconclusive with adults and CHF patients reporting no change (7, 16, 25) while a modest, transient decrease in MAP has been reported in diabetic patients and during L-NAME induced NOS blockade (20, 26).

Because enhanced NO bioavailability has the potential to both elevate Q̇o₂ and reduce V̇o₂, direct measurements of Po₂mv are necessary to evaluate the efficacy of $\mathrm{N}{\mathrm{O}}_2^{-}$ treatment to enhance blood-myocyte O₂ flux. Since changes in systemic blood pressure may influence Po₂mv measurements in the spinotrapezius muscle of the rat, we first hypothesized that directly applying sodium nitrite (NaNO₂) to the muscle via superfusion, when compared with intra-arterial (IA) infusion, would allow systemic pressure to remain stable. Moreover, because V̇o₂ demands change most rapidly within the first minute or so of contractions we argue that assessing the efficacy of $\mathrm{N}{\mathrm{O}}_2^{-}$ to raise Po₂mv across this interval with high temporal fidelity is crucial. We also tested the hypothesis that local $\mathrm{N}{\mathrm{O}}_2^{-}$ superfusion under normoxic conditions would elevate Po₂mv significantly and, importantly, would do so in the absence of systemic hypotension.

METHODS

Three male Sprague-Dawley rats (body wt = 449 ± 16 g; Charles River Laboratories, Wilmington, MA) were utilized in protocol 1 and 12 male Sprague-Dawley rats (body wt = 421 ± 23 g) were used in protocol 2. Rats were provided food and water ad libitum while housed in a 12/12 h light-dark cycle facility at Kansas State University. 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.

Surgical Preparation

Rats were initially anesthetized with a 5% isoflurane-O₂ mixture (isoflurane vaporizer; Ohio Medical Products) and subsequently maintained on 3% isoflurane-O₂. An incision was made on the ventral side of the neck and the right carotid artery was isolated and cannulated with PE-10 connected to PE-50 (Intra-Medic polyethylene tubing; Clay Adams Brand, Becton, Dickinson, Sparks, MD) for measurements of mean arterial pressure (MAP) and pulse rate (HR), and infusion of the phosphorescent probe (see below). The Digi-Med Blood Pressure Analyzer (model 400; Micro-Med, Louisville, KY) was utilized to measure MAP and pulse rate, where pulse rate was interpreted and recorded as heart rate (HR). A second catheter was inserted into the caudal artery for the infusion of pentobarbital sodium anesthesia and arterial blood sampling. After the incisions for the carotid and caudal catheters were closed, rats were transitioned to pentobarbital sodium anesthesia (~20 mg/kg body wt) given intra-arterially and concentrations of isoflurane were decreased and subsequently discontinued. The level of anesthesia was regularly monitored via toe pinch and palpebral reflex, with pentobarbital anesthesia supplemented (3.5–7.0 mg/kg) as necessary. Rats were placed in the prone position on a heating pad to maintain a core temperature of ~38°C (measured via rectal probe). Incisions were then made to carefully expose the right spinotrapezius muscle with overlying skin and fascia reflected such that the integrity of the neural and vascular supply was maintained (2). With the use of 6-0 silk sutures, silver wire electrodes were secured to the rostral (cathode) and caudal (anode) regions of the muscle (protocol 2 only). Exposed muscle tissue was superfused with warmed (38°C) Krebs-Henseleit bicarbonate-buffered solution equilibrated with 5% CO₂-95% N₂ (pH 7.4). Surrounding exposed tissue was covered with Saran wrap (Dow Brands, Indianapolis, IN) to minimize solution contact between nonspinotrapezius tissue and superfused solutions. The spinotrapezius muscle was selected based on its mixed muscle fiber-type composition and citrate synthase activity, which resembles the quadriceps muscle in humans (15, 37).

Experimental Protocol 1: NaNO₂ Superfusion vs. Intra-Arterial Infusion

The phosphorescent probe palladium meso-tetra (4 carboxyphenyl)tetrabenzoporphyrin dendrimer (G2: 15–20 mg/kg dissolved in 0.4 ml saline) was infused in bolus form (~10 s) via the carotid artery catheter followed by a saline flush. Following 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 as described previously (2). Fields containing large vessels were avoided to ensure that the measurements obtained were of principally capillaries and associated microvessels.

Po₂mv was measured during 180-s continuous superfusion of NaNO₂ (30 mM in 3.0 ml Krebs-Henseleit bicarbonate buffered solution) along the exposed muscle and 600 s of postsuperfusion rest via phosphorescence quenching (see below) and recorded at 2-s intervals. This NaNO₂ dose and a 180-s continuous method of delivery were chosen due to the curved nature of the spinotrapezius muscle overlying the rib cage. Once the superfused solution was applied, the portion that did not diffuse into the muscle ran down the tissue covered by Saran wrap and onto a waste collection site. A minimal amount (2–5 drops) of Krebs-Henseleit bicarbonate-buffered solution was applied periodically to ensure that the exposed spinotrapezius muscle surface did not become dry. Central hemodynamics (MAP and HR) were continuously measured and recorded at 30-s intervals throughout the 780-s protocol. Following a 30-min stabilization, the aforementioned protocol was used with the administration of a bolus intra-arterial infusion of NaNO₂ (~10 s; 7.0 mg/kg body wt in 0.4 ml heparinized saline solution).

Experimental Protocol 2: Muscle Contractions During Control and Following NaNO₂ Superfusion

In the second set of experiments, Po₂mv was measured at rest and during the 180-s contraction protocol (1 Hz, ~6 V, 2-ms pulse duration; Grass stimulator model S88, Quincy, MA) via phosphorescence quenching (see below) and recorded at 2-s intervals. Following the contraction period, blood samples were taken for analysis and Po₂mv was monitored to ensure that microvascular control was preserved and values returned to baseline. After a 30-min stabilization period, NaNO₂ (30 mM in 3.0 ml Krebs-Henseleit bicarbonate buffered solution) was continuously superfused for 180 s along the exposed muscle. Approximately 1 min following superfusion, after Po₂mv stabilized, the aforementioned contraction protocol was repeated. Rats were then euthanized via pentobarbital sodium overdose (≥50 mg/kg administered into the carotid artery catheter).

Po₂mv Measurement and Curve Fitting

Po₂mv was calculated using the Stern-Volmer relationship. Direct measurement of phosphorescence lifetime utilized in the following equation yields Po₂mv (51);

$$P{O}_{2}mv=\left[\tau °/\tau -1\right]/(k_Q\times \tau °)$$

where k_Q is the quenching constant and τ° and τ are the phosphorescence lifetimes in the absence of O₂ and at the ambient O₂ concentration, respectively. For G2, k_Q is 273 mmHg⁻¹·s⁻¹and τ° is 251 μs (18). Since the G2 phosphorescent probe binds to blood proteins, G2 is evenly distributed throughout plasma and compartmentalized to the vascular space (18, 45). Therefore, in vivo, the phosphorescence lifetimes are determined directly by O₂ pressure because k_Q and τ° do not change appreciably over the physiological ranges of temperature and pH (18, 51).

With the use of computer software (SigmaPlot 11.0; Systat Software, San Jose, CA), Po₂mv responses were curve-fitted from the collected Po₂mv data points. Monoexponential responses to muscle contractions were fit using a one-component model whereas three control (CON) and two $\mathrm{N}{\mathrm{O}}_2^{-}$ profiles expressed biexponential responses (exponential elevation of Po₂mv following the initial contracting nadir) requiring a two-component model to best fit the data. The models used to fit either one-component or two-component models are described below:

$$One\hspace{0.17em}component:P{O}_{2}m{v}_{(t)}=P{O}_{2}m{v}_{BL}–\mathrm{\Delta }P{O}_{2}mv\left(1-e^{–\left(t–TD\right)/\tau }\right)$$

$$Two\hspace{0.17em}component:P{O}_{2}m{v}_{(t)}=P{O}_{2}m{v}_{BL}–{\Delta }_{1}P{O}_{2}mv\left(1-e^{–\left(t–{TD}_1\right)/{\tau }_1}\right)+{\Delta }_{2}P{O}_{2}mv\left(1-e^{–\left(t–{TD}_2\right)/{\tau }_2}\right)$$

where Po₂mv_(t) represents the Po₂mv at any given time t, Po_2mvBL 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 reach 63% of the final response value) for each component.

Appropriate fits using either one-component or two-component models were 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. To provide an index of the overall principal kinetics response, the first and second components were used to calculate MRT₁ and MRT₂, respectively. Mean response time (MRT) was calculated using the following equations:

$$One\hspace{0.17em}component:MR{T}_1=T{D}_1+{\tau }_1$$

$$Two\hspace{0.17em}component:MR{T}_2=T{D}_2+{\tau }_2$$

where TD₁, TD₂, τ₂, and τ₂ are as described above.

Statistical Analysis

Data are presented as means ± SE. Superfusion vs. Intra-arterial results were compared using two-way repeated-measures ANOVA (MAP) with Tukey’s post hoc analyses. Muscle contraction results were compared within (pre- vs. postsuperfusion) groups using one-way repeated measures ANOVA (MAP and HR) or paired 1- and 2- tail Student’s t-tests as appropriate for a priori directional hypotheses (blood gases, blood [lactate], arterial pH, and Po₂mv kinetics variables). Significance was accepted at P ≤ 0.05.

RESULTS

Experimental Protocol 1: NaNO₂ Superfusion vs. Intra-Arterial Infusion

Results in Figs. 1 and 2 indicated that NaNO₂ (30 mM) during 180-s superfusion elevated Po₂mv without significantly changing MAP. In marked contrast, bolus (~10 s) intra-arterial infusion of NaNO₂ (7 mg/kg in 0.4 ml heparinized saline solution) in the caudal artery decreased MAP significantly and transiently decreased Po₂mv within the initial 180 s.

Fig. 1.

Averaged microvascular O₂ driving pressure (Po₂mv) from results (n = 3) during 180 s of NaNO₂ superfusion (30 mM in Krebs-Henseleit solution) and a bolus (~10 s) intra-arterial infusion of NaNO₂ (7 mg/kg in 0.4 ml heparinized saline solution). Inset: black box denotes the superfusion period. Measurements were recorded for an additional 600 s to monitor the effects of direct vs. systemic administration in the absence of muscle contraction. Following the bolus intra-arterial infusion Po₂mv declined at a rate of −0.098 mmHg/s (regression not shown) before returning to baseline levels. In the superfusion condition, following the initial increase and plateau in Po₂mv, there was a decline in Po₂mv at the rate of −0.012 mmHg/s. Data are presented as means ± SE every 30 s throughout each condition.

Fig. 2.

Mean arterial pressure (MAP) during 180 s of NaNO₂ superfusion (30 mM in Krebs-Henseleit solution) remained constant whereas a bolus (~10 s) intra-arterial infusion of NaNO₂ (7 mg/kg in 0.4 ml heparinized saline solution) produced a sustained reduction in MAP by as much as ~14–15 mmHg on average (n = 3; first experimental group of rats). Data are presented as means ± SE.

Experimental Protocol 2: Muscle Contractions During Control and Following NaNO₂ Superfusion

Microvascular oxygen pressures (Po₂mv).

Within 180-s superfusion of NaNO₂ Po₂mv was significantly elevated (Po_2mvBL; CON: 28.4 ± 1.1 vs. $\mathrm{N}{\mathrm{O}}_2^{-}$: 31.6 ± 1.2 mmHg; P ≤ 0.05) (Table 1 and Fig. 3). Following the onset of contractions, $\mathrm{N}{\mathrm{O}}_2^{-}$ exhibited a significantly larger Po₂mv amplitude (CON: 10.5 ± 0.9 vs. $\mathrm{N}{\mathrm{O}}_2^{-}$: 12.7 ± 0.8 mmHg; P ≤ 0.05), slower τ (CON: 12.3 ± 1.2 vs. $\mathrm{N}{\mathrm{O}}_2^{-}$: 19.7 ± 2.2 s; P ≤ 0.05), and increased MRT (CON: 19.3 ± 1.9 vs. $\mathrm{N}{\mathrm{O}}_2^{-}$: 25.6 ± 3.3 s; P ≤ 0.05; Fig. 4A). As shown in Fig. 4B, the difference in driving pressure between conditions continued for the first 40 s of electrically stimulated contractions.

Table 1.

Po₂mv variables in the spinotrapezius muscle at rest and following the onset of contractions under control and NaNO₂ conditions

	CON	$\mathrm{N}{\mathrm{O}}_2^{-}$
Po2mvBL, mmHg	28.4 ± 1.1	31.6 ± 1.2*
Δ1Po2mv, mmHg	10.5 ± 0.9	12.7 ± 0.8*
Po2mv (steady state), mmHg	18.0 ± 0.9	18.9 ± 1.2
TD1, s	7.0 ± 1.2	6.0 ± 1.6
τ1, s	12.3 ± 1.2	19.7 ± 2.2*
MRT1, s	19.3 ± 1.9	25.6 ± 3.3*
Δ2Po2mv, mmHg	3.4 ± 0.5	2.9 ± 0.1
TD2, s	78.5 ± 22.3	68.9 ± 9.5
τ2, s	67.9 ± 7.7	77.1 ± 14.2
MRT2, s	146.4 ± 25.7	97.3 ± 48.8

Values are means ± SE. Po_2mvBL, resting Po₂mv; Δ₁Po₂mv and Δ₂Po₂mv, amplitude of the first and second components; Po_{2mv(steady state)}, contracting steady-state Po₂mv; TD₁ and TD_2, time delay of the first and second component; τ₁, and τ₂, time constant of the first and second component; MRT₁ and MRT₂, mean response time of the first and second component. Primary components were calculated for all rats (n = 12). Secondary components were present in 3 control (CON) and 2 $\mathrm{N}{\mathrm{O}}_2^{-}$ rats.

^*P ≤ 0.05 vs. CON.

Fig. 3.

Microvascular oxygen pressure (Po₂mv) of the spinotrapezius muscle for the second experimental group of rats (n = 12) was measured every 2 s during the 180-s superfusion of 30 mM NaNO₂ in Krebs-Henseleit solution. Inset: black box denotes the superfusion period. Data are presented as means ± SE.

Fig. 4.

A: average spinotrapezius Po₂mv kinetics for the second experimental group of rats (n = 12) during 180 s electrically stimulated contractions in the control condition (closed circles) and following NaNO₂ superfusion (open circles). Mean arterial pressure was recorded at the beginning (105 ± 5 mmHg) and end (105 ± 5 mmHg) of the contraction protocol. Data are presented as means ± SE. B: Po₂mv differences for each 2 s measurement for the experimental group of rats (n = 12) between NaNO₂ and CON conditions during spinotrapezius muscle contractions. Note that before (i.e., left of) the dashed line, NaNO₂ is significantly different from CON (P < 0.05).

Central hemodynamics and blood gases.

Superfusion of NaNO₂ did not significantly alter MAP or HR from presuperfusion values (MAP: 103 ± 4 vs. 105 ± 4 mmHg, HR: 330 ± 10 vs. 321 ± 11 beats/min, pre and post, respectively; both P > 0.05). Likewise, there was no significant difference in MAP or HR between CON and $\mathrm{N}{\mathrm{O}}_2^{-}$ conditions following the contraction protocol (P > 0.05). Finally, there were no significant differences in %O₂ saturation (CON: 91.5 ± 1.5 vs. $\mathrm{N}{\mathrm{O}}_2^{-}$: 93.1 ± 0.7%), Pco₂ (CON: 42 ± 2 vs. $\mathrm{N}{\mathrm{O}}_2^{-}$: 41 ± 2 mmHg), blood [lactate] (CON: 1.6 ± 0.1 vs. $\mathrm{N}{\mathrm{O}}_2^{-}$: 1.6 ± 0.2 mM), or arterial pH (CON: 7.36 ± 0.01 vs. $\mathrm{N}{\mathrm{O}}_2^{-}$: 7.37 ± 0.01) between conditions (P > 0.05).

DISCUSSION

The principal findings of the present investigation demonstrate that NaNO₂ superfusion enhances the muscle Po₂mv at rest (i.e., elevated Po_2mvBL) and during the transition from rest-to-contractions, in part, by slowing the Po₂mv fall (i.e., increased τ and MRT). Importantly, these effects occurred concomitant with stable MAP. The ability of $\mathrm{N}{\mathrm{O}}_2^{-}$ to enhance the driving pressure of O₂ before and for the 40 s after the onset of muscle contractions (Fig. 4, A and B) would be expected to potentially decrease reliance on immediate, nonoxidative energy pathways and delay the production of fatigue-inducing metabolic products (31, 57). In disease conditions attended by cardiovascular complications, the inability to deliver O₂ may induce ischemic damage, impair recovery, and, for active skeletal muscle, constrain exercise performance and perhaps daily activities essential to life quality. The delivery of exogenous $\mathrm{N}{\mathrm{O}}_2^{-}$ is known to improve the central and systemic derangements prevalent in CHF (7, 39, 44) and we now demonstrate that it rapidly enhances peripheral control directly by circumventing the NOS pathway.

Microvascular Oxygen Pressure (Po₂mv) Dynamics

In skeletal muscle at rest, NaNO₂ has the potential to elevate the driving pressure of O₂ from blood to myocyte (Po_2mvBL) under normoxic conditions. This NOS-independent effect coheres with previous investigations that have administered NO precursors (22, 24, 27). With respect to the Po₂mv elevations, these may be the consequence of increased Q̇o₂, decreased V̇o₂, or a combination of both. Q̇o₂ increases with increasing [$\mathrm{N}{\mathrm{O}}_2^{-}$] because the one-step reduction to NO occurs in the low Po₂ environment of resistance vessels in healthy individuals (20, 21). The advantage of the $\mathrm{N}{\mathrm{O}}_2^{-}$-NO reduction is that NOS reliance is avoided, with $\mathrm{N}{\mathrm{O}}_2^{-}$ being reduced to NO in environments of low O₂/pH via interactions with deoxyhemoglobin, tissue and vascular myoglobin, xanthine oxidoreductase, and aldehyde dehydrogenase. Furthermore, by experimental blockade of endogenous production of NO from eNOS and nNOS pharmacologically [nitro-l-arginine methyl ester (l-NAME; eNOS and nNOS) and S-methyl-l-thiocitrulline (SMTC; nNOS)], blood flow is reduced independent of any increases in sympathetic activity/vasoconstriction (11, 12, 29, 30). The present results clearly demonstrate that NaNO₂ increases baseline (resting) Po₂mv and suggest strongly that the $\mathrm{N}{\mathrm{O}}_2^{-}$-NO reduction compliments any NO emanating from the intact NOS system to augment Po₂mv. The direct measurement of Po₂mv herein extends previous observations of $\mathrm{N}{\mathrm{O}}_2^{-}$-induced vasorelaxation and increased Q̇o₂ in healthy subjects (13, 14, 40).

Potentially, the increased Po₂mv could also be explained by mitochondrial inhibition (↓V̇o₂) where NO competitively binds to complexes I and IV of the respiratory chain (8, 9). However, if the primary effect of $\mathrm{N}{\mathrm{O}}_2^{-}$ on Po₂mv is mitochondrial inhibition, then a continuous increase in steady-state Po₂mv during muscle contractions would be expected but clearly this is not found (Fig. 4A). It is pertinent that using a similar protocol with SNP, Hirai et al. (27) demonstrated the integrity of mitochondrial respiration (and thus V̇o₂) concomitant with elevated $\mathrm{N}{\mathrm{O}}_2^{-}$ and NO. Thus the elevation in baseline Po₂mv seen herein is most likely the consequence of enhanced blood flow and Q̇o₂ rather than decreased V̇o₂.

The increases in Po₂mv presumed to be produced by an increased Q̇o₂ with NaNO₂ superfusion (13, 30, 52) persists into the onset of contractions (slowed Po₂mv kinetics) as demonstrated in Fig. 4, A and B. In Fig. 4B the rate at which Po₂mv is falling is slowed by disproportionate changes in Q̇o₂ and V̇o₂ across the initial 40 s. Enhancing the Q̇o₂-to-V̇o₂ relationship at this crucial transition would potentially improve energy production via oxidative metabolism, decrease the acidic sequelae of glycolytic pathways, and delay the onset of fatigue processes (31, 57).

Potential Therapeutic Significance

$\mathrm{N}{\mathrm{O}}_2^{-}$ precursors such as $\mathrm{N}{\mathrm{O}}_3^{-}$ containing beetroot juice effectively increase plasma [$\mathrm{N}{\mathrm{O}}_2^{-}$] and can improve exercise performance in healthy individuals (1, 34, 60) and patient populations where cardiovascular function is impaired such as CHF (61) and peripheral artery disease (PAD) (33). Specifically, CHF patients have an increased cardiac output reserve secondary to the reduction of vascular resistance without any changes in MAP (61). Likewise, PAD patients exhibit improved exercise/walking performance and delayed onset of claudication following beetroot juice supplementation (33). However, elevating circulating [$\mathrm{N}{\mathrm{O}}_2^{-}$] via dietary means takes 2–3 h, which may constitute too great a delay for an effective therapeutic outcome. A potential alternative is oral administration of NaNO₂, which elevates circulating [$\mathrm{N}{\mathrm{O}}_2^{-}$] within 30 min in older adults (16) and diabetic patients (26). More direct routes of NaNO₂ (i.e., intravascular infusion or oral administration) can bypass the need for bacterial breakdown of $\mathrm{N}{\mathrm{O}}_3^{-}$ and absorption in the gut and increase vascular [$\mathrm{N}{\mathrm{O}}_2^{-}$] rapidly. Thus intravenous $\mathrm{N}{\mathrm{O}}_2^{-}$ infusions have improved cardiac function and exercise performance in CHF patients (7). Additionally, $\mathrm{N}{\mathrm{O}}_2^{-}$ infusion during l-NAME-induced NOS blockade or CHF (20, 25) reverses the consequences of absent endogenous NO bioavailability and provides an avenue for enhanced Q̇o₂-to-V̇o₂ matching.

Administration of NaNO₂ protects against hypoxic damage to liver, cerebral, and myocardial tissue via the reduction of $\mathrm{N}{\mathrm{O}}_2^{-}$ to NO (19, 32, 54, 56) but possibly not the kidney (3, 55). Therefore topical administration of NaNO₂, potentially via implantable osmotic micro-pump, may provide tissue protection during acute surgical or other medical conditions by elevating Q̇o₂ and Po₂mv within 180 s of application (see Fig. 3) without confounding peripheral vascular effects [i.e., reductions in tissue Po₂mv (Fig. 1) that are coincident with reductions in MAP (Fig. 2)]. Local administration may open up the avenue for long-term administration at a desired location enhancing Q̇o₂ and Po₂mv for extended durations. The progressive decay of $\mathrm{N}{\mathrm{O}}_2^{-}$ effects on Po₂mv (Figs. 1 and 4B) indicates that setting the required [NaNO₂] and timing of application would be crucial.

Experimental Considerations

Although Po₂mv was significantly different at rest and the beginning of muscle contractions, the effect of $\mathrm{N}{\mathrm{O}}_2^{-}$ is largely diminished by the time the steady-state Po₂mv is reached (Fig. 4, A and B). It remains unclear whether this is a direct effect of $\mathrm{N}{\mathrm{O}}_2^{-}$ wearing off irrespective of muscle contractions or whether contractions and the attendant hypoxia are elevating $\mathrm{N}{\mathrm{O}}_2^{-}$ utilization. Figure 1 demonstrates that in the absence of muscle contractions, Po₂mv plateaus and then declines at a rate of ~0.7 mmHg/min after superfusion has ceased. This would indicate, in part, why NaNO₂ superfusion does not enhance steady-state Po₂mv relative to the control condition. Importantly, the linear decay in Po₂mv sheds light on the utilization of $\mathrm{N}{\mathrm{O}}_2^{-}$ in a localized volume of skeletal muscle when systemic levels of $\mathrm{N}{\mathrm{O}}_2^{-}$ have not been elevated upstream (i.e., drug infusion or oral administration). Future investigations into various delivery methods of NaNO₂ (i.e., injectable pellets or cutaneous patches) may be warranted considering that the administration of $\mathrm{N}{\mathrm{O}}_2^{-}$ augments Po₂mv at rest and during the rest-exercise transition.

Muscle contractile function was not directly measured in the current investigation yet previous studies have utilized the same contraction protocol to elicit a moderate metabolic stress of approximately three- and sevenfold elevation in blood flow and V̇o₂, respectively (4, 5). At this level of exercise contractile performance and V̇o₂ (or V̇o₂ kinetics) are not O₂ delivery limited. Accordingly, contractile performance and V̇o₂ would not be expected to increase with $\mathrm{N}{\mathrm{O}}_2^{-}$ administration but may be manifested with higher metabolic rates or under conditions of pathologically-reduced O₂ delivery as seen in heart failure patients during high-intensity exercise, for example (61). A higher rate of stimulation could have been used to create higher metabolic stress; however, any evoked increases in MAP would have complicated the interpretation of the effect of $\mathrm{N}{\mathrm{O}}_2^{-}$ on cardiovascular control. Thus the current contraction protocol and level of metabolic stress was selected to demonstrate proof-of-principle that the local NaNO₂ application to the spinotrapezius muscle would elevate Po₂mv significantly in the absence of altered MAP. Additionally, while $\mathrm{N}{\mathrm{O}}_2^{-}$ was investigated herein due to the selective reduction to NO in low Po₂ and pH environments found in ischemic muscle and elsewhere, other endogenous and exogenous vasodilators (i.e., ADP, cAMP, lidocaine, cyclooxygenase, or indomethacin) may potentially exert an elevation of Po₂mv without impacting central hemodynamic regulation (MAP or HR). Future investigations into these vasodilators may also serve to benefit patient populations.

A potential limitation to the Po₂mv measurement herein is assuring that the entirety of phosphorescent signal emanates from capillaries and associated microvessels. Micrograph images demonstrate that greater than 90% of the vascular volume in skeletal muscle [rat soleus and plantaris (49, 50), diaphragm (48), and mouse tibialis anterior (47)] is comprised of capillaries. Additionally, the light guide is positioned away from large vessels to minimize the potential for macrovascular influence.

Conclusion

NaNO₂ serves therapeutically as a hypoxic vasodilator with efficacy for improving exercise performance in patients with cardiovascular disease (1, 7, 38, 43, 44). The present investigation demonstrates the ability of NaNO₂ to locally elevate skeletal muscle Po₂mv at rest and following the onset of contractions in the healthy rat without altering MAP. Enhancing the muscle vascular O₂ driving pressure via NaNO₂ would provide a fast-acting modality to potentially improve metabolic control and thus delay fatigue and/or hypoxic damage. Improving Po₂mv dynamics alongside what remains of the endogenous NOS system under these conditions, NaNO₂ may ameliorate perturbations in the Q̇o₂-to-V̇o₂ ratio commonly found in disease states such as CHF and PAD. Fast-acting improvements in both resting and exercise Po₂mv dynamics may add to the current standard of care in populations with limited exercise tolerance along with individuals that may be suffering from focal tissue hypoxia and/or ischemia (i.e., frostbite, PAD, stroke).

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-108328 (to D. C. Poole) and American Heart Association (AHA) Grant 10GRNT4350011 (to D. C. Poole).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

T.D.C., S.K.F., C.T.H., J.C.C., T.I.M., and D.C.P. performed experiments; T.D.C., S.K.F., C.T.H., J.C.C., T.I.M., and D.C.P. analyzed data; T.D.C., S.K.F., C.T.H., J.C.C., T.I.M., and D.C.P. interpreted results of experiments; T.D.C., S.K.F., C.T.H., J.C.C., T.I.M., and D.C.P. prepared figures; T.D.C., C.T.H., J.C.C., T.I.M., and D.C.P. drafted manuscript; T.D.C., C.T.H., J.C.C., T.I.M., and D.C.P. edited and revised manuscript; T.D.C., S.K.F., C.T.H., J.C.C., T.I.M., and D.C.P. approved final version of manuscript.