Sex and nitric oxide bioavailability interact to modulate interstitial Po₂ in healthy rat skeletal muscle

Abstract

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 (Po_2is, determined by Q̇o₂/V̇o₂). This was performed under three conditions: control (CON), 300 µM sodium nitroprusside (SNP; NO donor), and 1.5 mM N^ω-nitro-l-arginine methyl ester (l-NAME; NOS blockade) superfusion. No sex differences were found for the Po_2is 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 ΔPo_2is 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 Po_2is 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 (Po_2is; determined by O₂ delivery-to-utilization matching) plays an important role for O₂ flux into skeletal muscle. We show that both sexes regulate Po_2is 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.

INTRODUCTION

Women have lower incidence of hypertension and cardiovascular disease up to the onset of menopause, at which point they catch up to, or surpass, that of age-matched men (44). This phenomena has been attributed to the protective effect of estrogen on the cardiovascular system (41), which is linked to lower muscle sympathetic nerve activity (42, 43, 55), increased expression of endothelial (31, 66) and neuronal (14) nitric oxide synthases, and thus lower blood pressure (9, 30, 41). The functional effects of estrogen on cardiovascular and metabolic regulation in young, healthy subjects are less clear. Although differences in blood pressure are commonly found between men and women (9, 30), whether this results in contrasting patterns of blood flow (Q̇) distribution to skeletal muscle remains equivocal, with some investigations finding differences between the sexes (29, 46) and others not (19, 35, 37, 60). Rogers and Sheriff (51) showed that estrogen plays a critical role in the regulation of terminal aortic Q̇ (i.e., bulk Q̇ to the hindlimb) during low- to moderate-intensity treadmill exercise in rats, but this effect was not necessarily linked to a sex difference. Fadel et al. (14) found that estrogen replacement in healthy, ovariectomized rats attenuated the ovariectomy-induced reduction in femoral artery Q̇ and vascular conductance during electrically induced contractions with the effect being principally mediated through nitric oxide (NO) pathways. Work from our laboratory has recently found no differences in respiratory muscle Q̇ between male and female rats during moderate- and near maximal-intensity treadmill exercise (56).

An important consideration when investigating sex differences is metabolic (i.e., V̇o₂) control, since Q̇ is tightly related to V̇o₂ across a range of exercise intensities (1). Males show greater maximal oxygen uptake (V̇o_2max) compared with their female counterparts which is largely attributed to greater muscle mass, hemoglobin volume, and maximal cardiac output. Normal female hormonal fluctuations (i.e., menstrual cycle) do not affect submaximal or maximal V̇o₂ (11, 28), but oral contraceptive-induced supraphysiological levels of female sex hormones may reduce V̇o_2max (7, 33). Measurements of Q̇ and V̇o₂ are often taken at set time points or during steady-state exercise and may overlook potential sex differences in the transition from rest to exercise (i.e., the dynamic response). Attempts to quantify and compare the dynamics of Q̇ and V̇O₂ following the onset of exercise in healthy women and men are limited.

The measurement of the partial pressure of oxygen (Po₂) within skeletal muscles is a powerful tool that assesses Q̇o₂-to-V̇o₂ matching close to the site of O₂ usage with excellent spatial and temporal fidelity, particularly during the transition from rest to skeletal muscle contractions in healthy (8, 10, 23) and diseased (15, 22, 26) rats. Therefore, the purpose of the present investigation was to determine the role of sex and NO bioavailability on skeletal muscle Q̇o₂-to-V̇o₂ matching at rest and following the onset of submaximal muscle contractions. Specifically, we tested the hypotheses that female rats would 1) elicit an elevated muscle O₂ delivery-to-utilization ratio (and thus higher interstitial Po₂) during contractions and 2) demonstrate a greater responsiveness to altered NO bioavailability.

MATERIALS AND METHODS

Sixteen young adult (~3–4 mo old) age-matched Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) including 8 male (body wt 384 ± 19 g) and 8 female (body wt 283 ± 9 g) rats were maintained in accredited animal facilities at Kansas State University on a 12:12-h light-dark cycle with food and water provided ad libitum in isolated cages. All procedures were approved by the Institutional Animal Care and Use Committee of Kansas State University and conducted according to the National Research Council Guide for the Care and Use of Laboratory Animals. All experiments were conducted between 1 and 3 wk of the animals’ arrival to the facilities. This allowed sufficient time for the animals to acclimate to their new settings and ensured the groups remained age matched.

Surgical preparation.

On the day of the experiment, rats were initially anesthetized with a 5% isoflurane-O₂ mixture and subsequently maintained on 2–3% isoflurane-O₂. Following the isolation of the carotid artery, a catheter (PE-10 connected to PE-50, Intra-Medic polyethylene tubing, Clay Adams Brand, Becton, Dickinson, Sparks, MD) was inserted into the carotid artery for measurement of mean arterial pressure (MAP) and heart rate (HR). A second catheter was introduced into the caudal artery for the administration of pentobarbital sodium anesthesia and arterial blood sampling. Upon closing the incisions for the carotid and caudal catheters, rats were progressively transitioned to pentobarbital sodium anesthesia. Depth of anesthesia was continuously monitored via the toe pinch and blink reflexes, with additional anesthesia administered as necessary. Rats were placed on a heating pad to maintain a core temperature of ~38°C (measured via rectal probe). Incisions were then made to expose the left 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, platinum iridium wire electrodes were secured to the rostral (cathode) and caudal (anode) regions of the muscle to facilitate electrically induced contractions. Surrounding exposed tissue was covered with Saran wrap (Dow Brands, Indianapolis, IN) to minimize the exposure of superfused solutions to bordering tissues and reduce tissue dehydration. Exposed muscle was superfused frequently with warmed (38°C) Krebs-Henseleit bicarbonate buffered solution equilibrated with 5% CO₂-95% N₂. The spinotrapezius muscle was selected based on its mixed muscle fiber-type composition and citrate synthase activity, which resembles the quadriceps muscle in humans (12, 34) and convenience with respect to minimally invasive exposure (2).

Experimental protocol.

Three separate contraction bouts were performed under control (CON), sodium nitroprusside (SNP; NO donor, 300 μM), and N^ω-nitro-l-arginine methyl ester (l-NAME; nonselective NO synthase (NOS) inhibitor, 1.5 mM) conditions. The initial two conditions (either CON or SNP) were randomly determined, whereas the final condition was always l-NAME. This was necessary due to the long half-life of l-NAME. The drugs were administered via superfusion (3 ml total volume) on the spinotrapezius over 180 s of continuous interstitial Po₂ (Po_2is) recording. The recording was extended for an additional 180 s to confirm that baseline Po_2is had stabilized before the onset of muscle contractions and for 180 s of muscle contractions. Contractions were evoked via electrical stimulation (1 Hz, 6–7 V, 2-ms pulse duration) with a Grass S88 Stimulator (Quincy, MA). This contraction protocol increases spinotrapezius muscle blood flow four- to fivefold and metabolic rate six- to sevenfold without altering blood pH and is consistent with moderate-intensity exercise (3, 23). Between contraction bouts, rats were given 20–30 min of recovery with regular superfusion of Krebs-Henseleit solution. Our laboratory has previously shown this duration of recovery elicits reproducible microvascular Po₂ (Po_2mv) (10, 22) responses. Upon completion of the protocol, rats were euthanized with intra-arterial potassium chloride overdose (1 ml/kg of 4 M KCl).

Spinotrapezius interstitial Po₂ measurement.

Phosphorescence quenching was used to measure Po_2is in the spinotrapezius at rest and during contractions using a frequency domain phosphorimeter (PMOD 5000; Oxygen Enterprises, Philadelphia, PA) as previously described (25). Briefly, the Oxyphor G4 [Pd-meso-tetra-(3,5-dicarboxyphenyl)-tetrabenzo-porphyrin] was injected locally (3–4 10-μl injections at 10 μM concentration) using a 29-gauge needle with care taken to avoid damaging any visible vasculature. After injection, the spinotrapezius was covered with Saran wrap and given at least 20 min to allow the G4 to diffuse throughout the interstitial space. This Oxyphor is well-suited for use in biological tissues due to its inability to cross membranes and stability across physiological pH ranges (13); it is, however, temperature sensitive and therefore spinotrapezius temperature was measured using a noncontact infrared thermometer. Mean spinotrapezius temperature was 32.4 ± 0.2°C, with no differences between sexes or change during contractions.

Phosphorescence quenching applies the Stern-Volmer relationship (13, 52), which describes the quantitative O₂ dependence of the phosphorescent probe G4 via the equation:

$${PO}_{2\text{is}}=\left[\left({\tau }_0/\tau \right)-1\right]/\left(k_{\text{q}}{\tau }_0\right)$$

where k_q is the quenching constant and τ and τ₀ are the phosphorescence lifetimes at the ambient O₂ concentration and in the absence of O₂, respectively. For G4 in tissue at 32.5°C, k_q is 258 mmHg⁻¹·s⁻¹ and τ₀ is 226 μs (13). Since muscle temperature does not appreciably change over the duration of the contraction protocol used herein, the phosphorescence lifetime is determined exclusively by the O₂ partial pressure. After injection of G4, the common end of the bifurcated light guide was positioned 3–4 mm above the dorsal surface of the exposed spinotrapezius. The phosphorimeter modulates sinusoidal excitation frequencies between 100 Hz and 20 kHz and allows phosphorescence lifetime measurements from 10 μs to ~2.5 ms. Po_2is was measured continuously and recorded at 2-s intervals throughout the duration of the experimental protocol.

Analysis of spinotrapezius interstitial Po₂ kinetics.

The kinetics analyses of the Po_2is responses were conducted using 30 s of resting data and the 180-s contraction bout using a monoexponential plus time delay model:

$${PO}_2\left(t\right)={PO}_{2(\text{BL)}}-{\Delta }_1{PO}_2\left(1-e^{-\left(t-\text{TD}\right)/\tau }\right)$$

or a monoexponential plus time delay with a secondary component when necessary:

$${PO}_2\left(t\right)={PO}_{2(\text{BL)}}-{\Delta }_1{PO}_2\left(1-e^{-\left(t-\text{TD}\right)/\tau }\right)+{\Delta }_2{PO}_2\left(1-e^{-\left(t-\text{TD2}\right)/\tau 2}\right)$$

where Po₂(t) represents the Po_2is at any point in time, Po_2(BL) is the baseline before the onset of contractions, Δ₁Po₂ and Δ₂Po₂ are the primary and secondary amplitudes, TD and TD2 are the time delays before the drop and secondary rise in Po₂, and τ and τ₂ are the time constants (i.e., the time required to reach 63% of the amplitude) for the primary and secondary amplitudes. The mean response time (MRT) was calculated as the sum of the model-derived TD and τ. When the secondary component model was necessary, the primary amplitude was constrained to not exceed the nadir value to maximize the accuracy of the primary response kinetics (see Fig. 1 for example). The goodness of model fit was determined using the following criteria: 1) the coefficient of determination, 2) sum of the squared residuals, 3) visual inspection and analysis of the model fits to the data and the residuals, and 4) manual calculation of the time taken to reach 63% of the primary response (T₆₃) compared with the model-derived MRT. Since Δ₂Po₂ (i.e., undershoot of Po₂) was often nonexponential in nature, Δ₂Po₂ was determined manually, when necessary, by calculating the difference between the steady-state Po₂ at the end of contractions minus the nadir value of Po₂ during contractions.

Fig. 1.

A representative interstitial Po₂ profile (●) during 180 s of muscle contractions. The solid line overlaid on the data represents the fit determined by the modeling procedure. Modeling was performed such that emphasis was put on the primary response of the Po₂ profile (see text for details). The bold solid line below represents the residuals of the fit. Inset text reports kinetics parameters determined by this procedure.

Central hemodynamics and blood samples.

MAP and HR were measured during the experiment via the right carotid artery catheter connected to a pressure transducer and Digi-med Blood Pressure Analyzer (model 400; Micro-Med, Louisville, KY). Approximately 0.4 ml of blood was sampled from the caudal artery catheter at the end of the experiment for the determination of arterial blood lactate concentration ([La⁻]), pH, Pco₂, %O₂ saturation, and hematocrit (Nova Stat Profile M; Nova Biomedical, Waltham, MA).

Statistical analysis.

All curve fitting and statistical analyses were performed using a commercially available software package (SigmaPlot 12.5, Systat Software, San Jose, CA). Sex differences for rat descriptive variables, blood gases, and effects of superfusion on resting variables [i.e., ΔMAP, ΔHR, and ΔPo_2(BL)] were compared using unpaired Student’s t-tests. Po_2is kinetics parameters were compared among conditions using two-way repeated-measures ANOVA (sex × superfusion) with Tukey’s post hoc tests as necessary. Goodness of model fit (i.e., fitting-derived MRT vs. model-independent T₆₃) was compared using paired Student’s t-tests with both sexes represented in each group. Pearson’s product-moment correlations and linear regressions were used to determine relationships among variables. Data are presented as means ± SD unless otherwise noted. Significance was accepted at P < 0.05.

RESULTS

Male rats were heavier than their age-matched female counterparts (384 ± 55 vs. 283 ± 25 g; P < 0.001) and spinotrapezius mass paralleled these differences (0.38 ± 0.04 vs. 0.26 ± 0.03 g; P < 0.001). The spinotrapezius mass-to-body mass ratio was not different between sexes (P = 0.61). No sex differences were found for arterial [La⁻] (1.7 ± 0.4 vs. 1.2 ± 0.4 mM, P = 0.12), %O₂ saturation (94 ± 3 vs. 92 ± 4%; P = 0.25), or hematocrit (34 ± 3 vs. 34 ± 3%; P = 0.86) for males and females, respectively. Males had lower arterial Pco₂ (34 ± 6 vs. 44 ± 6 mmHg; P = 0.01) and higher pH (7.42 ± 0.04 vs. 7.36 ± 0.02; P < 0.01), indicative of moderate hyperventilation. There were no sex differences for resting MAP (106 ± 12 vs. 99 ± 14 mmHg; P = 0.23) or HR (371 ± 31 vs. 365 ± 25 beats/min; P = 0.76) for male and female rats, respectively.

A representative Po_2is profile is presented in Fig. 1 to highlight the modeling fit and signal-to-noise of the Po_2is measurement. Both sexes showed an exponential drop in Po_2is following the onset of contractions that led to a Po_2is “undershoot” before reaching a steady-state Po_2is of 15.8 ± 2.8 and 13.1 ± 3.6 mmHg (P = 0.22) for males and female rats, respectively. There were no sex differences for Po_2is before or during contractions in the control condition (Fig. 2, Table 1). The model-independent estimation of T₆₃ did not differ from the model-derived MRT (16.5 ± 5.0 vs. 16.5 ± 5.5 s; P = 0.92) supporting the robustness of the model fitting procedures.

Fig. 2.

Control condition group average spinotrapezius interstitial Po₂ temporal response during 180 s of muscle contractions. No differences were found between males (n = 8) and females (n = 8) at rest or during contractions. Data are means ± SE.

Table 1.

Interstitial Po₂ kinetics parameters of the spinotrapezius at rest and during 180 s of contractions following control, SNP, and l-NAME superfusion

	Control		SNP		l-NAME
	Male	Female	Male	Female	Male	Female
Po2(BL), mmHg	20.4 ± 2.5	18.2 ± 4.9	41.9 ± 9.5*†‡	35.8 ± 6.6†‡	15.9 ± 4.4	14.4 ± 4.6
Δ1Po2, mmHg	11.6 ± 2.2	12.0 ± 4.4	21.6 ± 7.1*†‡	16.6 ± 4.2†‡	10.4 ± 3.5	9.5 ± 2.8
τ, s	9.2 ± 2.8	10.5 ± 4.0	23.5 ± 16.0†‡	20.2 ± 15.5‡	9.8 ± 2.5	9.9 ± 2.3
TD, s	6.7 ± 3.0	6.4 ± 3.4	2.0 ± 1.7*†	5.1 ± 3.9	3.3 ± 1.1†	3.9 ± 1.6
MRT, s	16.0 ± 4.2	17.0 ± 6.9	25.5 ± 15.5†‡	25.3 ± 13.6‡	13.1 ± 2.5	13.8 ± 2.1
Δ2Po2, mmHg	7.0 ± 2.9	7.0 ± 2.1	0.6 ± 1.6†‡	1.5 ± 3.1†‡	7.6 ± 2.2	7.9 ± 3.5
Δ1Po2/τ, mmHg/s	1.4 ± 0.8	1.2 ± 0.5	1.1 ± 0.5	1.1 ± 0.5	1.1 ± 0.6	1.0 ± 0.5

Values are means ± SD. Po_2(BL), baseline interstitial Po₂; Δ₁Po₂, Po₂ primary amplitude; τ, time constant; TD, time delay; MRT, mean response time; Δ₂Po₂, Po₂ undershoot during contractions.

^*P < 0.05 vs. female within superfusion.

^†P < 0.05 vs. control within sex.

^‡P < 0.05 vs. l-NAME within sex.

The effects of SNP superfusion on resting Po_2is are shown in Fig. 3, left. Following SNP superfusion, both sexes demonstrated an increase of Po_2is, but this was of lesser magnitude for the female rats (P < 0.01). There was no between-sex difference for ΔHR (P = 0.95; Table 2) although female rats showed a greater drop in MAP (P = 0.04; Table 2). Female rats expressed a lower postsuperfusion baseline Po_2is than males (P = 0.04) and a smaller primary amplitude of Po_2is decrease during contractions (P = 0.03; Fig. 4, Table 1). The Po_2is undershoot during contractions was reduced in both sexes compared with control and l-NAME (all P < 0.001; Fig. 5) and was not different between the sexes (P = 0.47). SNP slowed Po_2is kinetics as evidenced by the increased MRT (driven by an increased τ) compared with control and l-NAME for both sexes (all P < 0.05) and was not different between the sexes (P = 0.96). The steady-state Po_2is at the end of contractions was increased following SNP compared with control and l-NAME (all P < 0.02) and was not different between males and females (20.8 ± 4.7 vs. 20.7 ± 4.2 mmHg; P = 0.93). The model-independent estimation of T₆₃ was not different from the model-derived MRT (24.6 ± 12.6 vs. 25.4 ± 14.1 s; P = 0.34).

Fig. 3.

Absolute change for resting spinotrapezius interstitial Po₂ following superfusion of sodium nitroprusside (SNP, 300 μM) and N^ω nitro-l-arginine methyl ester (l-NAME, 1.5 mM) for male (closed bars) and female (open bars) rats. ΔPo₂ was calculated as the difference between 30 s of baseline Po₂ before superfusion and 30 s of Po₂ following the superfusion and stabilization period. Data are means ± SE *Significantly different from males (P < 0.05). All plots represent 8 animals per condition, except for male l-NAME, which is 7 animals.

Table 2.

Effect of SNP and l-NAME superfusion on resting central hemodynamics

	SNP		l-NAME
	Male	Female	Male	Female
ΔMAP, mmHg	−1 ± 1	−6 ± 2*	2 ± 3	1 ± 2
ΔHR, beats/min	26 ± 5	25 ± 4	3 ± 5	−9 ± 3*

Values are means ± SD.

^*P < 0.05 vs. male within superfusion.

Fig. 4.

Left: SNP condition group average spinotrapezius interstitial Po₂ temporal response during 180 s of muscle contractions. Males (n = 8) had a significantly greater baseline Po₂ than females (n = 8) following SNP superfusion. The ΔPo₂ during contractions was also greater in males (see text for details). Data are means ± SE. Right: l-NAME condition group average spinotrapezius interstitial Po₂ temporal response during 180 s of muscle contractions. No differences were found between males (n = 7) and females (n = 8) at rest or during contractions, although the ΔPo₂ in response to l-NAME superfusion was greater in females (see Fig. 2). Data are means ± SE.

Fig. 5.

Left: female rat individual interstitial Po₂ (Po_2is) undershoots as a function of the Po_2is nadirs achieved during spinotrapezius contractions. Right: male rat individual Po_2is data. In both groups, SNP superfusion (triangles; n = 8 for both) significantly increased the Po_2is nadir and reduced the Po_2is undershoot compared with the control condition (circles; n = 8 for both) and l-NAME (inverted triangles; n = 8 for females, n = 7 for males). Linear regression revealed significant relationships for both female (r² = 0.42, P < 0.01) and male (r² = 0.64, P < 0.01) rats. Visual inspection of the data reveals that there may be a “threshold” effect in the nadir Po_2is (see text for details).

l-NAME superfusion decreased resting Po_2is to a greater extent in female rats when compared with males (P = 0.04, Fig. 3, right). The ΔMAP following l-NAME superfusion was not different between males and females (P = 0.96), but females showed a greater reduction in HR (P = 0.04; Table 2). Similar to the control condition, no differences between the sexes for Po_2is on-kinetics were found during contractions following l-NAME (Fig. 4, Table 1) with a steady-state Po_2is of 13.1 ± 3.1 and 12.8 ± 6.1 mmHg (P = 0.93), for male and female rats, respectively. The model-independent estimation of T₆₃ was not different from the model-derived MRT (13.3 ± 2.8 vs. 13.5 ± 2.3 s; P = 0.61).

DISCUSSION

The primary original findings of the present investigation show that skeletal muscle Po_2is (determined by the matching of Q̇o₂-to-V̇o₂) does not differ between females and males during muscle contractions under CON conditions; however, alterations in NO bioavailability (via SNP and l-NAME) expose differences in Po_2is regulation at rest and during contractions. Specifically, SNP elicited a smaller effect on Po_2is (i.e., smaller increase) in female rats at rest and during contractions compared with males, whereas l-NAME showed a greater impact (i.e., larger reduction) in females at rest compared with males. These results provide partial support for our hypothesis that females would exhibit a greater responsiveness to alterations in NO bioavailability and suggest that females might rely more on NO to maintain skeletal muscle Q̇o₂-to-V̇o₂ matching at rest as evidenced by the greater transient reduction in Po_2is expressed by females following l-NAME superfusion. Females may also have a lower “capacity” to augment Po_2is in response to a NO donor due to a greater bioavailability (and thus role in basal regulation) of endogenous NO compared with males. Another explanation for the reduced responsiveness to SNP in females could relate to a ceiling effect caused by some anatomical difference in the vasculature or muscle. The total NO effect (SNP minus l-NAME; see Fig. 3) was not different between the sexes; however, the relationship was shifted to a lower PO_2is range in the females. An understanding of these effects of NO bioavailability on Po_2is is important because this carrier-free space is considered to represent a substantial barrier to oxygen flux from the blood to the mitochondria (64), with Po_2is and alteration thereof potentially impacting metabolic control.

Female and male Po_2is similarities for control.

Revealing the (dis)similarities between females and males for O₂ delivery-to-utilization matching at the level of the skeletal muscle interstitial space is a potentially powerful means to inform and help resolve the conflicting findings of sex differences in Q̇ and V̇o₂. The present investigation revealed that during the CON condition male and female rats both regulated Po_2is at a similar level at rest and during contractions. This commonality of regulation between male (24) and female (5) rats is also apparent in the upstream Po_2mv compartment, despite a differing temporal response (i.e., TD, τ, MRT) and qualitative shape (i.e., undershoot amplitude) compared with the Po_2is compartment.

Controversy exists with respect to potential sex differences in the individual components of PO_2is (i.e., Q̇o₂ and V̇o₂), particularly with regard to the regulation of active muscle Q̇ (and Q̇o₂) during exercise. Some studies have found that females have a greater exercising vasodilator response (and thus, higher Q̇) than males (29, 46), others have found no differences (19, 35, 37, 50, 56, 60) across a range of exercise intensities and modalities. Males may elicit higher absolute muscle Q̇ values during exercise, but those differences are abolished when Q̇ is expressed relative to workload. The second determining variable of the Po_2is, V̇o₂, is less contentious. The maximal V̇o₂ in females is typically lower, in absolute terms, owing mainly to less lean muscle and lower achievable workloads. Examined at submaximal intensities, there does not appear to be any appreciable sex difference for pulmonary or limb V̇o₂ (50, 59) when related to workload. Taken together, the aforementioned studies and this present investigation suggest that the cardiorespiratory system and metabolic apparatus are tuned to regulate muscle Q̇o₂ and V̇o₂ during normal operating conditions regardless of sex; however, the specific nature of that control might differ between the sexes (see Female and male (dis)similarities following SNP and l-NAME). The present investigation advances this understanding by directly measuring Po₂ at the site where the cardiorespiratory system and metabolic apparatus interact, in close proximity to the site of O₂ utilization.

Interestingly, a prominent undershoot in Po_2is was observed which approximated 60% of the primary amplitude in both sexes (undershoot observed in 100% of rats). This undershoot has been attributed to, and is exacerbated by, mismatched Q̇o₂ and V̇o₂ due to aging (4, 39), disease (3, 16, 26), and changes in NO availability (16, 23) seen in the upstream microvascular space (i.e., Po_2mv). This study is the first to observe the undershoot phenomenon consistently in the muscle interstitial space in both healthy male and female rats. These data suggest that this undershoot phenomena is a normal response in the interstitial space. The interstitial space may exhibit unique Po₂ profiles because of the compartment’s proximity to both the microvascular Q̇ and intracellular myoglobin/mitochondria. The present investigation, and previous work from our laboratory (25), revealed a Po_2is that is substantially higher than the myoglobin Po₂ [2–5 mmHg (49)] and expected mitochondrial Po₂ (0–2 mmHg) during contractions. In this context, the Po_2is undershoot seen during contractions may be crucial for reestablishing the gradient needed for O₂ flux into the intracellular compartment. That SNP attenuated and l-NAME augmented the undershoot (see Female and male (Dis)similarities following SNP and l-NAME) supports that NO plays an important role for the establishment of adequate O₂ flux into the myocyte during contractions.

Female and male PO_2is (dis)similarities following SNP and l-NAME.

NO signaling is important within many physiological systems in the body and has the ability to modulate both components of Po₂ (i.e., Q̇o₂ and V̇o₂). As a powerful vasodilatory agent, NO can increase Q̇ via relaxation of smooth muscle which increases vascular conductance (57). NO also modulates oxidative respiration within the mitochondria (6) which serves to reduce V̇o₂. Increased NO bioavailability (SNP) should then increase Q̇ and/or decrease V̇o₂ while decreased NO bioavailability (l-NAME) would induce the converse. The expected outcome of SNP superfusion would be an increased Po₂ while l-NAME superfusion would decrease Po₂ [as seen in Po_2mv studies (16, 23)].

SNP increased the baseline Po_2is in both females and males to twice that of the presuperfusion baselines with females achieving a postsuperfusion Po_2is ~15% lower than the males [Po_2(BL), Table 1]. Neither the absolute values or Δ for Po₂ were correlated with MAP or HR in either sex following SNP superfusion (data not shown, r²: 0.05–0.2), suggesting that the differential response to SNP was not driven by differences in driving pressure or HR. Instead, these differences appear to be the result of direct effects of SNP on the peripheral vasculature and/or skeletal muscle. Previous work investigating the vascular reactivity response to SNP in humans found no sex differences in some studies (58, 65), but, in contrast, Kneale et al. (32) reported that females exhibited a reduced increase in forearm Q̇ compared with males across a range of SNP doses. Unfortunately, those studies were not equipped to measure the metabolic consequences of SNP or the potential interaction it may have with the increased Q̇. The results herein, when taken in consideration with Kneale et al. (32), and Po_2mv studies (16, 23), suggest that the smaller increase in Po_2is seen in the females at rest was driven by a attenuated increase in Q̇ rather than a modulation of V̇o₂ (due to the low metabolic rate of resting skeletal muscle).

The female Po_2is primary amplitude (Δ₁Po₂) with contractions was smaller than the males under SNP. However, the relative rate of change for Po_2is (represented by Δ₁PO₂/τ) was not different between females and males following SNP; which was also not different from CON or l-NAME for either sex. These results support that the muscle metabolic rate (i.e., V̇o₂) was not different between the sexes, if the rate of decrease in Po_2is is primarily driven by the intracellular metabolic apparatus and resulting O₂ flux into the cell. Although not unequivocal, V̇o₂ kinetics have been shown to be independent of augmented Q̇ in both healthy humans (21) and isolated muscle (20). Furthermore, since the model and muscle contractions used herein have supported this view (5), we argue for like metabolic profiles herein.

SNP reduced the incidence and amplitude of the Po_2is undershoot for both sexes compared with CON and l-NAME (Table 1 and Fig. 5). The augmented Q̇ induced by SNP increased the nadir Po_2is during contractions to values greater than the steady-state Po_2is achieved in CON and l-NAME, likely reflecting a Q̇ in excess of the metabolic demand. Thus the undershoot which may have been necessary to preserve the Po₂ gradient and O₂ flux in the CON condition was not observed following SNP. Another potential explanation for the reduction in the undershoot occurrence is that the excess Q̇ mitigated any delay in the Q̇o₂ matching to V̇o₂. Whatever the case, observation of these data in Fig. 5 suggests there may be a “threshold” type effect in the interplay between the nadir and undershoot amplitudes. The undershoot amplitudes were reduced to zero once the nadir Po_2is was greater than ~15 mmHg. Further work is necessary to determine if this observation has physiological underpinnings or is a product of the experimental protocol. This novel observation supports the hypothesis that a functional barrier to O₂ flux exists between the interstitial and intracellular spaces (see Female and male similarities for control).

Inhibition of NOS reveals that NO plays an important role in the control of resting skeletal muscle Q̇ in both animals (38, 45, 47, 53, 62) and humans (36, 48, 54, 58, 63). It has also been shown that premenopausal women, and postmenopausal women treated with estrogen, exhibit a greater vasoconstrictor response to NOS inhibition compared with age-matched males (32, 36, 58) and postmenopausal women without estrogen treatment (36). In the present investigation, l-NAME superfusion reduced resting Po_2is to a greater extent in females vs. males (Fig. 3, right), supporting the notion that the increased NOS-generated NO in females (14, 31, 66) leads to different regulation of Q̇o₂-to-V̇o₂ matching across sexes. However, at the onset of contractions [i.e., Po_2(BL)], neither sex evinced a lower Po_2is compared with the CON condition. The lack of an effect on resting Po_2is with NOS inhibition could be explained by the substantial microvascular-to-interstitial Po₂ gradient (25), which may serve to “buffer” the reduction of Po_2is in the face of reduced Po_2mv (17). Similarly, there exists redundancy in the pathways that regulate Q̇ (for review see 27), which could act to offset the attenuation of Po_2is imposed by l-NAME. Despite the lack of an effect on the baseline Po₂ or the other parameters of the kinetics response, l-NAME increased the undershoot as a proportion of the primary amplitude. The undershoot in Po_2is that was observed to approximate 60% of the primary amplitude in CON was increased to ~84% by l-NAME, supporting the role of NO in establishing the Po₂ gradient necessary for facilitating myocyte O₂ flux.

Experimental considerations.

The estrus cycle of the female rats was not controlled for in the present study leaving the possibility that sex differences in the primary measurements were obfuscated by varying levels of estrogen present. However, had this indeed been the case, we would have expected greater variance within the female rat group compared with the males, but this scenario was not found in any of the primary measurements. Additionally, the proestrus phase occupies ~10 h of the typical rat 4–5 day cycle (61), which reduces the chances these rats were currently in the high-estrogen phase. Future studies aiming to maximize the potential for sex hormone-driven differences in Po_2is should target the ovulation/proestrus phase. Linear regression analyses were performed to explore the notion that the differences in body weight between the female and male rats could somehow have accounted for some of the (dis)similarities seen in the present investigation. However, body mass and spinotrapezius mass were not related to the primary variables. The most likely instance of this confounding effect is with the depth of anesthesia and resultant ventilatory pattern of the rats (reflected in arterial Pco₂ and pH). With regard to the differences in pH, we cannot exclude the potential effect this could have on the vasoreactivity of the microvessels (40).

Conclusions.

The primary novel finding of the present investigation is that female and male rats regulate skeletal muscle interstitial space O₂ delivery-to-utilization matching (reflected as Po_2is) at a similar level under CON conditions. The ratio of Q̇o₂-to-V̇o₂ can be modulated by altering NO bioavailability, with SNP (NO donor) increasing and l-NAME (NOS blockade) decreasing the ratio. Interestingly, the relationship between resting Po_2is and NO bioavailability was altered based on the sex of the animal with females exhibiting a lower Po_2is range across the availability of NO while males evince a higher range. Shown herein for the first time in the interstitial space was the presence of a substantial Po₂ undershoot during CON that was expressed in all rats (regardless of sex), which suggests that the interstitial site is important for controlling blood-myocyte O₂ flux. The occurrence and amplitude of this Po_2is undershoot was shown to be reduced by a NO donor.

GRANTS

This work was supported in part by National Heart, Lung and Blood Institute Grant HL-2-108328.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

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