Skeletal muscle interstitial Po₂ kinetics during recovery from contractions

Abstract

The oxygen partial pressure in the interstitial space (Po_2 is) drives O₂ into the myocyte via diffusion, thus supporting oxidative phosphorylation. Although crucial for metabolic recovery and the capacity to perform repetitive tasks, the time course of skeletal muscle Po_2 is during recovery from contractions remains unknown. We tested the hypothesis that Po_2 is would recover to resting values and display considerable on-off asymmetry (fast on-, slow off-kinetics), reflective of asymmetric capillary hemodynamics. Microvascular Po₂ (Po_2 mv) was also evaluated to test the hypothesis that a significant transcapillary gradient (ΔPo₂ = Po_2 mv − Po_2 is) would be sustained during recovery. Po_2 mv and Po_2 is (expressed in mmHg) were determined via phosphorescence quenching in the exposed rat spinotrapezius muscle during and after submaximal twitch contractions (n = 12). Po_2 is rose exponentially (P < 0.05) from end-contraction (11.1 ± 5.1), such that the end-recovery value (17.9 ± 7.9) was not different from resting Po_2 is (18.5 ± 8.1; P > 0.05). Po_2 is off-kinetics were slower than on-kinetics (mean response time: 53.1 ± 38.3 versus 18.5 ± 7.3 s; P < 0.05). A significant transcapillary ΔPo₂ observed at end-contraction (16.6 ± 7.4) was maintained throughout recovery (end-recovery: 18.8 ± 9.6; P > 0.05). Consistent with our hypotheses, muscle Po_2 is recovered to resting values with slower off-kinetics compared with the on-transient in line with the on-off asymmetry for capillary hemodynamics. Maintenance of a substantial transcapillary ΔPo₂ during recovery supports that the microvascular-interstitium interface provides considerable resistance to O₂ transport. As dictated by Fick’s law (V̇o₂ = Do₂ × ΔPo₂), modulation of O₂ flux (V̇o₂) during recovery must be achieved via corresponding changes in effective diffusing capacity (Do₂; mainly capillary red blood cell hemodynamics and distribution) in the face of unaltered ΔPo₂.

NEW & NOTEWORTHY Capillary blood-myocyte O₂ flux (V̇o₂) is determined by effective diffusing capacity (Do₂; mainly erythrocyte hemodynamics and distribution) and microvascular-interstitial Po₂ gradients (ΔPo₂ = Po_2 mv − Po_2 is). We show that Po_2 is demonstrates on-off asymmetry consistent with Po_2 mv and erythrocyte kinetics during metabolic transitions. A substantial transcapillary ΔPo₂ was preserved during recovery from contractions, indicative of considerable resistance to O₂ diffusion at the microvascular-interstitium interface. This reveals that effective Do₂ declines in step with V̇o₂ during recovery, as per Fick’s law.

INTRODUCTION

The ability to perform multiple bouts of muscle work (such as repetitive activities of daily living) is largely dependent on the dynamic matching between skeletal muscle oxygen delivery and utilization (Q̇o₂ and V̇o₂, respectively) (63). Limited O₂ availability following the cessation of contractions may impair muscle metabolic recovery and thereby subsequent contractile performance (34, 45). Within the skeletal muscle microcirculation, O₂ transport across the capillary wall is dictated by Fick’s law of diffusion: V̇o₂ = Do₂ × ΔPo₂; where V̇o₂ is oxygen flux, Do₂ is the diffusing capacity (determined primarily by red blood cell hemodynamics and distribution) (21, 32), and ΔPo₂ the oxygen partial pressure gradient between the microvascular and interstitial spaces (Po_2 mv and Po_2 is, respectively). This relationship establishes that transitions in metabolic demand (V̇o₂) require corresponding changes in effective Do₂ and/or ΔPo₂ to adequately support oxidative phosphorylation.

Our laboratory has recently employed dual-probe phosphorescence quenching techniques to interrogate both skeletal muscle Po_2 mv and, for the first time, Po_2 is dynamics from rest to submaximal contractions (39). This is important because it allowed the resolution of transcapillary ΔPo₂ and, therefore, examination of the mechanistic bases of O₂ diffusion from microvascular blood to interstitium during the onset of contractions. Interestingly, the significant transcapillary ΔPo₂ found at rest was largely maintained (as opposed to increased) during contractions (39, 64, 65). These findings not only support that the blood-myocyte interface provides a substantial effective resistance to O₂ diffusion (21, 32, 40, 73), but also underline that modulations in effective Do₂ (41, 44, 46) are critical in increasing V̇o₂ across the capillary wall during contractions. However, to date, the mechanisms regulating transcapillary O₂ transport during recovery from contractions remain to be elucidated. It is unknown whether the off-kinetic Po_2 is profile would track that of Po_2 mv and produce an invariant transcapillary ΔPo₂ during recovery. Differences in skeletal muscle Q̇o₂/V̇o₂ matching between on- and off-transients (22, 48, 53, 55, 56, 71) complicate attempts to make inferences from the on-responses. Resolution of the mechanisms underpinning diffusive V̇o₂ during recovery has direct relevance to the restoration of cellular energetic status and thus the ability to sustain multiple bouts of exercise (34, 45, 63).

In this context, the purpose of the present study was twofold. First, to resolve the temporal profile and determine model parameters of Po_2 is off-kinetics after termination of submaximal contractions in healthy skeletal muscle. We tested the hypothesis that Po_2 is would recover back to resting values and display considerable on-off asymmetry (i.e., fast on- and slow off-kinetics), similar to that observed previously within the microvascular space (7, 56) and in line with asymmetric capillary hemodynamics (24, 44). Second, based on our recent report of sustained transcapillary ΔPo₂ during the on-transient (39), skeletal muscle Po_2 mv off-kinetics were also evaluated to test the hypothesis that a significant transmural pressure gradient (i.e., ΔPo₂ = Po_2 mv − Po_2 is) would be maintained throughout the recovery period. Confirmation of these hypotheses would support 1) that the blood-myocyte interface provides significant effective resistance to O₂ diffusion; and 2) the critical role of erythrocyte hemodynamics and distribution (effective Do₂) in preserving the driving force for V̇o₂ across the capillary wall (ΔPo₂) during recovery from submaximal muscle contractions.

METHODS

Phosphorescence quenching experiments were performed on a total of 12 young male Sprague-Dawley rats (~3–4 mo old; 388 ± 74 g; Charles River Laboratories; Boston, MA) to examine skeletal muscle Po_2 is responses during both the on- and off-transients (part A). Five of these animals underwent additional phosphorescence quenching measurements for determination of off-transient Po_2 mv and, therefore, transcapillary ΔPo₂ responses during recovery from contractions (part B). Five out of the twelve animals investigated herein represent a subset of rats from a larger cohort (39) in which data related to the recovery period and the assessment of the on-off asymmetry have not been reported. Rats were maintained in accredited facilities (Association for the Assessment and Accreditation of Laboratory and Animal Care) under a 12:12-h light-dark cycle with food and water provided ad libitum. All procedures and protocols were approved by the Institutional Animal Care and Use Committee of Kansas State University and followed guidelines established by the National Institutes of Health.

Surgical Procedures and Experimental Protocol

All rats were anesthetized initially with a 5% isoflurane-O₂ mixture and maintained subsequently on 2–3% isoflurane-O₂ (Butler Animal Health Supply, Dublin, OH). Anesthetized rats were kept on a heating pad to maintain core temperature at ~37–38°C as measured via rectal probe. The right carotid artery was cannulated (PE-10 connected to PE-50; Intra-Medic polyethylene tubing; BD, Franklin Lakes, NJ) for continuous measurements of mean arterial pressure and heart rate (MAP and HR, respectively; BPA model 200; Digi-Med, Louisville, KY) and infusion of the phosphorescent probe Oxyphor G2 (see below). The caudal artery was cannulated (PE-10 connected to PE-50) for blood sampling and infusion of anesthetic agents. Blood samples were obtained at the end of each experimental protocol in subsets of animals (part A, n = 8; part B, n = 4) for determination of arterial Po₂ and Pco₂, O₂ saturation, systemic hematocrit, pH, and plasma lactate (Nova Stat Profile M; Nova Biomedical, Waltham, MA). Following catheter placement procedures, isoflurane inhalation was discontinued progressively, and rats were kept under anesthesia with pentobarbital sodium (50 mg/kg ia) throughout the remainder of the experiment. Anesthesia level was monitored at frequent and regular intervals via the toe-pinch and blink reflexes and supplemented as necessary.

As a result of overlapping spectral features of Oxyphor G2 and G4 probes (18, 20), separate measurements of skeletal muscle Po_2 is (left spinotrapezius; G4 probe; part A) and Po_2 mv (right spinotrapezius; G2 probe; part B) were performed in the same animals, as reported previously (39) and described below. Notably, our laboratory’s previous investigations documented no significant differences in blood flow or Po_2 mv between measurements performed in the left versus right spinotrapezius of the same animals (1). The spinotrapezius preparation also exhibits reproducible resting and contracting muscle blood flow, O₂ utilization, and Po_2 mv responses, with no time-related or ordering effects (36). Phosphorescent probes were protected from light sources, and experiments were performed in a darkened room to prevent contamination from ambient light.

Part A: Determination of Po_2 is on- and off-kinetics.

Overlying skin and fascia from the middorsal region were reflected to expose initially the left spinotrapezius muscle. The exposed muscle was moistened frequently via superfusion of Krebs–Henseleit bicarbonate-buffered solution (4.7 mM KCl, 2.0 mM CaCl₂, 2.4 mM MgSO₄, 131 mM NaCl, and 22 mM NaHCO₃; pH 7.4; equilibrated with 5% CO₂ and 95% N₂ at ~38°C), and the surrounding tissue was covered with Saran wrap (Dow Brands, Indianapolis, IN). Platinum iridium electrodes were sutured to the rostral (cathode) and caudal (anode) regions of the muscle to facilitate electrically induced contractions. Previous reports from our laboratory demonstrate that these surgical procedures do not impair the microvascular integrity or responsiveness of the spinotrapezius muscle (1). The Oxyphor probe G4 [Pd-meso-tetra-(3,5-dicarboxyphenyl)-tetrabenzoporphyrin; 10 µM solution] was delivered directly to the tissue compartment of the left spinotrapezius muscle via the microinjection technique (70) for Po_2 is measurements. Approximately four separate G4 microinjections (5–10 µL each) were performed with a 29-gauge needle and a 1-mL syringe (Exelint International, Redondo Beach, CA) along the length of the muscle dorsal aspect. A minimum of 15 min was allowed for uniform distribution of the injected probe within the muscle. Subsequently, submaximal twitch contractions were evoked for 3 min via electrical stimulation (1-Hz, 6-V, and 2-ms pulse duration; model s48; Grass Technologies, Quincy, MA). This stimulation protocol evokes an approximately four- to fivefold increase in muscle blood flow together with an approximately six- to sevenfold increase in muscle metabolic rate above resting, with either minor or no significant alterations in blood pH, consistent with moderate-intensity exercise (7, 36). The present anesthetized preparation retains vasomotor control, such that muscle blood flow increases in the same proportion with O₂ utilization as found in the exercising human (i.e., 5–6 L/min:1 L/min) (23, 63).

Part B: Determination of Po_2 mv and ΔPo₂ responses during the off-transient.

On completion of the first protocol (i.e., evaluation of Po_2 is in the left spinotrapezius muscle with the G4 probe; part A), electrodes were removed carefully, and the muscle was covered with Saran wrap. The right spinotrapezius muscle was then exposed, and electrodes sutured as described above. The Oxyphor probe G2 [Pd-meso-tetra-(4-carboxyphenyl)-porphyrin; 15–20 mg/kg dissolved in 0.4-ml saline] was infused into the carotid artery catheter as a bolus for Po_2 mv evaluation of the right spinotrapezius muscle (part B). G2 contains Pd-porphyrin cores that bind to biological macromolecules (principally albumin in plasma) (74). This facilitates its uniform distribution in the plasma, providing a signal corresponding to the volume-weighted O₂ pressure in the microvascular compartment at the primary site of diffusive O₂ transport in skeletal muscle (i.e., mainly the Po₂ within capillaries) (7, 62). A stabilization period of at least 15 min was allowed before muscle electrical stimulation using the same parameters as described above for part A. Rats were killed at the end of the experimental protocol with intra-arterial pentobarbital sodium overdose (>50 mg/kg).

Muscle Po₂ Measurements

Spinotrapezius muscle Po_2 is and Po_2 mv were measured via phosphorescence quenching using a frequency domain phosphorometer (PMOD 5000; Oxygen Enterprises; Philadelphia, PA) and the phosphorescent probes G4 (20) and G2 (18), respectively. Both probes are highly soluble in aqueous media (e.g., interstitial fluid, blood plasma) and do not permeate biological membranes within skeletal muscle (18, 20, 62). While G2 binds to albumin in the blood-forming complexes that serve as O₂ sensors, G4 operates in aqueous environments independently of albumin due to its unique polyethylene glycol surface layer. Although G4 allows Po₂ measurements within either the interstitial or microvascular compartments, it has been employed predominantly in the evaluation of Po_2 is, given that relatively small amounts are required for such measurements (20, 77, 78).

The principles of the phosphorescence quenching technique have been described in detail previously (7). Briefly, the technique applies the Stern-Volmer relationship to describe quantitatively the O₂ dependence of the phosphorescent probes as follows:

$${\text{P}\text{O}}_2=\left[\left(\tau °/\tau \right)-1\right]/\left(k_{\text{Q}}\times \tau °\right)$$

where k_Q is the quenching constant and τ° and τ are the phosphorescence lifetimes in the absence of O₂ and at a given Po₂, respectively (68). Probe-specific values of k_Q and τ° in the physiological range (pH ~7.4 and temperature of 38°C) are 273 mmHg/s and 251 µs for G2; and 304 mmHg/s and 218 µs for G4, respectively (18, 20). Spinotrapezius muscle surface temperature was measured using a noncontact infrared thermometer, and k_Q and τ° were adjusted accordingly. Phosphorescence lifetimes τ are independent of local probe concentration and insensitive to endogenous chromophores (74, 77, 78). The common end of the bifurcated light guide was placed ~2–4 mm superficial to the dorsal surface of the exposed spinotrapezius muscles. The phosphorometer modulates sinusoidal excitation frequencies between 100 Hz and 20 kHz, which allow phosphorescence lifetime measurements from 10 µs to ~2.5 ms. The excitation light (635-nm wavelength; penetration depth of ~500 µm) was focused on a randomly selected surface area of ~2 mm diameter of exposed muscle, devoid of large vessels, to minimize the potential for macrovascular influences. Po₂ was recorded at 2-s intervals at rest (i.e., precontracting baseline) and throughout the duration of the contraction and recovery periods (each lasting 3 min).

Analysis of Muscle Po₂ Kinetics

The kinetics of Po_2 is and Po_2 mv following the onset and offset of contractions were described by nonlinear regression analysis using the Marquardt–Levenberg algorithm (SigmaPlot 11.2; Systat Software; San Jose, CA). Transient Po₂ profiles were fit with either a one- or two-component model as follows:

• On-transient, one-component

  $${\text{P}\text{O}}_{2\left(t\right)}={\text{P}\text{O}}_{2\left(\text{rest}\right)}-A_1\left[1-e^{-\left(-t-{\text{TD}}_1\right)/{\tau }_1}\right]$$
• On-transient, two-component

  $${\text{P}\text{O}}_{2\left(t\right)}={\text{P}\text{O}}_{2\left(\text{rest}\right)}-A_1\left[1-e^{-\left(-t-{\text{TD}}_1\right)/{\tau }_1}\right]+A_2\left[1-e^{-\left(-t-{\text{TD}}_2\right)/{\tau }_2}\right]$$
• Off-transient, one-component

  $${\text{P}\text{O}}_{2\left(t\right)}={\text{P}\text{O}}_{2\left(\text{end-con}\right)}+A_1\left[1-e^{-\left(-t-{\text{TD}}_1\right)/{\tau }_1}\right]$$
• Off-transient, two-component

  $${\text{P}\text{O}}_{2\left(t\right)}={\text{P}\text{O}}_{2\left(\text{end-con}\right)}+A_1\left[1-e^{-\left(-t-{\text{TD}}_1\right)/{\tau }_1}\right]+A_2\left[1-e^{-\left(-t-{\text{TD}}_2\right)/{\tau }_2}\right]$$

where Po_2(t) is the Po₂ at any given time t; Po_2(rest) corresponds to the precontracting resting Po₂; Po_2(end-con) is the end-contraction Po₂ preceding the recovery period; A₁ and A₂ are the amplitudes for the first and second components, respectively; TD₁ and TD₂ are the independent time delays for each component; and τ₁ and τ₂ are the time constants (i.e., time taken to achieve 63% of the response) for each component. Goodness of fit and model parsimony were determined using three criteria: the coefficient of determination, sum of squared residuals, and visual inspection. The overall dynamics of the Po₂ profile, represented by the mean response time, was determined for both the on- and off-transients (MRT_on and MRT_off, respectively) (54):

• One-component

  $$\text{MRT}={\text{TD}}_1+{\tau }_1$$
• Two-component

  $$\text{MRT}=\left(A_1/A_{\text{total}}\right)\left({\text{TD}}_1+{\tau }_1\right)+\left(A_2/A_{\text{total}}\right)\left({\text{TD}}_2+{\tau }_2\right)$$

where A₁ and A₂, TD₁ and TD₂, and τ₁ and τ₂ are defined above, and A_total corresponds to the total change in Po₂ (i.e., A₁ + A₂) when using the two-component model during the off-transient. Because inclusion of the emergent second component of the Po₂ on-transient response underestimates the actual speed of Po₂ fall, the MRT_on analysis was constrained to the first component of the response (38). In addition, the time taken to reach 63% of A₁ (on-transient) and A_total (off-transient) was determined independent of modeling procedures (T₆₃). The area under the Po_2 mv and Po_2 is curves plotted as a function of time [Po_2(area); mmHg·s] was calculated during the 3-min recovery period to provide an index of the overall muscle Po₂ throughout the off-transient within each compartment (i.e., incorporating end-contraction and end-recovery Po₂, time delays, amplitudes, and time constants of the responses) (37). Transcapillary ΔPo₂ during recovery from contractions was determined as the difference between microvascular and interstitial values [i.e., ΔPo_2(t) = Po_2 mv − Po_2 is], as reported previously (39).

Statistical Analyses

Statistical analyses were performed using a commercially available software package (SigmaPlot 11.2; Systat Software; San Jose, CA). Differences between Po₂ kinetics parameters (part A: Po_2 is on- versus off-transient; part B: Po_2 mv versus Po_2 is during recovery) and recovery Po_2(area) (microvascular versus interstitial; part B) were determined using paired Student’s t tests. Serial MAP and HR measurements were compared across time (rest, end-contractions, and end-recovery) using one-way repeated-measures ANOVA (part A). Comparisons of MAP and HR across probe condition (G4: interstitial, G2: microvascular) were performed using two-way repeated-measures ANOVA [probe condition × time (end-contractions and end-recovery); part B]. Data that failed the normality (Shapiro-Wilk) were compared using Friedman’s repeated-measures ANOVA on ranks. Post hoc analyses were performed using Tukey’s test when a significant F ratio was detected. Significance was accepted at P < 0.05. Values are reported as means ± SD.

RESULTS

Part A: Muscle Po_2 is On- and Off-Kinetics

There were no differences in MAP (rest: 106 ± 16, end-contractions: 107 ± 13, end-recovery: 107 ± 14 mmHg; P > 0.05) or HR (rest: 357 ± 36, end-contractions: 360 ± 38, end-recovery: 359 ± 37 beats/min; P > 0.05) throughout the protocol. Arterial Po₂ was 85 ± 9 mmHg, Pco₂ 37 ± 4 mmHg, O₂ saturation 95 ± 2%, systemic hematocrit 34 ± 3%, pH 7.4 ± 0.1, and plasma lactate concentration 1.5 ± 0.3 mM.

Mean temporal muscle Po_2 is responses during both the on- and off-transients are shown in Fig. 1, and kinetics parameters derived from model fits are presented in Table 1. The Po_2 is value attained at the end of the recovery period [Po_2(end-rec)] was not different from Po_2(rest) (P > 0.05). The speed of Po_2 is fall during contractions (τ₁ and MRT) was faster than the speed of Po_2 is increase during recovery (Table 1; P < 0.05 for both). The normalized change in Po_2 is shown in Fig. 2 for both the on- and off-transients illustrates this temporal asymmetry. Model-independent assessment of the speed of Po_2 is (T₆₃) following the onset and cessation of contractions also supports these differences in on- versus off-kinetics (Table 1; P < 0.05).

Fig. 1.

Temporal muscle interstitial Po₂ (Po_2 is) profile following the onset of and recovery from contractions. Electrical stimulation was initiated at time 0 and ceased at time 180 s. Values are means ± SD. For both Po_2 is on- and off-transients, n = 12 animals.

Table 1.

Spinotrapezius muscle interstitial Po₂ kinetics following the onset of and recovery from contractions

	Means ± SD
On-transient
Po2(rest), mmHg	18.5 ± 8.1
A1, mmHg	11.4 ± 4.2
Po2(nadir), mmHg	7.1 ± 4.8
A2, mmHg	4.2 ± 2.9
TD1, s	3.7 ± 2.9
TD2, s	45.0 ± 25.0
τ1, s	14.8 ± 7.3
τ2, s	60.3 ± 23.8
MRTon, s	18.5 ± 7.3
T63, s	15.6 ± 5.3
Off-transient
Po2(end-con), mmHg	11.1 ± 5.1
A1, mmHg	6.7 ± 1.3*
A2, mmHg	6.1
Po2(end-rec), mmHg	17.9 ± 7.9
TD1, s	4.0 ± 4.0
TD2, s	70.5
τ1, s	40.9 ± 36.7*
τ2, s	46.2
MRToff, s	53.1 ± 38.3*
T63, s	48.8 ± 34.7*

Values are means ± SD. For both Po_2 is on- and off-transients, n = 12 animals. τ₁, Time constant for the first component; τ₂, time constant for the second component; A₁, amplitude of the first component; A₂, amplitude of the second component; MRT_on, mean response time for the on-transient; MRT_off, mean response time for the off-transient; Po_2(end-con), end-contraction Po₂ preceding the recovery period; Po_2(end-rec), end-recovery Po₂; Po_2(nadir), lowest Po₂ during the on-transient; Po_2(rest), precontracting resting Po₂; T₆₃, time to 63% of the final response (i.e., model-independent analysis of Po₂ dynamics); TD₁, time delay for the first component; TD₂, time delay for the second component. The majority of on-transient profiles were analyzed with the two-component model (11/12 rats), whereas most off-transient profiles were described with the one-component model (11/12 rats). See text for further details.

^*P < 0.05 vs. on-transient.

Fig. 2.

Normalized change in the mean interstitial Po₂ (Po_2 is) temporal profiles following the onset and cessation of contractions. Time 0 denotes onset of contractions for the on-transient and cessation of contractions for the off-transient. Note the marked asymmetry between Po_2 is on-off responses (i.e., fast on- vs. slow off-kinetics). For both Po_2 is on- and off-transients, n = 12 animals.

Part B: Transcapillary ΔPo₂ Responses During Recovery from Contractions

No differences in MAP (microvascular, end-contractions: 106 ± 11, end-recovery: 106 ± 13; interstitial, end-contractions: 99 ± 8; end-recovery: 98 ± 8 mmHg; P > 0.05) or HR (microvascular, end-contractions: 383 ± 38, end-recovery: 377 ± 40; interstitial, end-contractions: 377 ± 25, end-recovery: 373 ± 24 beats/min; P > 0.05) were observed between microvascular and interstitial measurements throughout the protocol. Arterial Po₂ was 80 ± 8 mmHg, Pco₂ 40 ± 3 mmHg, O₂ saturation 94 ± 2%, systemic hematocrit 36 ± 1%, pH 7.4 ± 0.1, and plasma lactate concentration 1.1 ± 0.3 mM. Analysis of Po_2 is off-kinetics in the five animals that underwent experimental procedures to assess Po_2 mv off-kinetics (part B) revealed no significant differences compared with the remaining seven animals from part A (data not shown; P > 0.05 for all kinetics parameters).

Mean temporal muscle Po_2 mv and Po_2 is responses during recovery from contractions (part B) are shown in Fig. 3. Po_2 mv was higher than Po_2 is throughout the protocol from end-contractions (25.6 ± 3.9 versus 9.1 ± 5.1 mmHg) to end-recovery (33.8 ± 3.6 versus 15.2 ± 6.9 mmHg; P < 0.05), such that the mean transcapillary ΔPo₂ during the entire recovery period was 17.6 ± 8.6 mmHg (Fig. 3, bottom). This gradient was maintained during the off-transient, given that no differences were observed between end-contraction and end-recovery ΔPo₂ values (16.6 ± 7.4 versus 18.8 ± 9.6 mmHg, respectively; P > 0.05). As depicted in Fig. 3 (inset), the overall compartmental oxygenation, as evaluated by the Po_2(area) was more than twice as large in the microvascular compared with the interstitial space during recovery from contractions (P < 0.05).

Fig. 3.

Top: temporal muscle microvascular Po₂ (Po_2 mv) and interstitial Po₂ (Po_2 is) profiles following cessation of contractions. Bottom: transcapillary Po₂ gradient [i.e., ΔPo_2(t) = Po_2 mv − Po_2 is] during recovery. Inset: mean values for the area under the Po₂ curves [Po_2(area)] in the microvascular and interstitial spaces (M and I, respectively). Po_2(area) was determined through integration of the area under the Po_2 mv and Po_2 is curves during the 3-min recovery phase. Note the pronounced ΔPo₂ between muscle microvascular and interstitial spaces during recovery from contractions. Electrical stimulation was terminated at time 0. Values are means ± SD. For both Po_2 mv and Po_2 is off-transient responses, n = 5 animals. *P < 0.05 vs. M.

The normalized change in temporal Po_2 mv and Po_2 is responses during recovery from contractions is presented in Fig. 4. There were no differences in the overall speed of the Po₂ profile during the off-transient between the microvascular and interstitial spaces (MRT, 61.7 ± 12.0 versus 55.7 ± 34.9 s; T₆₃, 61.2 ± 21.6 versus 54.8 ± 32.5 s, respectively, P > 0.05 for both).

Fig. 4.

Normalized change in mean microvascular Po₂ (Po_2 mv) and interstitial Po₂ (Po_2 is) temporal profiles following cessation of contractions. Electrical stimulation was terminated at time 0. For both Po_2 mv and Po_2 is off-transient responses, n = 5 animals.

DISCUSSION

This investigation addresses for the first time the dynamics of skeletal muscle Po_2 is and transcapillary ΔPo₂ during recovery from submaximal contractions. The principal novel findings are as follows. First, consistent with our hypothesis, Po_2 is recovered to resting values with slower off-kinetics compared with the on-transient. This is similar to the dynamic on-off asymmetry reported previously within the microvascular space (7, 56) and in agreement with that of capillary hemodynamics (24, 44). Second, and also consistent with our hypothesis, a substantial transcapillary ΔPo₂ was preserved throughout recovery from contractions. The latter resulted from a lack of difference between the overall speed of Po_2 mv and Po_2 is off-transient profiles. Collectively, these data provide important insights into the mechanistic bases for diffusive V̇o₂ across the capillary wall during recovery. Similar to the on-transient phase (39), modulations in effective Do₂ (mainly red blood cell hemodynamics and distribution) are vital to preserve transcapillary ΔPo₂ and support oxidative phosphorylation following cessation of muscle contractions.

Part A: Muscle Po_2 is On- and Off-Kinetics

Given its anatomical arrangement at the interface between the microvascular and intracellular spaces, the interstitium is directly exposed to physiological processes taking place in both of those compartments. Specifically, upstream events related to Q̇o₂ mechanisms (capillary red blood cell hemodynamics and distribution) and downstream events related to O₂ utilization mechanisms (oxidative phosphorylation) interact to determine O₂ pressures within the interstitium (35). For this reason, phosphorescence quenching-based measurements of Po_2 is kinetics are ideal to evaluate skeletal muscle Q̇o₂/V̇o₂ matching during transitions in metabolic demand. After termination of muscle contractions, adequate Q̇o₂/V̇o₂ matching (and thus Po_2 is) is essential to support metabolic recovery and sustain repeated bouts of contractile activity (34, 45).

While Po_2 mv profiles during both on- and off-transients have been reported previously in young and healthy (7, 56), aged (38), and diseased (e.g., heart failure) (12, 15) muscle, Po_2 is kinetics have been described recently only during the onset of contractions (39). The present study thus reports the novel Po_2 is dynamic profile during the off-transient in healthy skeletal muscle. As illustrated in Fig. 1, Po_2 is returned to resting values within the 3-min recovery period employed herein (see also Table 1 for kinetics parameters). Notably, a marked dynamic on-off asymmetry was observed where the speed of Po_2 is fall during contractions was approximately three times faster than the speed of Po_2 is increase during recovery (τ₁, MRT, and T₆₃; Table 1). Similar responses have been noted previously during the onset and offset of contractions within the microvascular space (i.e., fast on- and slow off-kinetics of Po_2 mv) (7, 56). The temporal asymmetry in the Po_2 is response likely results from different local Q̇o₂/V̇o₂ regulation between the contraction and recovery periods. Following the onset of muscle contractions, Q̇o₂ kinetics are generally faster than or coupled with those of V̇o₂ (22, 33, 53, 69, 71). Conversely, during the recovery phase, muscle Q̇o₂ kinetics have been found to be slower than or match those of V̇o₂ (26, 31, 48, 55). As Barstow et al. (4) indicated earlier via elegant computer simulations, slower Q̇o₂ off-kinetics relative to that of V̇o₂ ensures that there is no Q̇o₂ limitation to the restoration of muscle metabolism during recovery.

It is interesting that the on-off asymmetry evident in local Q̇o₂/V̇o₂ matching (and reflected here in the Po_2 is data) might be driven primarily by distinct (asymmetric) capillary Q̇o₂ kinetics during the onset versus offset of contractions. Previous investigations of key components of capillary gas exchange revealed that the time course of red blood cell flux (f_RBC; an important determinant of convective Q̇o₂ within the microcirculation) during recovery is considerably slower than that following the onset of muscle contractions (24, 44). This behavior is consistent with the latency to the reduction in arterial and arteriolar diameter during recovery (5, 30, 72) and implies that different mechanisms regulate capillary blood flow on- and off-kinetics (e.g., muscle pump contribution to capillary hyperemia only during the onset of contractions) (24). On the other hand, no differences are typically apparent when comparing the dynamics of muscle V̇o₂ during transitions to and from submaximal contractions (i.e., symmetrical on-off muscle V̇o₂ kinetics) (6, 43, 49, 75). Divergent results have been found, however, with respect to the symmetry of skeletal muscle phosphocreatine concentrations (a putative controller of muscle V̇o₂) during the onset and offset of submaximal exercise (66, 67). This disparity has been attributed to different exercise modes (single leg plantarflexion versus bilateral knee extension) and relative exercise intensities employed. Importantly, a distinction between muscle and pulmonary V̇o₂ must be made, given their potential temporal dissociation (asynchrony) during recovery due to circulatory transit delays (49). The latter could explain, at least in part, the divergent findings of symmetry (or lack thereof) between on- and off-transient responses of pulmonary V̇o₂ to moderate exercise (9–11, 19, 33, 50, 51, 60, 61, 67, 76). Other potential reasons for this discrepancy include different modeling procedures used to describe pulmonary V̇o₂ (19, 43), exercise modalities (3), active versus passive recovery (2), and fitness levels (28, 42). It must also be emphasized that pulmonary V̇o₂ data reflect an aggregate of muscle fibers and microvascular units with distinct individual Q̇o₂/V̇o₂ matching and recruitment profiles that may ultimately conceal (or distort) local mechanisms operating at the onset of, and recovery from, muscle contractions (8, 47, 49). The wealth of evidence presented above thus indicates that, in the presence of symmetric muscle V̇o₂ responses during the on- and off-transients, asymmetric capillary Q̇o₂ kinetics likely produce different Po₂ profiles during the onset versus offset of contractions (i.e., distinct dynamics and number of exponential components) (25, 37).

Part B: Transcapillary ΔPo₂ Responses During Recovery from Contractions

Dual-probe phosphorescence quenching was used herein to assess both Po_2 mv and Po_2 is during recovery from submaximal contractions (Fig. 3, top). This allowed us to resolve, for the first time, transcapillary ΔPo₂ (i.e., ΔPo₂ = Po_2 mv − Po_2 is; Fig. 3, bottom) and thus the driving force for V̇o₂ across the capillary wall following cessation of contractions. Consistent with our previous investigations of on-responses (39) and as mandated by Fick’s law (i.e., pressure gradients are required to drive diffusion), Po_2 mv was higher than Po_2 is during the recovery period. As reviewed recently (35), the presence of a significant, relatively large transmural ΔPo₂ throughout the recovery period supports that the microvascular blood-myocyte interface is the site of substantial resistance to diffusive O₂ transport (21, 32, 40, 73). Notably, the lack of differences between Po_2 mv and Po_2 is off-kinetics (Fig. 4) produced an invariant ΔPo₂ during recovery (Fig. 3, bottom). Integration of the current phosphorescence quenching data (mean recovery ΔPo₂; Fig. 3, bottom) with our previous anatomical assessment of the rat spinotrapezius via transmission electron microscopy (Fig. 5) (39) reveals a relatively steep ΔPo₂ from the red blood cell to sarcolemma during the off-transient (~12.1 mmHg/µm). This value is similar to that found during moderate-intensity contractions of the same muscle (~11.6 mmHg/µm) (39) and consistent with previous estimates during low- and high-intensity muscle contractions (i.e., ranging from ~7 to 15–20 mmHg/µm) (32, 40).

Fig. 5.

The oxygen transport pathway within the skeletal muscle microcirculation. This cross-sectional transmission election microscopy (TEM) picture of the rat spinotrapezius muscle shows the short diffusion distance from the red blood cell (RBC) surface to the sarcolemma (s), known as the carrier-free region (CFR; as denoted by the arrow). As reported previously, the magnitude of the rat spinotrapezius CFR is ~1.46 ± 0.55 µm (n = 16) (39). Note the thin plasma layer (p) between the RBC and capillary wall (w). f, muscle fiber; i, interstitial space; m, mitochondrion; pe, pericyte. TEM magnification: ×6,000. Scale bar: 1 µm. TEM image was acquired as described previously (39). See text for further details.

Maintenance of transcapillary ΔPo₂ during the off-transient is similar to what was found during the onset of contractions (39) and indicates that Do₂ must decline in step with V̇o₂ during recovery, as per Fick’s law (i.e., ↓V̇o₂ = ↓Do₂ × ↔ΔPo₂) (64, 65). It is remarkable that transcapillary ΔPo₂ is preserved during both the on- and off-transitions, given 1) the prevailing high-flux density along the diffusion path from the red blood cell to sarcolemma (i.e., encompassing the pathway segment that lacks an O₂ carrier and is thus known as the “carrier-free region” (see Fig. 5) (40); and 2) disparate mechanisms regulating capillary red blood cell hemodynamics and distribution (i.e., primary factors determining Do₂) during the onset and offset phases (24, 44). It thus seems that, as with the on-transient (39), the rate of blood-myocyte O₂ transport during recovery is an important regulated variable within the skeletal muscle microcirculation, as proposed earlier by Duling (16, 17).

Do₂ is thought to be determined largely by capillary hematocrit (Hct_cap) and the volume density of red blood cell-flowing capillaries (21, 32). This stems from the low O₂ diffusivity in plasma and particulate nature of microvascular blood, which render only the capillary surface area in close proximity to the red blood cell functional for transmural V̇o₂ at any given time. As noted above, the invariant transcapillary ΔPo₂ during recovery occurs at a time when V̇o₂ is declining toward resting values. This implies that Do₂ (mainly Hct_cap) decreases in proportion to V̇o₂ during the off-transient. Although the mechanisms determining changes in Hct_cap during transitions in metabolic demand remain to be elucidated (24, 44), analysis of its mathematical description may provide some clues in this regard:

$${\text{Hct}}_{\text{cap}}=\frac{{\text{Vol}}_{\text{RBC}}{f}_{\text{RBC}}}{\text{π}{({\text{Diam}}_{\text{cap}}/2)}^2{V}_{\text{RBC}}}$$

where Vol_RBC is red blood cell volume, f_RBC is red blood cell flux as defined above, Diam_cap is capillary diameter, and V_RBC is red blood cell velocity. Considering constant Diam_cap and Vol_RBC during recovery from submaximal contractions, changes in Hct_cap will be proportional to the f_RBC-to-V_RBC ratio. Ferreira et al. (24) demonstrated previously that the relationship between V_RBC and f_RBC during recovery is close to linear with an intercept different from zero. This suggests that different time courses of capillary f_RBC and V_RBC modulate the decrease in Hct_cap after cessation of contractions. As such, it appears that the interaction of factors regulating both f_RBC and V_RBC (e.g., progressive reduction in flow-mediated and metabolic vasodilation) likely dictates the decrease in Hct_cap and, ultimately, Do₂ during recovery.

It is likely that intracellular mechanisms regulating Do₂ also contribute to the maintenance of transcapillary ΔPo₂ during transitions to and from submaximal contractions (present results and Ref. 39). As reviewed recently (35), myoglobin resaturation during recovery may reinstate (increase the thickness of) the “functionally O₂ carrier-depleted region,” thereby reducing the potential for myoglobin-facilitated diffusion and intramyocyte Do₂ (40). Furthermore, whether termination of contractions removes other potential mechanism(s) promoting O₂ diffusion into the myocyte (e.g., mechanical effects altering the shape/distribution of the “carrier-free region”) requires further investigation.

Future Directions and Clinical Implications

Po_2 is off-kinetics.

The spinotrapezius muscle utilized herein exhibits a mixed fiber-type composition and oxidative capacity similar to the human quadriceps (14, 52), thus representing a useful analog of human locomotor muscle. It is expected that variations in Q̇o₂/V̇o₂ matching across the spectrum of muscle fiber types result in distinct Po_2 is off-kinetics, as observed previously for the Po_2 mv response (57). Alterations in capillary hemodynamics regulation with aging, disease (e.g., heart failure), and experimental interventions (e.g., pharmacological inhibition of nitric oxide synthase) are anticipated to reduce Po_2 is during the contraction offset (12, 13, 37, 38, 58). Given that Po_2 is represents the upstream driving force for transarcolemmal O₂ transport (35, 39), reductions in Po_2 is following cessation of contractions likely compromise O₂ diffusion into the myocyte and, therefore, metabolic recovery and the ability to perform repetitive tasks (34, 45).

Transcapillary ΔPo₂ during recovery.

The present results shed light into the determinants of transcapillary V̇o₂ during recovery from muscle contractions. As discussed above, modulation of red blood cell hemodynamics and distribution (Do₂) is crucial to preserve the pressure gradient for diffusive O₂ transport out of the capillary (ΔPo₂). Structural and functional microcirculatory impairments with aging and chronic disease likely disrupt this fine regulation, thereby reducing transcapillary O₂ transport and availability to the mitochondria. It is interesting that the healthy, young skeletal muscle can, at least to a certain extent, compensate for reductions in Q̇o₂ during hypoxia via further elevations in Do₂ during submaximal contractions (65). This compensation may occur via greater myoglobin desaturation (i.e., further reductions in the functionally O₂ carrier-depleted region, which increase the magnitude of myoglobin-facilitated O₂ diffusion) (40, 65) and modulations in capillary f_RBC, V_RBC, and/or Hct_cap (27). However, microcirculatory dysregulation characteristic of aged and diseased muscle likely limits the effectiveness of extracellular (capillary) compensatory mechanisms. Reliance on intracellular Do₂ compensation (i.e., via increased myoglobin desaturation) is also problematic, given the very low intramyocyte Po₂ values observed during submaximal and maximal contractions in healthy skeletal muscle (i.e., ~5 to 2- to 3-mmHg myoglobin Po₂) (65). In this context, both microvascular structural/functional impairments and finite intracellular O₂ conductance play a key role in determining blood-myocyte V̇o₂ and exercise tolerance in aged and diseased conditions (63). Improving or restoring skeletal muscle transcapillary ΔPo₂ may, therefore, constitute an important therapeutic goal (35).

Experimental Considerations

The potential for O₂ photoconsumption (photooxidation) with phosphorescence quenching-based measurements of Po_2 is has been discussed in detail previously (29, 39). Briefly, it is pertinent that our recent report showed no reductions in resting skeletal muscle Po_2 mv or Po_2 is over time with the current preparation (39). This refutes the possibility that an artifact created or amplified the transmural ΔPo₂ observed in the present study.

The current Po_2 mv data represent an average signal from arterioles, capillaries, and venules in proportion to their volumetric contribution within the interrogated field (35, 62). This property precludes insights into 1) intraluminal or longitudinal distributions within the microvascular network; and 2) heterogeneities among distinct vessels or branch orders. Similar considerations also apply to the Po_2 is signal acquired with the current phosphorescence quenching technique. Furthermore, given that only moderate-intensity muscle contractions were evoked in the present study, assessment of Po_2 is and transcapillary ΔPo₂ responses to heavy-intensity and maximal contractions is warranted (cf., Ref. 59).

Summary and Conclusions

The present study describes for the first time the dynamics of skeletal muscle Po_2 is and transcapillary ΔPo₂ during recovery from submaximal contractions. Consistent with our hypothesis, spinotrapezius Po_2 is recovered back to resting values with slower off-kinetics compared with the on-transient in line with the on-off asymmetry for capillary hemodynamics (i.e., fast-on and slow-off kinetics of f_RBC) (24, 44). A substantial transcapillary ΔPo₂ was preserved throughout the recovery phase, thus supporting that the microvascular-interstitium interface provides considerable resistance to O₂ diffusion. As mandated by Fick’s law, reductions in V̇o₂ following the cessation of contractions must be achieved via corresponding changes in effective Do₂ in the face of invariant transcapillary pressure gradients (ΔPo₂).

GRANTS

This work was supported, in part, by a Post-Doctoral Fellowship from the College of Human Ecology, Kansas State University, and 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

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