Transcapillary PO₂ Gradients in Contracting Muscles Across the Fibre Type and Oxidative Continuum

Abstract

In mixed fibre type skeletal muscle transcapillary PO₂ gradients (PO₂mv−PO₂is; microvascular and interstitial, respectively) drive O₂ flux across the blood-myocyte interface where the greatest resistance to that O₂ flux resides. Herein we assessed a broad spectrum of fibre type and oxidative capacity rat muscles across the rest-to-contractions (1 Hz, 120 s) transient to test the novel hypotheses that: i) slow-twitch PO₂is would be greater than fast-twitch, ii) muscles with greater oxidative capacity have greater PO₂is than glycolytic counterparts, and iii) whether PO₂mv−PO₂is at rest is maintained during contractions across all muscle types. PO₂mv and PO₂is were determined via phosphorescence quenching in soleus (SOL; 91% type I+IIa fibres and CSa: ~21 μmol min⁻¹ g⁻¹), peroneal (PER; 33% and ~20 μmol min⁻¹ g⁻¹), mixed (MG; 9% and ~26 μmol min⁻¹ g⁻¹) and white gastrocnemius (WG; 0% and ~8 μmol min⁻¹ g⁻¹) across the rest-contractions transient. PO₂mv was higher than PO₂is in each muscle (~6–13 mmHg; p<0.05). SOL PO₂is _area was greater than the fast-twitch muscles during contractions (p<0.05). Oxidative muscles had greater PO₂is _nadir (9.4 ± 0.8, 7.4 ± 0.9, and 6.4 ± 0.4; SOL, PER, MG respectively) than WG (3.0 ± 0.3 mmHg, p<0.05). The magnitude of PO₂mv−PO₂is at rest decreased during contractions in MG only (~11 to 7 mmHg; Time × (PO₂mv−PO₂is) Interaction, p<0.05). These data support that, since transcapillary PO₂ gradients during contractions are maintained in all muscle types, increased O₂ flux must occur via enhanced intracapillary diffusing conductance, which is most extreme in highly oxidative fast-twitch muscle.

Introduction

Sustained skeletal muscle contractile function and, thus, exercise tolerance, requires adequate energy production via oxidative metabolism. Most mammalian species maintain minimal muscle O₂ stores (i.e., myoglobin concentration <1 mM, Reynaferje, 1962; Hickson et al. 1981; Nemeth & Lowry, 1984; Terrados et al. 1990; Bekedam et al. 2009). Therefore, a rapid increase of pulmonary O₂ uptake (V̇O₂) coupled to red blood cell (RBC)-mediated transport to muscle tissue and existence of an appropriate driving pressure across the microvascular-myocyte interface is crucially important. The successful integration of these systems (pulmonary-cardiovascular-metabolic) to match O₂ utilization with O₂ delivery in the muscle (i.e, Q̇O₂/V̇O₂ ratio which establishes the partial pressure of O₂, PO₂), is dependent, in part, on the transmural PO₂ gradient and the diffusive properties of the blood-myocyte interface.

Thus, according to Fick’s law of diffusion, V̇O₂ = DO₂ (ΔPO₂), where V̇O₂ corresponds to the O₂ flux across a given membrane/barrier, O₂ movement is dictated by changes in the O₂ diffusing conductance of that barrier (DO₂) and the pressure difference between the relevant compartments (ΔPO₂). In skeletal muscle there are structural and functional barriers to transcapillary O₂ flux. The particulate nature of blood combined with the modest fraction of capillary wall that facilitates O₂ flux at any given instant results in the effective capillary surface area being at least two orders of magnitude less than that of mitochondria (Federspiel & Popel, 1986; Groebe & Thews, 1990; Honig & Gayeski, 1993; Golub & Pittman, 2005). This will necessitate a significant transcapillary PO₂ gradient (i.e., PO₂mv−PO₂is, microvascular and interstitial, respectively) (Hirai et al. 2018, 2019). Importantly, there is no O₂ carrier to facilitate transportation from the microvascular space into the interstitial space that immediately surrounds the muscle sarcolemma (known as the carrier-free region, CFR) (Honig & Gayeski, 1993). Given that blood-myocyte O₂ flux only occurs via that portion of the capillary wall in intimate approximation to the RBC (Federspiel & Popel, 1986) there is an attendant high O₂ flux density (i.e. flux per unit area) that increases with muscle V̇O₂ during contractions. Thus, examining the extra-myocyte PO₂ profile from RBC to sarcolemma will provide important information regarding the effective resistance to trans-membrane and trans-compartmental O₂ resistance.

Our laboratory has utilised the latest phosphorescence quenching techniques to reveal a significant PO₂ drop in that small physical space between the microvascular and interstitial compartments of the mixed fibre type spinotrapezius muscle (i.e., PO₂mv−PO₂is; Hirai et al. 2018). Thus, rapid contraction-induced increases in myocyte V̇O₂ incur commensurate falls in PO₂is and PO₂mv, such that increases in transcapillary O₂ flux (V̇O₂) must be achieved via elevated effective DO₂. However, in rat hindlimb muscles spanning the fibre type continuum (slow-twitch and fast-twitch) but with differential oxidative capacities it remains unknown whether, at rest or during contractions: 1) slow-twitch fibres support a greater interstitial-myocyte driving pressure (PO₂is) compared to fast-twitch fibres, 2) muscles with greater oxidative capacity have greater PO₂is than their predominantly glycolytic counterparts, and 3) whether PO₂mv−PO₂is is maintained throughout contractions in a similar fashion (i.e., the magnitude of PO₂mv−PO₂is is not different during contractions from that at rest) across all muscle fibre types.

Therefore, given the pronounced fibre type PO₂mv (Behnke et al. 2003; Ferguson et al. 2015; McDonough et al. 2005) and vasomotor control (Behnke et al. 2011) differences in rat hindlimb muscles, we hypothesized (Hypothesis #1) that PO₂is of slow-twitch muscle would be greater at rest and during contractions demonstrating superior Q̇O₂/V̇O₂ matching (i.e., slower rate of fall (τ; time constant) and mean response time (MRT)) compared to fast-twitch muscle. We further hypothesized (Hypothesis #2) that, in fast-twitch muscles, those with higher oxidative capacity would maintain greater PO₂is throughout contractions despite having faster kinetics (τ and MRT) characteristic of greater O₂ utilization. Lastly, by comparison with extant PO₂mv data (Behnke et al. 2003; Ferguson et al. 2015), we tested the hypothesis (Hypothesis #3) that a significant transcapillary PO₂ gradient (PO₂mv−PO₂is) would be maintained throughout contractions in muscles spanning the fibre-type and oxidative spectrum. This latter hypothesis, if correct, would support a mechanistic link between oxidative potential and transcapillary DO₂ within individual muscles (i.e., increased transcapillary DO₂ to meet increasing metabolic demands (V̇O₂) of contractions in the absence of increased PO₂mv−PO₂is compared to rest, per Fick’s law of diffusion, V̇O₂ = DO₂ × (PO₂mv−PO₂is)).

Methods

Ethical Approval

All procedures and protocols were approved by the Kansas State University Institutional Animal Care and Use Committee (IACUC No. 3762) following guidelines established by the National Institutes of Health. Experiments were also conducted in accordance with the ethical standards mandated by the Journal of Physiology (Grundy, 2015). Rats were maintained in Association for the Assessment and Accreditation of Laboratory and Animal Care accredited animal facilities under a 12:12 h light:dark cycle with food and water provided ad libitum.

Muscles selected for Fibre-type and Oxidative capacity continuum

The interstitial space PO₂ data presented in the current manuscript is the culmination of multiple ongoing investigations. All data were collected with the same procedures and described in detail below (see Phosphorescence quenching determination of PO₂mv and PO₂is). Selection of muscles in the present investigation (soleus, SOL; peroneal, PER; mixed gastrocnemius, MG; and white gastrocnemius, WG) was based on their fibre-type composition and oxidative enzyme capacity (Delp & Duan, 1996) and muscle recruitment patterns from low-to-high intensity exercise (SOL < PER/MG < WG). The SOL muscle is comprised principally of slow-twitch fibres (84% type I, 7% type IIa and 9% type IId/x) with a citrate synthase activity of ~21 μmol min⁻¹ g⁻¹ (CSa; used herein as a marker of oxidative capacity) that is utilized for posture, plantar flexion and ankle stabilization. PER and MG muscles are comprised of predominately fast-twitch fibres with a high oxidative capacity. PER (14% type I, 19% type IIa, 22% type IId/x and 45% type IIb fibres) is an ankle everter with a CSa of ~20 μmol min⁻¹ g⁻¹. The MG (3% type I, 6% type IIa, 34% type IId/x and 57% type IIb fibres) is a powerful plantar flexion muscle with a CSa of ~26 μmol min⁻¹ g⁻¹. Lastly, the WG represents fast-twitch glycolytic muscle (8% type IId/x and 92% type IIb fibres; CSa: ~8 μmol min⁻¹ g⁻¹) that is more heavily recruited at high speed/intensity (Armstrong & Laughlin, 1985; Copp et al. 2010).

The PO₂mv data herein come from previous studies within our laboratory investigating fibre-type differences in the rat hindlimb musculature where electrically induced muscle contractions were applied with electrodes sutured to the muscle surface (Behnke et al. 2003 and Ferguson et al. 2015; with WG data collected during Ferguson et al. 2015); as was performed for all novel PO₂is data.

Phosphorescence quenching determination of PO₂mv and PO₂is

In all experiments, phosphorescence quenching was performed in young Sprague-Dawley rats (<7 months old; Charles Rivers Laboratories; Boston, MA, USA) to assess microvascular and interstitial PO₂. The composition of male and female rats are presented in Figures 1 and 2A-D. Previous data investigating sex differences in PO₂is revealed no differences in the control condition (Craig et al. 2018, 2019b); therefore, male/female PO₂ data were combined for any given data set. Phosphorescence signal overlap precludes the simultaneous measurement of PO₂mv and PO₂is in the same muscle (Dunphy et al. 2002; Esipova et al. 2011) and pharmacological protocols assessing channel blockade following the current PO₂is control data (blockade data not shown) precluded the surgical exposure of hindlimb muscles for both limbs and assessment of each muscle (i.e. SOL, PER, MG and WG) within the same animal. Therefore, the current study reports data as separate animals with unpaired statistical comparisons.

Figure 1. Interstitial PO₂ from rest to contractions in muscles of differing fibre type and oxidative capacity.

Note the significantly greater PO₂is of slow-twitch oxidative soleus (SOL; circle; CSa ~21 μmol min⁻¹ g⁻¹) muscle compared to fast-twitch oxidative peroneal (PER; downward triangle; CSa ~20 μmol min⁻¹ g⁻¹) and mixed gastrocnemius (MG; square; CSa ~26 μmol min⁻¹ g⁻¹) muscles. Additionally, within fast-twitch muscles, PER and MG remained greater than the glycolytic white gastrocnemius (WG; diamond; CSa ~8 μmol min⁻¹ g⁻¹). Time zero depicts the onset of twitch contractions. Data are mean ± SD. Citrate synthase activity (CSa) data are from Delp & Duan (1996).

Figures 2A-D. Microvascular and Interstitial PO₂ and Transcapillary PO₂ from rest to contractions.

The transcapillary pressure gradient for O₂ (PO₂mv−PO₂is) at rest is maintained throughout twitch contractions. According to Fick’s Law of Diffusion [V̇O₂ = DO₂ × (PO₂mv−PO₂is)], increased metabolic demand (V̇O₂) necessitates increased diffusing conductance (DO₂) across the capillary wall during maintained, or decreased, PO₂mv−PO₂is. Time zero depicts the onset of twitch contractions. The bolded line (PO₂mv−PO₂is) is the difference between mean PO₂ of microvascular (closed circle) and interstitial (open circle) compartments. Note the reduction in PO₂mv−PO₂is following the onset of contractions in MG (Time × (PO₂mv−PO₂is) Interaction), highlighting further increases in transcapillary DO₂ to accommodate the increased myocyte V̇O₂. Data are mean ± SD with the PO₂mv and PO₂is of each muscle compared across time via Two-Way ANOVA (Time × Compartment) with Tukey’s post hoc analyses.

Surgical instrumentation

Rats were initially anesthetized with a 5% isoflurane-O₂ mixture and maintained on ~2% isoflurane-O₂ mixture (Butler Animal Health Supply) throughout the surgical exposure of hindlimb muscles. Core body temperature was maintained ~38°C, assessed via rectal thermometer, on a heating pad. Following an incision on the ventral lateral surface of the neck, the right carotid artery was isolated and cannulated (PE-10 connected to PE-50; Intra-Medic polyethylene tubing; BD, Franklin Lakes, NJ, USA) and connected to a pressure transducer for continuous mean arterial pressure (MAP; PowerLab/LabChart data acquisition system, AD Instruments) measurements and infusion of microvascular phosphorescence probes (R2 and G2). Following an incision on the ventral surface of the tail, the caudal artery was isolated and cannulated (PE-10 connected to PE-50) for blood sampling and infusion of pentobarbital sodium anesthesia. Arterial blood samples were collected following the final contraction protocol of each muscle for determination of O₂ saturation, systemic haematocrit and plasma lactate (Nova Stat Profile M; Nova Biomedical, Waltham, MA, USA).

Following catheter placement, incisions were made to carefully remove overlying skin and fascia to expose the biceps femoris. The semitendinosus was separated from the biceps femoris and the lateral saphenous vein was sutured at the ankle before reflecting the biceps femoris, exposing MG and WG muscles. For SOL and PER measurements, the MG was reflected while maintaining origin and insertion attachments. Rats were then transitioned to pentobarbital sodium anesthesia (~20 mg/kg body wt) given arterially while concentrations of isoflurane were decreased and subsequently discontinued. Toe pinch and palpebral reflexes were checked regularly to monitor the level of anesthesia, supplementing pentobarbital sodium as necessary (0.03–0.05 mL diluted to 0.3 mL with heparinized saline). The left foot was braced and secured to fix the ankle and knee joints at 90° angles. Using 6–0 silk sutures, platinum iridium wire electrodes were secured to the proximal (cathode) and distal (anode) surface regions of each muscle. Exposed muscle tissue was superfused with warmed (~38°C) Krebs-Hensleit bicarbonate-buffered solution equilibrated with 5% CO₂-95% N₂ (pH 7.4). All exposed tissue surrounding the sutured electrodes were covered with Saran Wrap (Dow Brands, Indianapolis, IN) to minimize tissue dehydration.

Experimental protocol

The microvascular phosphorescent probes Oxyphor R2 (Pd-meso-tetra(4-carboxyphenyl)porphyrin dendrimer) and Oxyphor G2 (Pd-meso-tetra-(4-carboxyphenyl)-porphoryin) were infused arterially (15–20 mg kg⁻¹ dissolved in 0.4 mL of saline) while the interstitial space probe Oxyphor G4 (Pd-meso-tetra-(3,5-dicarboxyphenyl)-tetrabenzoporphyrin) was injected into the muscle (~10 μl/injection; 10 μM) and subsequently covered with Saran Wrap to protect the muscle from ambient air. Notably, these highly soluble probes do not permeate biological membranes in skeletal muscle (Dunphy et al. 2002; Poole et al 2004; Esipova et al. 2011) and therefore remain in the compartment of interest for PO₂ measurements.

At least 15 min was allowed for Oxyphors R2 and G2 to bind to albumin (Lo et al. 1997; Wilson et al. 2006) and distribute evenly in the plasma of systemic vasculature as well as for G4 to diffuse and stabilize within the interstitial space following microinjections (Craig et al. 2018, 2019a,b; Hirai et al. 2018; Smith et al. 2007). The muscle surface temperature was measured via infrared surface thermometer during interstitial assesssments to ensure that proper k_Q and τ₀ settings of the frequency domain phosphorimeter (PMOD 5000; Oxygen Enterprises, Philadelphia, PA) were used. The common end of the light guide was positioned ~2–4 mm superficial to the lateral surface of exposed muscle tissue and in a field absent of large vessels to ensure the microvascular and interstitial regions being measured were principally of capillary-myocyte interface. Importantly, the largest contributor to vascular volume is capillary volume (~85%; Behnke et al. 2001; Poole et al. 1997).

PO₂mv (4–8 V) and PO₂is (6–8 V) were measured via phosphorescence quenching (see below) at rest and during 120-s of twitch contractions (1 Hz, 2-ms pulse duration; Grass stimulator model S88, Quincy, MA) and recorded at 2-s intervals. Following contractions PO₂ was monitored to ensure microvascular control was preserved and values returned to baseline. Following data collection, rats were euthanized via pentobarbital sodium overdose (> 100 mg kg⁻¹ i.p. or > 50 mg kg⁻¹ i.a.) followed by pneumothorax.

PO₂ Measurement and Curve-fitting

The Stern-Volmer relationship was used in calculating PO₂. Direct measurement of phosphorescence lifetime yielded PO₂ via the following equation:

$$\mathrm{P}{\mathrm{O}}_2=[\mathrm{\tau }°/\mathrm{\tau }-1]/({\mathrm{k}}_{\mathrm{Q}}\mathrm{x}\mathrm{\tau }°)$$

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. In tissues at 32.3°C (muscle surface temperature: ~31°C) the parameters for G4 were as follows: k_Q of 258 mmHg⁻¹ s⁻¹ and τ° of 226 μs (Esipova et al 2011). Parameters for PO₂mv were utilized presuming blood to be temperature regulated by core temperature (38°C). R2 parameters were k_Q: 409 mmHg⁻¹ s⁻¹ and τ°: 601 μs (Lo et al. 1997). G2 parameters were k_Q: 273 mmHg⁻¹ s⁻¹ and τ°: 251 μs (Dunphy et al. 2002). Muscle temperature does not change appreciably during the contraction protocol used herein (Craig et al. 2019), therefore the phosphorescence lifetime is affected exclusively by the O₂ partial pressure.

Data for the initial fall in PO₂ (i.e. primary PO₂ response) were obtained via curve-fitting PO₂is data points with computer software (SigmaPlot 12.5, Systat Software, San Jose, CA) while data regarding secondary responses (undershoot of PO₂ (Δ₂PO₂)) were calculated manually (see below). Using the two-component model below, the primary PO₂ responses were constrained to the primary amplitude (Δ₁) as to not overestimate magnitude and rate of PO₂ fall. The following two-component model was used to fit data over time:

$$\mathrm{Two-component}:\mathrm{P}{\mathrm{O}}_{2(\mathrm{t})}=\mathrm{P}{\mathrm{O}}_{2\mathrm{BL}}-{\mathrm{\Delta }}_1\mathrm{P}{\mathrm{O}}_2(1-{\mathrm{e}}^{-(\mathrm{t}-\mathrm{T}{\mathrm{D}}_1)/{\mathrm{\tau }}_1})+{\mathrm{\Delta }}_2\mathrm{P}{\mathrm{O}}_2(1-{\mathrm{e}}^{-(\mathrm{t}-\mathrm{T}{\mathrm{D}}_2)/{\mathrm{\tau }}_2})$$

Where PO_{2 (t)} is the PO₂ at any given time point t, PO_{2 (BL)} corresponds to pre-contracting resting baseline PO₂, 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 were determined via: 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. Mean response time (MRT) is the overall kinetics of the primary response and calculated as TD₁ + τ₁. The secondary response (Δ₂PO₂) was taken as the difference between end contractions (PO_{2 end}) and nadir (PO_{2 nadir}), where PO_{2 nadir} was calculated as [PO_{2 BL} – Δ₁PO₂] and PO_{2 end} was a 10 s average of raw data (i.e. 112–120 s). Area under the curves (PO_{2 area}) were integrated by summing each 2 s measurement across the 120 s contraction protocol.

Statistical Analyses

Arterial blood samples were compared between groups via One-Way ANOVA with Bonferroni correction for multiple comparisons. Central haemodynamic and PO₂ kinetic parameters were compared using unpaired Student’s t-tests. PO₂ profiles were assessed via Two-Way ANOVA (Time × Compartment) with Tukey’s post hoc analyses. Data are reported as mean ± standard deviation (SD) with statistical significance accepted at p < 0.05.

Results

Of the six PER PO₂mv measurements obtained, one animal was removed due to a coefficient of determination greater than 2 standard deviations from the group mean. The remaining PO₂mv and PO₂is of all animals were used in making statistical comparisons between muscles (SOL, PER, MG, and WG) and compartments (PO₂mv vs PO₂is).

Arterial Blood Samples and Central Haemodynamics

Data for arterial O₂ saturation, systemic hematocrit and lactate concentration for PO₂mv and PO₂is are presented in Table 1. MAP was significantly higher during PO₂mv measurements compared to PO₂is measurements in SOL (116 ± 13 vs 101 ± 14), MG (113 ± 14 vs 102 ± 11) and WG (114 ± 9 vs 99 ± 10 mmHg; p < 0.05 for all). Nonetheless, there is no significant effect of MAP on PO₂mv until MAP falls below 70 mmHg (Behnke et al. 2006) and equivalent ΔPO₂mv−PO₂is has been shown with these MAPs (Hirai et al. 2018). Therefore, no differences in central haemodynamic responses were expected to influence PO₂ comparisons between the microvascular and interstitial compartments.

Table 1.

Arterial Blood Samples following Microvascular and Interstitial PO₂ Measurements

	Soleus		Peroneal		Mixed Gastrocnemius		White Gastrocnemius
	PO2mv	PO2is	PO2mv	PO2is	PO2mv	PO2is	PO2mv	PO2is
Arterial pH	7.40 ± 0.06	7.41 ± 0.05	7.38 ± 0.05	7.44 ± 0.04	7.42 ± 0.06	7.41 ± 0.03	7.40 ± 0.08	7.42 ± 0.04
O2 Saturation (%)	93.6 ± 4.8	87.2 ± 12.9	91.6 ± 6.2	91.1 ± 2.1	95.3 ± 3.2 ‡	91.4 ± 3.5	93.9 ± 5.3	89.7 ± 4.2
Systemic Haematocrit (%)	35.4 ± 5.8	31.1 ± 4.3	34.5 ± 6.1	35.6 ± 1.1	36.3 ± 6.2	34.1 ± 4.3	40.0 ^	34.1 ± 2.9
Lactate (mM)	1.4 ± 0.4	1.0 ± 0.5	0.9 ± 0.3 ‡	2.4 ± 0.5 *	1.6 ± 0.2	1.4 ± 0.3 #	1.8 ± 0.2	1.4 ± 0.6 #

Data are mean ± SD and compared via One-Way ANOVA with Bonferroni correction for multiple comparisons. See Figures 2A-D for the sample size of each group.

^‡p < 0.05 vs PO₂is

^*p < 0.05 vs SOL

^#p < 0.05 vs PER

^†p < 0.05 vs MG.

^{^}Systemic Haematocrit analysis was only available for one White Gastrocnemius PO₂mv.

Interstitial PO₂: Influence of Muscle Fibre Type and Oxidative Capacity

Mean PO₂is profiles (Figure 1) and kinetics parameter data (Table 2) are presented for SOL, PER, MG and WG at rest and during 120 s of twitch contractions. Resting PO₂is was greater in slow-twitch SOL and the postural fast-twitch oxidative PER muscles compared to fast-twitch MG and WG muscles (SOL and PER > MG > WG; p < 0.05). Following the onset of contractions, the postural SOL and PER muscles fell to PO₂is _nadir and PO₂is _end levels not different from each other (p > 0.05 for both), however SOL fell at a slower rate (τ and Δ₁PO₂is/τ; p < 0.05). SOL PO₂is also fell at a slower rate (τ and MRT) compared to fast-twitch MG and WG muscles while maintaining a significantly higher interstitial-myocyte O₂ driving pressure throughout (i.e., PO₂is _nadir and PO₂is _end; p < 0.05).

Table 2.

Microvascular and Interstitial PO₂ Kinetic Parameters Following the Onset of Twitch Contractions

	Soleus		Peroneal		Mixed Gastrocnemius		White Gastrocnemius
	PO2mv	PO2is	PO2mv	PO2is	PO2mv	PO2is	PO2mv	PO2is
PO2 BL (mmHg)	29.8 ± 5.1 ‡	20.0 ± 5.3	23.9 ± 4.4 ‡*	18.3 ± 6.2	24.2 ± 3.6 ‡*	13.3 ± 2.7 *#	21.8 ± 3.6 ‡*	8.9 ± 3.3 *#†
Δ1PO2 (mmHg)	9.9 ± 2.7	10.5 ± 3.3	12.2 ± 3.2	10.9 ± 3.8	10.7 ± 3.9 ‡	7.0 ± 2.2 *#	5.2 ± 1.1 *#†	5.9 ± 2.7 *#
TD (s)	14.6 ± 5.6	13.7 ± 3.7	9.0 ± 3.5 ‡*	3.9 ± 3.0 *	5.8 ± 5.4 *	8.1 ± 2.5 *#	4.2 ± 1.2 *#	4.1 ± 1.6 *†
τ (s)	23.6 ± 7.5 ‡	16.8 ± 1.3	14.1 ± 1.8 ‡*	10.8 ± 2.6 *	13.2 ± 5.0 *	12.7 ± 3.8 *	14.7 ± 5.0 *	16.9 ± 4.6 #†
MRT (s)	38.3 ± 9.7 ‡	30.5 ± 7.5	23.1 ± 3.8 ‡*	14.7 ± 3.1 *	19.0 ± 6.5 *	20.8 ± 5.6 *#	18.9 ± 6.1 *	21.0 ± 5.2 *#
PO2 nadir	19.9 ± 4.6 ‡	9.4 ± 3.9	11.7 ± 5.0 ‡*	7.4 ± 3.3	3.5 ± 1.3 ‡*	6.3 ± 1.5 *	16.7 ± 2.5 ‡†	3.0 ± 1.4 *#†
Δ2PO2 (mmHg)	0.6 ± 0.5 ‡	1.1 ± 0.8	1.7 ± 1.9 *	2.1 ± 1.4 *	− 0.1 ± 0.2 ‡*#	0.8 ± 0.6 #	0.7 ± 0.9 †	0.5 ± 0.4 *#†
PO2 end (mmHg)	20.4 ± 4.6 ‡	10.5 ± 4.2	13.4 ± 5.2 ‡*	9.5 ± 3.4	13.4 ± 1.4 ‡*	7.1 ± 1.7 *#	17.4 ± 1.7 ‡†	3.5 ± 1.6 *#†
Δ1PO2/τ (mmHg/s)	0.47 ± 0.25 ‡	0.71 ± 0.36	0.87 ± 0.17 *	1.05 ± 0.40 *	0.82 ± 0.20 ‡*	0.60 ± 0.26 #	0.38 ± 0.12 #†	0.35 ± 0.14 *#†

PO₂mv, microvascular PO₂; PO₂is, interstitial PO₂; PO_{2 BL}, resting baseline PO₂; Δ₁PO₂ and Δ₂PO₂, amplitude of the first and second components, respectively; TD, time delay; τ, time constant; MRT, mean response time; PO_{2 nadir}, lowest response prior to secondary rise in PO₂; PO_{2 end}, PO₂ at the end of contractions; Δ₁PO₂/τ, rate of PO₂ fall. Data are mean ± SD and compared via unpaired Student’s t-tests. See Figures 2A-D for the sample size of each group.

^‡p < 0.05 vs PO₂is

^*p < 0.05 vs SOL

^#p < 0.05 vs PER

^†p < 0.05 vs MG.

Within fast-twitch fibre muscles, the more oxidative PER and MG had a greater interstitial-myocyte O₂ driving pressure at rest and throughout contractions (PO₂is _BL, PO₂is _nadir, and PO₂is _end; p < 0.05 for all) than the glycolytic WG, with greater absolute and relative fall in PO₂is (Δ₁PO₂is and Δ₁PO₂is/τ, respectively; p < 0.05 for all). In addition, integrating the area under the curves (Figure 1, PO_{2 area} shown in Figure 4) revealed a graded reduction in total PO₂is during contractions when transitioning from slow-twitch oxidative to fast-twitch glycolytic muscle (SOL > PER > MG > WG; p < 0.05).

Figure 4. Transcapillary Pressure Gradient for O₂ flux.

PO_{2 area} was determined by integrating the area under the PO₂mv and PO₂is curves throughout the 120 s of twitch contractions. The difference between microvascular and interstitial PO_{2 area} denotes the total transcapillary driving pressure for O₂ flux (i.e. transcapillary PO₂) throughout the rest-contraction transient. Note the profound reduction in transcapillary PO₂ of fast-twitch oxidative PER and MG compared to slow-oxidative SOL (likely driven via high O₂ flux density) and fast-twitch glycolytic WG (via low intracapillary DO₂; Dawson et al. 1987). Data are mean ± SD and compared via unpaired Student’s t-tests. ‡ p < 0.05 vs PO₂is; * p < 0.05 vs SOL; # p < 0.05 vs PER; † p < 0.05 vs MG.

Transcapillary O₂ Gradients in Muscles of Differing Fibre-type and Oxidative Capacity

Mean PO₂mv of extant data (Behnke et al. 2003 and Ferguson et al. 2015) and PO₂is are presented in Figures 2A-D, along with the transcapillary pressure gradient between the microvascular and interstitial compartments (PO₂mv−PO₂is). In all muscles, there was a significant pressure gradient at rest (~6–13 mmHg) that was maintained throughout contractions (Figures 2A-D, Figure 3 and Table 2: PO_{2 BL}, PO_{2 nadir}, and PO_{2 end}, p < 0.05 for all). Interestingly, there were no significant differences in Δ₁PO₂ between microvascular and interstitial compartments (p > 0.05) except for the locomotive fast-twitch oxidative MG where there was a greater reduction in PO₂mv (10.7 ± 3.9 vs 7.0 ± 2.2 mmHg, p < 0.05). In addition, there was a Time × (PO₂mv−PO₂is) interaction during MG contractions where the pressure gradient, and thus driving pressure for transcapillary O₂ flux, was reduced (Figure 2C; p < 0.001). Accordingly, Figure 4 demonstrates that the difference in total available O₂ driving pressure (PO₂mv−PO₂is PO_{2 area}) during contractions between microvascular and interstitial compartments of MG and PER is approximately half that of their slow-twitch oxidative counterpart (i.e. SOL).

Figure 3. Microvascular and Interstitial PO_{2 nadir} following the onset of contractions.

Even at the lowest portion of the rest-contraction transient, interstitial (open) PO₂ is significantly lower than the microvascular (closed) compartment in all muscles (p < 0.05). Interstitial PO_{2 nadir} exhibits a decline transitioning from slow-twitch oxidative to fast-twitch glycolytic muscle. Data are individual responses with box plots and compared via unpaired Student’s t-tests. * p < 0.05 vs SOL; # p < 0.05 vs PER; † p < 0.05 vs MG.

In Figure 4, the PO_{2 area} in the microvascular compartment demonstrates the significantly greater driving pressure, and thus O₂ availability, in the slow-twitch SOL muscle compared to the fast-twitch fibre muscles (SOL > PER = MG = WG). This single step reduction from slow-twitch to fast-twitch fibres contrasts the graded reduction present in the interstitial space (SOL > PER > MG > WG; all p < 0.05). The difference between PO₂mv and PO₂is (i.e. transcapillary PO₂) represents the contrasting muscle-specific O₂ pressure gradients across the capillary wall, with fast-twitch oxidative PER and MG muscles having ~31–54% lower transcapillary driving pressure than slow-twitch SOL and fast-twitch WG.

Discussion

The present investigation is the first to resolve the profile of skeletal muscle interstitial PO₂ during the rest-contractions transition across a broad spectrum of muscle fibre types and oxidative capacities. Consequently, these data resolve a significant transcapillary pressure gradient (PO₂mv−PO₂is) thereby revealing the presence of a significant resistance to O₂ flux in all muscles examined at rest and during contractions regardless of fibre type and oxidative capacity. Importantly, for oxidative metabolism to support contractile function and exercise tolerance, the tightly coordinated increase in O₂ flux across the microvascular-myocyte interface and into the mitochondria (Fick’s Law of Diffusion: V̇O₂ = DO₂ × ΔPO₂mv−mito), in health, is established through an effectively maintained microvascular PO₂ relative to mitochondrial PO₂ during contractions whilst V̇O₂ increases and PO₂ subsequently decreases in all compartments. Furthermore, these data demonstrate that: i) in oxidative muscles, slow-twitch fibre PO₂is falls at a slower rate (i.e. τ and MRT) than fast-twitch fibres while maintaining greater PO₂is, ii) within fast-twitch muscles, a higher oxidative capacity is supplied by a greater interstitial-mitochondrial driving pressure (i.e. PO₂is) than for glycolytic counterparts, and iii) despite varying magnitudes of transcapillary pressure gradients at rest (PO₂mv−PO₂is ~6–13 mmHg), increased O₂ utilization with contractions must be supported by effective increases in transcapillary DO₂ (i.e. RBC flux, velocity, and hematocrit (Hirai et al. 2018, 2019)) as per Fick’s law.

Slow-twitch vs. Fast-twitch Interstitial PO₂ (Hypothesis #1)

Slow-twitch fibres are primarily located in postural muscles and those recruited during lower-intensity exercise (i.e. below critical speed (Copp et al. 2010)). Having a greater capillary-to-fibre ratio and greater vasodilatory capacity compared to their fast-twitch counterparts, slow-twitch fibres evince a significantly longer PO₂mv time delay and MRT during contractions (Behnke et al. 2003, 2011; McDonough et al. 2005). Herein we demonstrate that this behavior is present within the interstitial space and precedes PO₂is falling towards its nadir/steady-state (Table 2; p < 0.05). Figure 3 shows that the PO₂is _nadir for fast-twitch muscles is also significantly lower than the slow-twitch SOL. This greater interstitial-myocyte driving pressure in slow-twitch muscle is interpreted as evidence of a better Q̇O₂is-to-V̇O₂is matching at rest and through coordinated changes in increased microvascular Q̇O₂ during contractions (muscle pump, immediate and prolonged vasodilation (Joyner & Casey, 2015; Laughlin et al. 2012; Thomas & Segal, 2004)) as well as myoglobin-mediated O₂ storage and PO₂ buffering (Type I > IIA > IIX, mostly absent in IIB fibres; Hickson 1981; Ordway & Garry, 2004). Although the fibre and oxidative distribution within and among muscles is generally more distinct in rodents compared to humans (Armstrong & Laughlin 1984, 1985, Edgerton et al. 1975, Johnson et al. 1973), there exists a substantial heterogeneity of fibre types across human muscles (i.e., type I fibres: soleus 88% vs rectus femoris 36%) as a function of depth within a given muscle (greater proportion of type I fibres in deeper regions; Johnson et al. 1973) and across elite athletic populations (i.e., sprinters <30% and distance runners >70% type I fibres; Saltin & Gollnick, 1983). Thus, although the more compartmentalized (rodent) compared to mosaic (human) distribution of fibres and their oxidative capacity, and consequent sharing of capillaries, affects energetic control to exercise (Forbes et al. 2008), the Q̇O₂is-to-V̇O₂is matching in rodent muscles provides context when interpreting human experiments where gathering such data is technically infeasible at present.

Oxidative Capacity and Fast-twitch Interstitial PO₂ (Hypothesis #2)

During voluntary exercise in humans mean intracellular PO₂ measured during contractions of major locomotor muscles falls to ~3–5 mmHg (Richardson et al. 1995; Mole et al. 1999). However, those measurements represent a mosaic of fibre types and it is known that individual muscles with higher oxidative capacity exhibit greater microvascular Q̇O₂ and microvascular-myocyte driving pressures (Behnke et al. 2003; McDonough et al. 2005). The current investigation illustrates that higher PO₂is’s are present within oxidative muscles compared to low-oxidative WG (Table 2 and Figure 1), and within fast-twitch muscles the oxidative PER and MG display faster PO₂is kinetics (i.e. τ; Table 2) compared to the primarily glycolytic WG. Faster PO₂is kinetics (PER/MG < WG) likely reflect a combination of i) higher oxidative potential (V̇O₂) compared to WG and ii) myoglobin-O₂ storage establishing greater PO₂is in PER and MG at rest with subsequent O₂ offloading during contractions enhancing the relative rate of PO₂is decline (i.e., Δ₁PO₂is/τ) in the initial ~30–60 seconds. Importantly, although myoglobin-mediated supply of O₂ for mitochondrial V̇O₂ may slow PO₂is kinetics upstream, the concentration of mammalian myoglobin is minimal (<1 mM, Reynaferje, 1962; Hickson et al. 1981; Nemeth & Lowry, 1984; Terrados et al. 1990; Bekedam et al. 2009) and the current data suggests that the magnitude of intracellular V̇O₂ in oxidative tissue outstrips myoglobin’s ability to slow the absolute rate of PO₂is decline (i.e., τ and MRT) compared to myoglobin-lacking WG (Hickson 1981; Ordway & Garry, 2004). Myoglobin is thus anticipated to enhance the amount of O₂ available for mitochondria during contractions whilst not contributing significantly to slowing the kinetic response as assessed in the interstitial space.

The initial PO₂is decline is followed by a subsequent rise in PO₂is resulting from an elevated vascular response (increased Q̇O₂; Behnke et al. 2011) and potentially transcapillary DO₂ (see Transcapillary O₂ Diffusing Capacity) on Q̇O₂is/V̇O₂is matching (Δ₂PO₂is; SOL/PER/MG > WG, Table 2). Ultimately, greater PO₂ in that compartment nearest the contracting myocyte (i.e. PO₂is) could potentially indicate a higher intramyocyte PO₂ and be crucial for limiting the amount of energy produced via glycolytic metabolism and the subsequent increase in fatigue-related metabolites (i.e., H⁺, Pi; Wilson et al. 1977; Hogan et al. 1992). In support of this notion, Richmond et al. (1999) determined, in the mixed fibre spinotrapezius muscle, that the ‘critical PO₂is’ is ~2.4–2.9 mmHg when aerobic metabolism becomes limited and glycolytic metabolism (assessed via NADH fluorescence) begins to increase. The WG in the present investigation reached ~3.0 mmHg during contractions which very likely decreased intracellular V̇O₂ reflecting the requisite preservation of the interstitial-myocyte driving pressure of O₂.

Transcapillary PO₂ Gradient (Hypothesis #3a)

To support oxidative metabolism O₂ must move from O₂-carrying RBCs across plasma, capillary wall, interstitial space, sarcolemma, cytoplasm and then across the outer mitochondrial membrane. The pathway elements and physical barriers from RBC to sarcolemma are considered to constitute the carrier free region (CFR) where PO₂ gradients are necessary to drive blood-myocyte O₂ flux. Extending upon previous work in mesentery and skin (Tsai et al. 1998; Cabrales et al. 2006; Golub et al. 2007, 2008), Hirai and colleagues (2018) recently demonstrated a transcapillary PO₂ gradient in mixed fibre-type skeletal muscle of moderate oxidative capacity (48% type I+IIa fibres with CSa of 14 μmol min⁻¹ g⁻¹; from Delp & Duan, 1996). Interestingly, the lowest PO₂is (~7 mmHg) was still greater than previously measured intramuscular PO₂ during contractions (~3–5 mmHg; Mole et al. 1999; Richardson et al. 1995), highlighting a potential intermediate step in the O₂ cascade where separate microvascular-interstitial and interstitial-myocyte pressure gradients exist. Data herein further supports the notion of an intermediate step in the O₂ cascade with the lowest PO₂is during the rest-contractions transient remaining above 6 mmHg in the more oxidative muscles (Figure 1).

Importantly, maintenance of a transcapillary and potentially interstitial-myocyte PO₂ gradient (see Figure 5) may be influenced by the metabolic demands of fast-twitch versus slow-twitch, and high-oxidative compared to low-oxidative muscles, relative to the supply of microvascular O₂ (Q̇O₂mv). Q̇O₂mv increases immediately following the onset of contractions (<4 s) (reviewed by Joyner & Casey, 2015; Laughlin et al. 2012; Thomas & Segal 2004). Specifically, RBC flux in mixed fibre muscle increases in concert with V̇O₂mv which maintains PO₂mv (~4–16 s) and even PO₂is (~6–8 s) close to resting before falling precipitously to its nadir/steady-state levels (Behnke et al. 2001, 2002; Craig et al. 2018; Hirai et al. 2018; Kindig et al. 2002). In the microvascular compartment across the differing muscle compositions herein, slow-twitch SOL had a significantly longer time delay (TD) compared to fast-twitch muscles (Table 2). Assessing the TD (i.e., maintained Q̇O₂/V̇O₂ ratio) in microvascular PO₂, compared to the interstitial compartment, may provide insight into resting arterial saturation/extraction and RBC dynamics (thus O₂ diffusing conductance) immediately following contractions onset, especially when increases in Q̇O₂ matches immediate (Behnke et al. 2002) or delayed (Grassi, 2005; Richardson et al. 2015) increases in V̇O_2. Specifically, the slow twitch SOL had equivalent delays in both compartments suggesting that O₂ delivery into the microvascular compartment (Q̇O₂mv) increased such that O₂ utilization from the blood (V̇O₂mv, equal to Q̇O₂is) increased in proportion to O₂ being utilized from the interstitial compartment (V̇O₂is). Within fast-twitch oxidative muscle, however, PO₂is fell before PO₂mv in the PER yet had a longer TD in the MG. The ability of PO₂is to be maintained close to resting longer than PO₂mv in the MG suggests that RBC flux (and thus DO₂ conductance) within the MG increased more rapidly than the PER. Beyond the initial few seconds, the MG had a significantly smaller transcapillary pressure gradient compared to rest (Time × (PO₂mv−PO₂is) interaction), further suggesting increased transcapillary DO₂.

Figure 5. The flow of O₂ from red blood cell to myocyte.

This schematic represents the flow of O₂ from the microvasculature down into skeletal muscle with O₂ delivery (Q̇O₂)-to-utilization (V̇O₂) matching establishing O₂ partial pressures (PO₂ α Q̇O₂ / V̇O₂). Red blood cells (RBC) transport O₂ (O₂ bound to haemoglobin) through the microvascular compartment (Q̇O₂mv) where, at the RBC-capillary interface, O₂ diffuses across the capillary wall into the interstitium (V̇O₂mv). As there is no haeme-storage for O₂ in the interstitial space, the O₂ flux into the interstitium (V̇O₂mv) must equal O₂ leaving the interstitium (Q̇O₂is) and also O₂ entering the myocyte (V̇O₂is; i.e., Q̇O₂mv > V̇O₂mv = Q̇O₂is = V̇O₂is). Total intramyocyte V̇O₂ is the sum of V̇O₂is and O₂ utilized from myoglobin-O₂ stores (I > IIA > IIX, absent in IIB; Hickson et al. 1981). The driving pressure for O₂ flux (i.e., PO₂) is the mass balance between Q̇O₂ and V̇O₂ for each compartment, with transcapillary O₂ flux at any given moment resulting from the coordinated balance between the pressure gradient (PO₂mv-PO₂is) and diffusing conductance (DO₂) between RBC and sarcolemma, per Fick’s Law of Diffusion: Transcapillary V̇O₂ = DO₂ × (PO₂mv−PO₂is). With a mosaic distribution pattern of myofibres and shared capillaries in vivo, fibre type differences are depicted regarding cross sectional area (Delp & Duan, 1996) and capillary:fibre ratio (Saltin & Gollnick, 1983) and capillary haematocrit when transitioning from rest to contractions (~16 to 21%; Kindig et al. 2002).

Figures 2A-D demonstrate that, even despite the transcapillary resistance to O₂ flux, the ability for Q̇O₂ into the microvascular compartment to match the increased V̇O₂ out of the microvascular compartment in individual contracting muscles is paralleled in the interstitial space regardless of muscle fibre type and oxidative capacity (i.e., PO₂mv−PO₂is is not significantly increased). Additionally, in fast-twitch oxidative muscle, the resistance to transcapillary O₂ flux may be reduced (Figures 2C and 4, also see Transcapillary O₂ Diffusing Capacity below) therefore revealing a greater proportional contribution of elevated transcapillary DO₂ to increase transcapillary O₂ flux during contractions. Previous studies have demonstrated augmented blood flow and elevated PO₂mv utilizing exogenous nitric oxide donors such as sodium nitroprusside and nitrate/nitrite supplementation in health and disease (Colburn et al. 2017, Craig et al. 2018, 2019b, Ferguson et al. 2013a,b, 2015, 2016a,b, Ferreira et al. 2006a, Glean et al. 2015). Whether enhancing Q̇O₂mv increases PO₂is to a similar degree or widens PO₂mv−PO₂is therefore increasing transcapillary driving pressure and decreasing the proportional reliance on transcapillary DO₂ for increasing O₂ flux, remains to be investigated. In studies where blood flow and O₂ delivery are altered (i.e., enzyme/channel inhibition studies and/or disease), the ability to measure blood flow and PO₂is simultaneously, and subsequently to estimate muscle V̇O₂, will enhance our understanding of the influence of extracellular PO₂ on controlling myocyte V̇O_2, metabolism and contractile function.

Transcapillary O₂ Diffusing Capacity (Hypothesis #3b)

Comparing total PO₂is following the onset of contractions with respect to PO₂mv (Figure 4) provides the opportunity to assess how V̇O₂ (increased consumption decreasing intracellular PO₂ and O₂ resistance), relative to Q̇O₂ (high flux density increasing O₂ resistance), impacts the pressure gradient between microvascular and interstitial compartments. PO₂mv falls more slowly than PO₂is (i.e. greater τ and lesser ΔPO₂is/τ) in slow-twitch SOL and fast-twitch PER of similar oxidative capacity (~20–21 μmol min⁻¹ g⁻¹; Table 2) transiently increasing the PO₂mv−PO₂is gradient (Figures 2A-B). The greater total transcapillary driving pressure difference in SOL (Microvascular-Interstitial PO_{2 area}, Figure 4) suggests that the transient increase in PO₂mv−PO₂is was consequent to faster vasodilation and high Q̇O₂mv flux density limiting transcapillary O₂ diffusing conductance (Behnke et al. 2011, Dawson et al. 1987). Fast-twitch oxidative muscles (PER and MG) conversely exhibited lower total transcapillary O₂ driving pressure emphasizing a proportionally greater transcapillary DO₂. However, the more oxidative fast-twitch MG (~26 μmol min⁻¹ g⁻¹) begins from a lower resting PO₂is and falls at the same rate as PO₂mv supporting a greater V̇O₂ throughout (Table 2 and Figure 2C), effectively reducing resistance to transcapillary O₂ flux immediately at contraction onset (i.e. decreased PO₂mv−PO₂is). Greater transcapillary PO₂ in the low-oxidative WG compared to PER and MG may mark the i) presence of low capillary:fibre surface area for transcapillary O₂ flux (Anderson & Henriksson, 1977; Dawson et al. 1987; Saltin & Gollnick, 1983) and/or ii) absence of a myoglobin O₂ store at rest (greater PO₂mv−PO₂is) which, if present, would be expected to desaturate rapidly during contractions to support mitochondrial O₂ provision and increase the relative fall in PO₂is (i.e., Δ₁PO₂is/τ) as it facilitates intramyocte DO₂. Therefore, increased DO₂mv-mito of contracting myoglobin-deficient fast-twitch muscle may extend the whole path of the carrier free region (CFR) in fast-twitch glycolytic (i.e. WG) yet be largely localized to the narrow distance between microvascular and interstitial compartments within its myoglobin-containing oxidative counterparts (PER and MG; smaller PO₂mv−PO₂is). These interpretations extend further our mechanistic interpretation of increased O₂ diffusing conductance within contracting fast-twitch compared to slow-twitch muscle using PO₂mv and blood flow measurements (Ferreira et al. 2006; McDonough et al. 2005).

Implications for Exercise Training and Disease Conditions

Maximal aerobic capacity (V̇O₂max) and submaximal exercise tolerance (critical speed) are reduced in disease (i.e., heart failure with reduced (HFrEF) and preserved (HFpEF) ejection fraction (Craig et al. 2019a; Mezzani et al. 2010, Poole et al. 2011, 2018), diabetes (Regensteiner et al. 1995), and COPD (Chiappa et al. 2008; Neder et al. 2000a)) and aging (Neder et al. 2000b; Ogawa et al. 1992). Specifically, PO₂mv (Behnke et al. 2004, 2005, 2007; Ferreira et al. 2006a; Padilla et al. 2007) and PO₂is (Craig et al. 2019b) are reduced in HFrEF and diabetes which lowers intramuscular PO₂ initiating increases in fatigue-related metabolites via increased reliance on glycolytic metabolism (Hogan et al. 1992; Wilson et al. 1977). Exercise training elevates mixed fibre PO₂mv (Hirai et al. 2014) and enhances Q̇O₂mv kinetics (Hirai et al. 2015; Laughlin & Roseguini, 2008) and myoglobin content in rats (Hickson et al. 1981) in a fibre type specific manner. These training-induced adaptations to O₂ delivery and storage likely permit tighter metabolic regulation of contracting PO₂is and intramuscular PO₂. Considering the reduction in PO₂is of slow-twitch, but not fast-twitch glycolytic, muscle in HFrEF (Craig et al. 2019a), whether disease or aging impairs, or exercise training enhances, transcapillary PO₂ gradients (i.e., impairs/improves the total driving pressure for transcapillary O₂ flux) and transcapillary DO₂ (i.e., removes/enhances the proportional reliance on transcapillary DO₂ for O₂ flux) in a fibre type-specific manner warrants further investigation.

Experimental Considerations

The current investigation employed a large established data set of microvascular and interstitial PO₂ measurements under the same anesthesia and contraction protocols that afforded three key advantages: limiting surgical time, avoiding signal overlap of phosphorescence probes and reduction of animal numbers consistent with IACUC mandates. Namely, the availability of PO₂ measurements from separate animals in existing studies ensured that the preparations in each instance remained fresh and stable and were not compromised by extended elapsed time. Specifically, PO₂is measurement across multiple muscles entails surgical exposure and localized interstitial injections and blood gas sampling. To follow this with surgical access of opposing limb muscles, systemic infusion of phosphorescence probes, PO₂mv measurements and further blood gas sampling (Hirai et al. 2018) would have extended the protocol such that cardiovascular stability may be compromised. In light of these considerations, the current methodology aligns best with the IACUC mandates of both reduction and refinement.

Since interpretation of PO₂ changes from rest to exercise in any given compartment (i.e., microvascular or interstitial) and among separate muscles are dependent on adjustments in Q̇O₂ and V̇O₂, changes in intracellular V̇O₂ during contractions could influence the measured PO₂ (i.e., WG). We therefore utilized previous blood flow data (Behnke et al. 2003, McDonough et al. 2005), capillary haematocrit at rest and during contractions (16 and 20%, respectively, Kindig et al 2002), together with current PO₂mv and O₂ saturation levels based on the O₂ dissociation curve (SOL: 35/17, PER: 23/6, MG: 24/6, and WG: 19/12%, rest/contractions, respectively) in order to infer, to the best of our ability, changes in muscle V̇O₂ during contractions (Table 3). Calculated increases in V̇O₂mv from rest to submaximal contractions were not different among fast-twitch muscles (ΔV̇O₂mv ~7 ml O₂ min⁻¹ 100g⁻¹), with slow-twitch SOL having the greatest increase (ΔV̇O₂mv ~11 ml O₂ min⁻¹ 100g⁻¹). Yet, the Q̇O₂mv/V̇O₂mv matching during contractions was greatest in slow-twitch SOL (1.084) versus fast-twitch muscles, with WG (1.058) numerically greater compared to PER/MG (1.031). As mentioned previously, the WG likely reached a ‘critical PO₂is’ which limited further increases in intracellular V̇O₂ and demonstrated the smallest reduction in O₂ saturation (−7% versus −17–18% of the oxidative muscles). However, it is the Q̇O₂mv/V̇O₂mv during contractions compared to that at rest which addresses vascular adjustments in supply (Q̇O₂mv) to different levels of demand (V̇O₂mv) between muscles. This assessment revealed that, in addition to limited intracellular V̇O₂ (and thus V̇O₂mv) of the WG, this low-oxidative muscle also had the smallest perturbation in ΔQ̇O₂mv/V̇O₂mv (−0.020 versus −0.065 in SOL/PER/MG) highlighting that, compared to the more oxidative muscles, O₂ in the WG microvascular compartment was unable to diffuse into the interstitial space even when the O₂ was available (i.e., low capillary:fibre ratio and surface area for O₂ flux keeping PO₂mv and O₂ saturation elevated despite very low PO₂is; Anderson & Henriksson, 1977; Dawson et al. 1987, Saltin & Gollnick, 1983). Thus, these calculations support the interpretations to the proportional reliance on sustained transcapillary O₂ driving pressures (PO₂mv−PO₂is of slow-twitch oxidative and fast-twitch glycolytic) and/or enhanced diffusive O₂ conductance (fast-twitch oxidative MG, and potentially PER) contributing to the present transcapillary PO₂ gradients throughout the transition to increased metabolic demands (Figure 4).

Table 3.

Microvascular Oxygen Transport from Rest to Contractions

	O2 Sat (%)	Hct (%)	Q̇m (ml min−1 100g−1)	Q̇O2mv (ml O2 min−1 100g−1)	V̇O2mv (ml min−1 100g−1)	DO2mv-mito (ml O2 min−1 mmHg−1 100g−1)	Q̇O2mv/V̇O2mv
SOL Rest	35	16	27	5.35	4.65	0.156	1.149
SOL End	17	20	85	16.83	15.52	0.761	1.084
PER Rest	23	16	8	1.58	1.45	0.061	1.095
PER End	6	20	46	9.11	8.83	0.659	1.031
MG Rest	24	16	6	1.19	1.08	0.045	1.098
MG End	6	20	42	8.31	8.06	0.602	1.031
WG Rest	19	16	8	1.58	1.47	0.067	1.078
WG End	12	20	45	8.91	8.42	0.484	1.058

Microvascular oxygen delivery (Q̇O₂mv), utilization (V̇O₂mv) and diffusing conductance (DO₂mv-mito) at rest and at the end of 120 s of twitch contractions. The Fick equation was used to calculate V̇O₂mv (i.e., V̇O₂mv = Q̇m × (CaO₂ − CvO2)) assuming the present PO₂mv is analogous to venous PO₂ (McDonough et al. 2001) and, by extension from the O₂ dissociation curve, venous blood O₂ content (Roca et al. 1992). Thus venous O₂ contents (CvO₂) were calculated [(1.34 ml O₂ (gHb)⁻¹ × (Hct/3) × %O₂ Saturation) + (PO₂mv × 0.003)] based on the constructed rat O₂ dissociation curve with Hill coefficient (n) of 2.6 to obtain O₂ saturation (O₂ Sat) from the present PO₂mv (see Table 2), an O₂ carrying capacity of 1.34 ml O₂ (gHb)⁻¹, haemoglobin (Hb) concentration using capillary haematocrit at rest and during contractions (Kindig et al. 2002), and a P₅₀ of 38 (the PO₂ at which Hb is 50% saturated with O₂). Q̇O₂mv (i.e., Q̇O₂mv = Q̇m × CaO₂) and V̇O₂mv utilizing extant blood flow (Q̇m; Behnke et al. 2003, McDonough et al. 2005) and capillary haematocrit during rest and contractions (Hct; Kindig et al. 2002). DO₂mv-mito was defined as V̇O₂mv/PO₂mv and provides an index of diffusive O₂ transport per unit of O₂ driving pressure. Q̇O₂mv/V̇O₂mv emphasizes the degree of O₂ delivery relative to O₂ utilization (i.e., higher values suggesting greater muscle perfusion per unit of intramyocyte V̇O₂).

Conclusion

These data are the first to demonstrate a significant transmural PO₂ gradient between microvascular and interstitial compartments in muscles spanning the fibre type and oxidative capacity continuum. The significant PO₂ gradient between red blood cell and adjacent sarcolemma (referred to as the “carrier free region”, CFR) present at rest is maintained throughout submaximal twitch contractions (Hypothesis #3). Since the magnitude of the transcapillary PO₂ gradient is maintained in slow-twitch SOL the interstitial-myocyte driving pressure for O₂ flux (i.e. PO₂is) of slow-twitch fibres is greater, and falls at a slower rate, compared to fast-twitch muscle (Hypothesis #1), as demonstrated previously in the microvascular compartment. Fast-twitch muscles with greater oxidative capacity maintain a higher PO₂is than their low-oxidative glycolytic counterpart to support greater O₂ metabolism (Hypothesis #2). Accordingly, there is a graded reduction in total interstitial O₂ driving pressure throughout the rest-contraction transient (PO_{2 area}) from slow-twitch oxidative to fast-twitch glycolytic. As dictated by Fick’s law of diffusion for O₂ flux across the capillary wall (V̇O₂ = DO₂ × (PO₂mv−PO₂is)), increases in O₂ flux (V̇O₂) must result from increases in effective diffusing conductance (DO₂; primarily capillary red blood cell haemodynamics and distribution). In the case of fast-twitch oxidative muscle, the transcapillary DO₂ must increase further in the face of decreased PO₂mv−PO₂is. Therefore, there is an apparent interplay between the functional influence of physical properties within muscles of differing fibre composition (i.e., capillarity and vascular reactivity) and intracellular oxidative potential (i.e., high V̇O₂is in fast-twitch muscle or decreased V̇O₂is in critically low O₂ environments) in establishing PO₂is during contractions. This dynamic interplay manifests in either sustaining the transcapillary O₂ driving pressures present at rest (PO₂mv−PO₂is of slow-twitch oxidative and fast-twitch glycolytic) and/or further enhancing diffusive O₂ conductance (decreasing PO₂mv−PO₂is in fast-twitch oxidative MG, and potentially PER) to increase O₂ flux into the space nearest the contracting myocyte.

Key Points.

• Within skeletal muscle the greatest resistance to oxygen transport is thought to reside across the short distance at the red blood cell-myocyte interface. These structures generate a significant transmural oxygen pressure (PO₂) gradient in mixed-fibre type muscle.

• Increasing O₂ flux across the capillary wall during exercise depends on i) transmural O₂ pressure gradient, which is maintained in mixed-fibre muscle, and/or ii) elevating diffusing properties between microvascular and interstitial compartments resulting, in part, from microvascular haemodynamics and red blood cell distribution.

• We evaluated PO₂s within microvascular and interstitial spaces of muscles spanning the slow- to fast-twitch fibre and high- to low-oxidative capacity spectrums, at rest and during contractions, to assess the magnitude of transcapillary PO₂ gradients in rats.

• Our findings demonstrate that, across the metabolic rest-contractions transition, the transcapillary pressure gradient for O₂ flux is i) maintained in all muscle types, and ii) the lowest in contracting highly oxidative fast-twitch muscle.