Intracellular oxygen tension limits muscle contraction-induced change in muscle oxygen consumption under hypoxic conditions during Hb-free perfusion.

Under acute hypoxic conditions, the muscle oxygen uptake (mV˙O2) during exercise is reduced by the restriction in oxygen-supplied volume to the mitochondria within the peripheral tissue. This suggests the existence of a factor restricting the mV˙O2 under hypoxic conditions at the peripheral tissue level. Therefore, this study set out to test the hypothesis that the restriction in mV˙O2 is regulated by the net decrease in intracellular oxygen tension equilibrated with myoglobin oxygen saturation (∆PmbO2) during muscle contraction under hypoxic conditions. The hindlimb of male Wistar rats (8 weeks old, n = 5) was perfused with hemoglobin-free Krebs-Henseleit buffer equilibrated with three different fractions of O2 gas: 95.0%O2, 71.3%O2, and 47.5%O2 The deoxygenated myoglobin (Mb) kinetics during muscle contraction were measured under each oxygen condition with a near-infrared spectroscopy. The ∆[deoxy-Mb] kinetics were converted to oxygen saturation of myoglobin (SmbO2), and the PmbO2 was then calculated based on the SmbO2 and the O2 dissociation curve of the Mb. The SmbO2 and PmbO2 at rest decreased with the decrease in O2 supply, and the muscle contraction caused a further decrease in SmbO2 and PmbO2 under all O2 conditions. The net increase in mV˙O2 from the muscle contraction (∆mV˙O2) gradually decreased as the ∆PmbO2 decreased during muscle contraction. The results of this study suggest that ΔPmbO2 is a key determinant of the ΔmV˙O2.

Introduction

Under acute hypoxic conditions, the decrease in O2 supply reduces muscle oxygen consumption (mO2) during exercise (Calbet et al. 2003; Lundby et al. 2004; Calbet et al. 2009). However, a series of O2 transport steps (ventilation, diffusion from the lung to the blood, bulk delivery by the cardiovascular system, and the transfer of O2 from the blood to the skeletal muscle) control the mO2 and oxidative ATP generation (Bassett and Howley 2000; Howlett et al. 2009). The way the various steps involved in the determination of the O2 contribute to the respiration during exercise under hypoxia conditions remains unclear. Recent research has reported that in hypoxia O2 might be limited by factors aside from arterial oxygen content (Lundby et al. 2004). Taken together, these facts suggest the existence of factors regulating the mO2 at the peripheral tissue level.

As the value of the mO2 depends on the O2 flux – that is, the O2 diffusion conductance (DO2) and the O2 gradient between the microvasculature and the myocytes – the decrease in mO2 under hypoxic conditions may be caused by decreases in both the DO2 and O2 gradients across the plasma membrane (Richardson et al. 1995; Takakura et al. 2010). However, previous studies have not considered the contribution of the O2 gradient as based on the intracellular oxygen tension equilibrated with myoglobin oxygen saturation (PmbO2) (Gonzalez et al. 2006). Currently, only the PcapO2 is regarded as an adequate measure of the O2 diffusion between the plasma membrane. The study assumed that the PmbO2 at rest was close to zero and that it remained unchanged during moderate‐ to high‐intensity exercise (Richardson et al. 1995). However, under hypoxic condition, Richardson et al. (2006) could not detect any deoxy myoglobin (Mb) signal at rest due to presumably a low signal to noise ratio. Therefore, it remains unclear whether muscle contraction under hypoxic conditions causes a further decrease in PmbO2. Other groups have reported that the PmbO2 decreases with the increase in exercise intensity under normoxic conditions, and that the O2 gradient from the vasculature to the cell could play a key role in the control of the O2 (Molé et al. 1999; Chung et al. 2005). These conflicting results necessitate an examination of the relationship between the mO2 and the intracellular O2 environment during muscle contraction under hypoxic conditions.

We recently demonstrated that the intracellular Mb dynamics during muscle contraction involved in enhancing the O2 flux to meet the increased muscle O2 demand (Masuda et al. 2010; Takakura et al. 2010). Indeed, the PmbO2 decreased with increasing exercise intensity under normoxic conditions (Molé et al. 1999; Takakura et al. 2010). This change in the O2 gradient as reflected by the decrease in the PmbO2 can contribute to the muscle O2 uptake. Therefore, this study considers the effect of hypoxia on the intracellular O2 environment and assesses whether the vasculature‐to‐cell O2 gradient contributes in regulating the mO2 during muscle contraction.

Materials and Methods

Experimental animals and preparation of hindlimb perfusion

Male Wistar rats were employed as subjects. All were housed in a temperature‐controlled room at 23 ± 2°C with a 12‐h light–dark cycle and maintained on a commercial diet with water ad libitum. The procedures conformed to the “Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions” (published by the Ministry of Education, Culture, Sports, Science and Technology, Japan) and was approved by the Ethics Committee for Animal Experimentation of Kanazawa University (Protocol AP‐101821).

The hindlimb perfusion was performed to the rats at 8 week of age (body weight at experiment; 256.0 ± 8.7 g). Preparation of isolated rat hindlimb and the perfusion apparatus are described in previous reports (Masuda et al. 2010; Takakura et al. 2010, 2015). All surgical procedures were performed under pentobarbital sodium anesthesia (64.8 mg/kg intraperitoneal). After finishing a surgical procedure, the rats were killed by injecting 1 mol/L KCl solution directly into the heart and a hemoglobin‐free Krebs–Henseleit buffer (NaCl, 118 mmol/L; KCl, 5.9 mmol/L; KH2PO4, 1.2 mmol/L; MgSO4, 1.2 mmol/L; CaCl2, 1.8 mmol/L; NaHCO3, 20 mmol/L; Glucose, 15 mmol/L) equilibrated with 95% O2 + 5% CO2 at 37°C was perfused into the abdominal aorta in flow through mode, at a constant flow rate. In order to adjust the perfusion pressure to approximately 80.0 mmHg, the flow rate was set to 22.2 ± 0.5 mL min−1 throughout the perfusion period. In this condition, the average perfusion pressures were 77.5 ± 3.1 mmHg, and the perfusion resistance was unchanged throughout the perfusion period. In addition, no sign of edema in the hindlimb was seen at the given flow rate. The effluent was collected from the inferior vena cava in order to measure mO2 and the lactate and pyruvate concentrations.

During the perfusion period, the oxygen supply was modulated by adjusting the O2 fraction of the equilibration gas using nitrogen gas, while 5% CO2 concentration was maintain to keep pH in perfusate. This study set three levels of O2 concentration: a 95.0% O2 fraction, a 71.3% O2 fraction, and a 47.5% O2 fraction. After establishing a sufficient equilibrium in each oxygen condition, muscle contraction was performed on a rat at the maximal twitch tension in all oxygen conditions. The order of the three oxygen conditions was randomized.

Measurement parameters

The twitch contraction protocol and measurement of Mb oxygenation and mO2 followed the previous methods (Masuda et al. 2010; Takakura et al. 2010). The sciatic nerve of the left hindlimb was then exposed and connected to two parallel stainless steel wire electrodes (Unique Medical, Tokyo, Japan) and the Achilles’ tendon was connected to a sensitive strain gauge with a string (MLT500/D, AD Instrument, Castle Hill, NSW, Australia). The stimulation pulse via the sciatic nerve derived by an electrostimulator system (Model RU‐72, Nihon Koden, Tokyo, Japan) was 1 Hz in frequency (delay, 10 μsec; duration, 1 msec) for 120 sec (120 twitch contractions). Target tension was controlled by changing the voltage of stimuli to obtain 100% of peak tension under buffer‐perfused conditions (3–8 volts). Twitch tension was calculated as the average of a series of contractions. The muscle also showed no sign of fatigue at the set stimulation intensity.

An NIRS instrument (NIRO‐300 + Detection Fibre Adapter Kit, Hamamatsu Photonics, Shizuoka, Japan) was employed to measure oxygenation of Mb at rest and during muscle contraction. The distance between the photodiode and the LED was fixed at 10 mm. The toe of the foot was secured by a clamp with the rat laid on its back. After that, the NIRS probes were firmly attached to the skin of the gastrocnemius muscle and were fixed by clamps on both sides of the muscle. During the initial period, for at least 30 sec before the start of contraction, the average fluctuation in the NIRS signals was adjusted to a reference value of zero. After the exercise protocol, the anoxic buffer (equilibrated with 95% N2 + 5% CO2 gas) was perfused for 30 min to obtain maximal Mb desaturation. The muscle then received electrical stimulation to contract for 2 min. No further increase in change in NIRS signal associated with concentration of deoxygenated Mb (∆[deoxy‐Mb]) signal was evident. The final ∆[deoxy‐Mb] signal intensity served as the normalization constant for 100% Mb deoxygenation.

The value of mO2 was calculated from the arteriovenous oxygen content difference multiplied by the flow rate, using the equation:

where flow rate is the flow in milliliters per minute, and PO2in and PO2out are the arterial and venous oxygen tensions after adjusting for the vapor pressure of water. Inflow PO2 and outflow PO2 were measured continuously using two O2 electrodes (5300A, YSI, Yellow Springs, OH) along tubing before and after perfusion of the hindlimb. The vapor pressure at 37°C was 47.03 mmHg. The solubility of oxygen in the buffer was 0.00135 μmol mL−1 mmHg−1 at 37°C (Philip and Dorothy 1971). The mO2 at rest and during muscle contraction was calculated by using the values of PO2in−PO2out averaged over 15 sec in the steady‐state conditions before and during muscle contraction.

The sampling rate for the NIRS data was 1 Hz. The other parameters (tension, perfusion pressure, O2 content at the inflow and outflow) were collected using a data acquisition system (PowerLab 8SP, AD Instruments, Australia) at a sampling rate of 1 kHz. All the data were transferred to a personal computer with acquisition software (Chart ver. 5.5.6. AD Instruments).

Data analysis

The data analysis followed our previous methods (Takakura et al. 2010, 2015). A simple moving average smoothed the ∆[deoxy‐Mb] NIRS signals using a rolling average of 5 points, which corresponds to a 5 sec timeframe(Box et al. 1978). The ∆[deoxy‐Mb] signals were calibrated against two different NIRS signal values: one at rest as 10% Mb deoxygenation and the other during steady state with anoxic buffer perfusion as 100% Mb deoxygenation. While the SmbO2 at rest could not be determined by NIRS, the value was assumed to be 90% based on previous studies reporting that the SmbO2 at rest was greater than 90% (Chung et al. 2005). The %∆[deoxy‐Mb] plots were converted to SmbO2 (%) plots using the following equation:

SmbO2 plots were fitted by the following single‐exponential equation to calculate kinetics parameters using an iterative least‐squares technique by means of a commercial graphing/analysis package (KaleidaGraph 3.6.1, Synergy Software, Reading, PA):

where BL is the baseline value, AP the amplitude between BL and the steady‐state value during the exponential component, TD the time delay between onset of contraction and appearance of SmbO2 signals, and τ the time constant of SmbO2 signal kinetics. MRT calculated by TD + τ was used as an effective parameter of the response time for Mb deoxygenation at onset of muscle contraction. Dividing 63% of AP by MRT yields a value for the time‐dependent change in Mb deoxygenation. The PmbO2 value (mmHg) at rest and steady state during muscle contraction was converted from the SmbO2 value using the following equation:

where P50 is the partial oxygen pressure required to half‐saturate Mb. A P50 of 2.4 mmHg was used for this equation, assuming a muscle temperature of 37°C (Schenkman et al. 1997). The calculated PmbO2 plots were evaluated to obtain an MRT of its kinetics using the same single exponential equation as for PmbO2. The 0.63AP/MRT for PmbO2 indicates a rate of decrease in PmbO2 at muscle contraction onset. PmbO2 at steady state was calculated by using the SmbO2 value at steady state. Since O2 partial pressure corresponds to a specific amount of dissolved O2, intracellular [O2] (μM) was calculated from the PmbO2 value at rest and at each O2 condition using the following equation:

with PmbO2 is in mmHg, and O2 solubility in buffer is 0.00135 μmol mL−1 mmHg−1 at 37°C (Philip and Dorothy 1971).

The relationship of mO2 to conductance and O2 gradient used the following equation:

where mO2 is muscle oxygen consumption, k is constant, D is conductance, PcapO2 is microvascular oxygen tension, PmbO2 is intracellular oxygen tension equilibrated with myoglobin oxygen saturation as determined from the Mb signal. Because study used a constant flow perfused hindquarter model, the analysis has assumed a proportional relationship between the outflow and capillary PO2 and has set 30 mmHg as the reference the normoxic PcapO2 value (Behnke et al. 2001; McCullough et al. 2011; Kano et al. 2014; Ferguson et al. 2015).

Statistical analyses

All data are expressed as mean ± SD. Statistical differences were examined using one‐way paired measures analysis of variance (ANOVA) (factor: O2 fraction). A Turkey–Kramer post hoc test was applied if the ANOVA indicated a significant difference. The level of significance was set at P < 0.05.

Results

In this study, the oxygen supply to the hindlimb muscle decreased from 17.1 ± 1.3 μmol min−1 at 95% O2 saturation to 12.0 ± 1.2 and 8.8 ± 0.9 μmol min−1 in the 71.3% and 47.5% O2 saturation, respectively. The correspondence between the O2 saturation and the measured O2 concentration in the perfusate confirmed the appropriateness of the experimental conditions. As the O2 supply volume decreased, the mO2 in resting muscle decreased gradually (Table 1). Even though SmbO2, and PmbO2 also decrease, the measurements could not detect a significant change in the estimated O2 gradient as reflected in (PcapO2‐PmbO2), and did not reveal any significant alteration. Under all hypoxia conditions, the lactate to pyruvate ratio (L/P) showed no increase (Table 1).

Table 1

Muscle oxygen consumption and intracellular O2 parameter at rest during hindlimb perfusion with different O2 fraction

Parameter	Unit	O2 fraction in pefusate
		47.5%	71.3%	95.0%
Inflow PO2	mmHg	291.1 ± 37.4a, b	399.1 ± 48.0a	566.9 ± 53.7
[O2] in perfusate	μmol/L	18.0 ± 2.1a, b	24.5 ± 2.7a	35.1 ± 3.0
Outflow PO2	mmHg	112.3 ± 20.7a, b	170.5 ± 31.8a	291.1 ± 37.4
Relative outflow PO2		0.41 ± 0.08a, b	0.63 ± 0.12a	1.00 ± 0.15
Estimated PcapO2	mmHg	12.4 ± 2.3a, b	18.8 ± 3.5a	30.0 ± 4.6
mV˙O2	μmol g−1 min−1	0.34 ± 0.05a, b	0.43 ± 0.07a	0.55 ± 0.12
SmbO2	%	55.0 ± 7.9a, b	74.1 ± 11.7a	90.0 ± 0.1
PmbO2	mmHg	3.1 ± 0.9a, b	8.5 ± 4.6a	21.7 ± 0.2
Intracellular [O2]	μmol/L	4.1 ± 1.2a, b	11.5 ± 6.2a	29.3 ± 0.3
PcapO2‐PmbO2	mmHg	9.3 ± 3.0	10.3 ± 5.2	8.3 ± 4.5
L/P		19.6 ± 3.0	17.8 ± 2.3	18.3 ± 2.0

Values are mean ± SD (n = 5 in each condition). Inflow PO2, oxygen tension before perfusion of the hindlimb; [O2] in perfusate, O2 concentration in perfusate; Outflow PO2, oxygen tension after perfusion of the hindlimb; Relative outflow PO2, relative value of outflow PO2 based on the outflow PO2 value at 95.0%O2 fraction; Estimated PcapO2, estimated microvasucular oxygen tension based on 30 mmHg of PcapO2 at 95.0%O2 fraction. mO2, muscle oxygen consumption; SmbO2, intracellular O2 myoglobin saturation; PmbO2: intracellular O2 tension equilibrated with myoglobin O2 saturation; Intracellular [O2], intracellular O2 concentration; PcapO2‐PmbO2, the difference in oxygen tension between PcapO2‐PmbO2; L/P, lactate to pyruvate ratio measured in effluent perfusate.

P < 0.05 versus 95.0% condition

P < 0.05 versus 71.3% condition.

Relative to the resting mO2, muscle contracting at a similar tension (77‐83 g) increased mO2 under all O2 supply conditions. The representative time course of the change in twitch tension at each O2 fraction condition was shown in Figure 1. However, the largest increase occurred at 95% O2, where the ∆mO2 rose by 0.28 μmol g−1 min−1. At 47.5% and 71.3% O2 saturation, ∆mO2 rose only by 0.11 and 0.16 μmol g−1 min−1, respectively. Even though mO2 increased under hypoxia conditions, it increased much less than muscle under normoxic condition (Table 2).

Figure 1

Representative muscle tension generation during muscle contraction for 120 sec under each O2 fraction conditions. Maximal twitch muscle contractions were elicited every 1 sec (1 Hz) by stimulating sciatic nerve. The time courses of the change in twitch tension at each O2 fraction condition are shown. No sign of fatigue was observed during muscle contraction regardless of the O2 fraction conditions.

Table 2

Muscle oxygen consumption and intracellular O2 parameter during muscle contraction during hindlimb perfusion with different O2 fraction

Parameter	Unit	O2 fraction in perfusate
		47.5%	71.3%	95.0%
Muscle tension	g	76.7 ± 15.2	81.5 ± 8.9	82.8 ± 12.9
mV˙O2	μmol g−1 min−1	0.45 ± 0.08a, †	0.60 ± 0.07a	0.87 ± 0.15
∆mV˙O2	μmol g−1 min−1	0.11 ± 0.04a, b	0.16 ± 0.07a	0.28 ± 0.05
Outflow PO2	mmHg	104.9 ± 20.6a, †	158.7 ± 30.1a	252.2 ± 41.1
Relative outflow PO2		0.42 ± 0.08a, b	0.63 ± 0.12a	1.00 ± 0.15
Estimated PcapO2	mmHg	12.5 ± 2.4a, b	18.9 ± 3.6a	30.0 ± 4.9
SmbO2 kinetics
Steady‐state value	%	12.3 ± 8.0a, b	42.0 ± 16.6a	68.7 ± 3.0
AP	%	−42.7 ± 7.5a	−32.1 ± 8.8	−21.4 ± 3.0
MRT	s	39.4 ± 7.8	37.3 ± 8.0	42.4 ± 11.8
0.63AP/MRT	% s−1	−0.69 ± 0.10a	−0.57 ± 0.23a	−0.34 ± 0.11
PmbO2 kinetics
Steady‐state Value	mmHg	0.4 ± 0.3a, b	2.1 ± 1.2a	5.5 ± 0.9
AP	mmHg	−2.7 ± 0.8a	−6.5 ± 3.8a	−16.2 ± 0.9
MRT	sec	30.6 ± 5.4	35.3 ± 8.2	33.4 ± 11.7
0.63AP/MRT	mmHg sec−1	−0.03 ± 0.07a	−0.13 ± 0.18a	−0.41 ± 0.14
Intracellular [O2]	μmol/L	0.5 ± 0.4a, b	2.8 ± 1.7a	7.4 ± 1.2
PcapO2‐PmbO2	mmHg	12.1 ± 2.5a, a	16.8 ± 3.8a	24.5 ± 2.5
∆L/P		3.6 ± 3.4a	2.5 ± 1.7	1.8 ± 1.0

Values are mean ± SD (n = 5 in each condition). mO2, muscle oxygen consumption; ∆mO2, the net increase in mO2 due to muscle contraction; Outflow PO2: oxygen tension after perfusion of the hindlimb, Relative outflow PO2, relative value of outflow PO2 based on the outflow PO2 value at 95.0 %O2 fraction; Estimated PcapO2, estimated microvasucular oxygen tension based on 30 mmHg of PcapO2 at 95.0% O2 fraction; SmbO2, intracellular O2 myoglobin saturation; AP is the amplitude between BL (baseline) and the steady‐state value during the exponential component; MRT is the time required to reach 63% of AP from the onset of muscle contraction. 0.63AP/MRT is calculated by dividing 0.63AP by MRT; PmbO2, intracelluar O2 tension equilibrated with myoglobin O2 saturation; Intracellular [O2], intracellular O2 concentration; PcapO2‐PmbO2, the difference in oxygen tension between PcapO2‐PmbO2; ∆L/P, the net increase in lactate to pyruvate ratio measured in effluent perfusate.

P < 0.05 versus 95.0% condition.

P < 0.05 versus 71.3% condition.

In contrast, L/P increased with declining O2 supply. L/P increased significantly higher at 47.5% O2 (3.6 ± 3.4) than at 95.0% O2 (1.8 ± 1.0). At 95.0% O2 the muscle contraction causes no significant change in the L/P ratio, consistent with previous observations (Takakura et al. 2010).

During muscle contraction, the steady SmbO2 and PmbO2 still decreased with decreasing oxygen supply. However, the SmbO2 and PmbO2 kinetics did not show a significant difference (Table 2). Figure 2 shows representative ∆[deoxy‐Mb] kinetics during different levels of O2 delivery conditions and during anoxic perfusion, as assessed by NIRS. The roman numerals I, II and III represent the NIRS signal response to 1 Hz maximal twitch contractions at 95.0%, 71.3% and 47.5% O2 fraction condition, respectively. In protocol IV, the noncontracting muscle received a perfusate equilibrated with 95% N2 + 5% CO2.

Figure 2

Representative time courses of the ∆[deoxy‐Mb] NIRS signals during muscle contraction under different O2 fraction conditions (I–III) and during anoxia perfusion (IV). The arrows indicate the onset of muscle contraction (I–III) and anoxic perfusion (95%N2 + 5%CO 2; IV). Protocols I, II and III show the ∆[deoxy‐Mb] signals during maximal twitch contraction under 95.0%, 71.3% and 47.5% O2 fraction conditions in the Hb‐free perfusion model. The ∆[deoxy‐Mb] signals changed immediately at the onset of contraction and reached the steady state. As protocol IV was conducted after finishing protocol III, desaturated Mb already existed at a certain level at rest before perfusing anoxia buffer.

Figure 3 shows the net increase in mO2 (∆mO2) relative to its resting value during muscle contraction in each O2 fraction condition. The ∆mO2 due to muscle contraction (0.11 ± 0.04, 0.16 ± 0.07, and 0.28 ± 0.05 μmol g−1 min−1 at 47.5%, 71.3%, and 95% O2 fraction) decreased progressively with hypoxia. Under resting conditions, the measurements indicate PmbO2 values of 3.1, 8.5, and 21.7 mmHg at 47.5, 71.3, and 95% O2. With muscle contraction, the PmbO2 values decrease correspondingly to 0.4, 2.1, and 5.5 mmHg. Figure 4 shows then net decrease in PmbO2 (∆PmbO2) due to muscle contraction (2.50 ± 0.81, 6.12 ± 3.52, and 16.10 ± 0.87 mmHg in 47.5, 71.3, and 95% O2 fraction conditions, respectively). Even though PmbO2 decreases under all exercise conditions, the value changes the most at 95% O2. Figure 5 shows the relationship between the ∆mO2 and the ∆PmbO2 in each O2 fraction condition. During muscle contraction, the decrease in oxygen supply caused a decrease in the ∆PmbO2, which led to a decrease in the ∆mO2.

Figure 3

Change in net increase in muscle O2 consumption (∆mO2) due to muscle contraction for each O2 fraction. The ∆mO2 due to muscle contraction decreased with the decrease in the O2 supply volume. ∆mO2: net increase in muscle oxygen consumption due to contraction. Values are expressed as means ± SD (n = 5). *P < 0.05 vs. 95.0% O2 fraction.

Figure 4

Net decrease in intracellular oxygen tension (∆PmbO2) due to muscle contraction for each O2 fraction. The ∆mO2 due to muscle contraction decreased with the decrease in the O2 supply volume. PmbO2 during contraction, hatched bars: net decrease in PmbO2. ∆PmbO2: net decrease in intracellular oxygen tension equilibrated with O2 saturation myoglobin. Values are expressed as means ± SD (n = 5). *P < 0.05 vs. 95.0% O2 fraction.

Figure 5

Relationship between net decrease in intracellular oxygen tension (∆PmbO2) and net increase in muscle oxygen consumption (∆mO2) during muscle contraction. The ∆PmbO2 and ∆mO2 gradually decreased as the O2 supply volume decreased. The relationship between the ∆PmbO2 and the ∆mO2 was represented with a line graph. ∆mO2: net increase in muscle oxygen consumption due to contraction. ∆PmbO2: net decrease in intracellular oxygen tension equilibrated with O2 saturation myoglobin. Each data point represents a mean ± SD. n = 5 × 3 points. *P < 0.05 for ∆mO2 parameter. † P < 0.05 for ∆PmbO2 parameter.

The linear relationship between ∆mO2 and ∆PmbO2 suggests that the O2 gradient as reflected in (PcapO2‐PmbO2) has changed. Using an estimate PCapO2, the analysis shows that indeed ∆mO2 changes linearly with (PcapO2‐PmbO2) (Fig. 6).

Figure 6

Relationship between delta change in muscle oxygen consumption (∆mO2) and estimated O2 gradient between microvascular oxygen tension (PcapO2) and intracellular oxygen tension (PmbO2) under exercising condition. Delta change in mO2 due to muscle contraction increased linearly as a function of estimated O2 gradient (PcapO2‐PmbO2). Regression line is based on mean values (mO2 = 0.020 × (PcapO2‐PmbO2), R  = 0.99, n = 5 in each point). Each data point represents a mean ± SD.

Discussion

Intracellular O2 environment under hypoxic conditions

As oxygen delivery decreases, the PmbO2 at rest decreased correspondingly from 21.7 to 8.5 to 3.1 mmHg. The mO2 decreases correspondingly from 0.55 to 0.43 to 0.34 μmol g−1 min−1. It might appear that the decreasing O2 supply leads consequently and directly to the decreasing mO2. Yet in normoxic muscle the decreasing intracellular O2 leads to an increasing mO2, which implies a critical role for the O2 gradient instead of the O2 supply alone. An analysis of the O2 gradient as reflected in (PcapO2‐PmbO2) shows that despite the decreasing intracellular O2, the O2 gradient appears to have increased during hypoxia. The low mO2 in resting muscle most likely creates a shallow gradient, which obscures measurement accuracy. Nevertheless, the constant L/P ratio would argue that the O2 supply and mO2 have not compromised oxidative metabolism, which would require glycolysis to compensate for any energy deficit. Consistent with a previous findings, the decreasing O2 supply did not affect the low mO2 at rest (Shiota and Sugano 1986).

However, when muscle begins to contract, mO2 rose and the PmbO2 decreased further from the resting levels. At 47.5% O2, the PmbO2 drops below the critical PO2 level that limits oxidative phosphorylation in mitochondria (1.5 mmHg) (Kreutzer et al. 1992). L/P rises more at 47.5% O2 than at 95.0% O2. However, the drop in PmbO2 also expands the O2 gradient under all O2 saturation conditions during muscle contraction. But the widening O2 gradient does not sufficiently enhance the O2 flux to meet the cell's oxidative needs. Anaerobic glycolysis commences to supplement the energy demand, especially as the buffer PO2 drops to 47%.

These findings indicate that even under hypoxia mO2 rises during contraction. Given the same O2 saturation conditions, the rise in mO2 required an increase in DO2 or (PcapO2‐PmbO2) as shown in equation (Richardson et al. 1995; Takakura et al. 2010). Even though the PmbO2 decreases, the O2 gradient has widened to increase the O2 flux to balance out the O2 supply and demand. A decreasing PmbO2 with a rising mO2 agree with the previously reported observations (Molé et al. 1999; Takakura et al. 2010). In contrast, Richardson et al. (1995) reported that under normoxic or hypoxic condition, the PmbO2 did not decrease proportionally to muscle contraction, once the contraction has exceeded about 40% of maximum work exercise workload. Even under hypoxic condition, the Mb desaturation level does increase as the leg O2 rose to its maximum level (Richardson et al. 1995). In addition, the PmbO2 reaches a low level (<5 mmHg) in the skeletal muscle, even when moderate levels of work were performed (Molé et al. 1999; Chung et al. 2005; Takakura et al. 2010). These findings have led researchers to ignore the PmbO2 variable from the formula for the calculation of the amount of O2 consumption in the muscle tissue in hypoxic environments (Gonzalez et al. 2006).

However, probably due to a signal‐to‐noise ratio limitation, Richardson et al. (2006) could not detect the PmbO2 at rest in hypoxic muscle. As a result, the study could not determine the true magnitude of the change in the intracellular oxygen environment during exercise. Our study's unique experimental model, with the subjects placed under Hb‐free perfusion, suggests that changes in the intracellular oxygen environment and the O2 gradient can contribute to the ΔmO2 during hypoxia. In addition, the PmbO2 at the resting muscle did not reach a nadir as a result of the hypoxia. Instead, the PmbO2 can decreased further during the muscle contraction even in the hypoxic conditions.

Change in PmbO2 from muscle contraction modulating change in mO2 under Hb‐Free perfusion

The study investigated whether the mO2 during muscle contraction in various oxygen concentration conditions depend on the changes in the PmbO2 or the O2 gradient. Because the oxygen delivery remained constant at any muscle contraction condition in the perfusion model at a constant flow, the decrease in PmbO2 due to muscle contraction widen the O2 gradient between the microvasculature and myocyte as reported in the our previous study (Takakura et al. 2010). This change contributes the increase in mO2 due to muscle contraction. This interpretation is proven by the following equation;

Because kDO2 does not significantly contribute to the increase in mO2 due to muscle contraction in the constant‐flow hindlimb perfusion model, the mO2 cannot increase unless the O2 gradient expands by decrease in PmbO2 in our previous studies (Takakura et al. 2010, 2015). In the present study, the results have confirmed the hypothesis that the delta change in PmbO2 and in the O2 gradient contributes to delta increase in mO2 during muscle contraction. Our results show that under all oxygenation conditions, mO2 rises but PmbO2 continues to decline by muscle contraction between 77% and 88% of maximal voluntary contraction (MVC). However, rise in the mO2 and O2 gradient expansion under hypoxia conditions are much less than the corresponding changes under normoxic conditions. The observation then agree with the studies by Molé et al. (1999) and Takakura et al. (2010), which indicate the progressive expansion of the O2 gradient between the PcapO2 and the PmbO2 during muscle contraction contribute to the O2 flux supporting the rising mO2. No PmbO2 plateau even under hypoxia condition seems apparent at MVC well above 40%.

Moreover, a changing mO2 in constant flow model also supports a PmbO2 contribution. In constant flow perfusion, a constant flow‐induced vasodilatation occurs at a given flow rate. As a consequence, the model maintains a constant diffusion conductance throughout the perfusion (Hepple et al. 2003). Consequently, any change in ∆mO2 must have a contribution from PmbO2. Therefore, the mechanism behind the mO2 in hypoxic environments, includes then a contribution PmbO2 (Molé et al. 1999; Takakura et al. 2010).

The mechanism of the oxygen transport to the mitochondria in the myocytes is dependent on the Mb‐mediated oxygen transport or the dissolved O2 flux. The contribution from the Mb‐mediated oxygen transport increases with decreasing PmbO2. Below the equipoise diffusion PO2 value (1.77 mmHg), 50% of the total oxygen transport to the mitochondria stems from the Mb‐mediated oxygen transport (Lin et al. 2007). The PmbO2 during muscle contraction in the 47.5% O2 fraction condition was 0.4 ± 0.3 mmHg. As this was below the equipoise diffusion PO2 value, the Mb‐mediated oxygen transport mechanism will dominate. In addition, any increase in Mb concentration will raise the equipoise PO2 (Lin et al. 2007; Takakura et al. 2015). Because people acclimated to high altitudes often have a high concentration in Mb, muscle contraction under hypoxic conditions in these people may also benefit from Mb‐mediated transport (Reynafarje 1962; Reynafarje and Morrison 1962).

Van der Laarse et al. (2005) have examined about the value of the diffusion coefficient for oxygen from extracellular region in muscle. The diffusion coefficient is important because it is a determinant of the extracellular oxygen tension at which the core of muscle fibers becomes anoxic (PO2crit). According to Hill (1965), PO2crit at the maximum rate of oxygen consumption (O2max; in nmol mm−3 sec−1) is given by

where CSA is the cross‐sectional area of the cell (mm2), D is the diffusion coefficient for oxygen in the muscle cell (mm2/sec), and α is the solubility of oxygen in muscle cell (mmol/L per mmHg). Dα is known as Krogh's diffusion coefficient. Dα seems to depend on fat content, extracellular space, and unknown factors (Jones and Kennedy 1986; Dutta and Popel 1995; Baranov et al. 2000). Indeed, Dα increased with enlargement of extracellular space in the preparation (van der Laarse et al. 2005). However, the results of the previous study indicated that Hill's model for oxygen diffusion in valid for single muscle fibers without myoglobin and a fairly homogeneous mitochondrial distribution consuming oxygen at the maximum rate. Because above Hill's equation is based on the following assumption: (1) the cross section of the fiber is a circle, and oxygen diffuses in the radial direction only; (2) O2 is distributed homogeneously in the cells; (3) O2 by mitochondria is independent of local intracellular PO2; and (4) myoglobin‐facilitated oxygen diffusion is negligible, the role of Mb was not taken account of above Dα. We have conducted the maximal twitch contraction under three O2 fraction conditions to the same individual in the present perfusion model. Moreover, as the order of the three O2 condition was randomized and no sign of edema in the hindlimb was seen at the given flow rate, change in extracellular space would be almost none. Thus, Dα in the above equation would not been affected throughout the perfusion experiment in this study. On the other hand, others and we reported the effect of Mb on the intracellular O2 diffusion to the mitochondria (Lin et al. 2007; Takakura et al. 2010, 2015). As mentioned above, Lin et al. (2007) reported that 50% of the total oxygen transport to the mitochondria stems from the Mb‐mediated oxygen transport below the equipoise diffusion PO2 value (1.77 mmHg). Also, we reported that decrease in PmbO2 directly contribute the expansion of the O2 gradient to lead the increased mO2 (Takakura et al. 2010, 2015). As for a key factor regulating O2 diffusion to intracellular mitochondria, the change in PmbO2 might be one of candidates, because the change in PmbO2 affects O2 gradient, Mb‐mediated O2 transport and mitochondria respiratory.

Conclusion

This study investigated the effects of reduced oxygen supply on the PmbO2 at rest and during muscle contraction, as well as the effect of the ΔPmbO2 on the ΔmO2 during muscle contraction. Our findings showed that the resting PmbO2 decreases with the decrease in oxygen supply. However, the decrease in PmbO2 expands the O2 gradient, which then supports the rising ∆mO2 during muscle contraction even under hypoxic conditions. The results suggest that the ΔPmbO2 is a key determinant factor of the ΔmO2.

Conflict of interest

There is no conflict of interest for this study.