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

Abstract 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 O 2 supply reduces muscle oxygen consumption (m _ VO 2 ) during exercise (Calbet et al. 2003;Lundby et al. 2004;Calbet et al. 2009). However, a series of O 2 transport steps (ventilation, diffusion from the lung to the blood, bulk delivery by the cardiovascular system, and the transfer of O 2 from the blood to the skeletal muscle) control the m _ VO 2 and oxidative ATP generation (Bassett and Howley 2000;Howlett et al. 2009). The way the various steps involved in the determination of the _ VO 2 contribute to the respiration during exercise under hypoxia conditions remains unclear. Recent research has reported that in hypoxia _ VO 2 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 m _ VO 2 at the peripheral tissue level. As the value of the m _ VO 2 depends on the O 2 fluxthat is, the O 2 diffusion conductance (DO 2 ) and the O 2 gradient between the microvasculature and the myocytesthe decrease in m _ VO 2 under hypoxic conditions may be caused by decreases in both the DO 2 and O 2 gradients across the plasma membrane (Richardson et al. 1995;Takakura et al. 2010). However, previous studies have not considered the contribution of the O 2 gradient as based on the intracellular oxygen tension equilibrated with myoglobin oxygen saturation (P mb O 2 ) (Gonzalez et al. 2006). Currently, only the P cap O 2 is regarded as an adequate measure of the O 2 diffusion between the plasma membrane. The study assumed that the P mb O 2 at rest was close to zero and that it remained unchanged during moderate-to highintensity 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 P mb O 2 . Other groups have reported that the P mb O 2 decreases with the increase in exercise intensity under normoxic conditions, and that the O 2 gradient from the vasculature to the cell could play a key role in the control of the _ VO 2 (Mol e et al. 1999; Chung et al. 2005). These conflicting results necessitate an examination of the relationship between the m _ VO 2 and the intracellular O 2 environment during muscle contraction under hypoxic conditions. We recently demonstrated that the intracellular Mb dynamics during muscle contraction involved in enhancing the O 2 flux to meet the increased muscle O 2 demand Takakura et al. 2010). Indeed, the P mb O 2 decreased with increasing exercise intensity under normoxic conditions (Mol e et al. 1999;Takakura et al. 2010). This change in the O 2 gradient as reflected by the decrease in the P mb O 2 can contribute to the muscle O 2 uptake. Therefore, this study considers the effect of hypoxia on the intracellular O 2 environment and assesses whether the vasculature-to-cell O 2 gradient contributes in regulating the m _ VO 2 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 AE 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 AE 8.7 g). Preparation of isolated rat hindlimb and the perfusion apparatus are described in previous reports Takakura et al. 2010Takakura et al. , 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; KH 2 PO 4 , 1.2 mmol/L; MgSO 4 , 1.2 mmol/L; CaCl 2 , 1.8 mmol/L; NaHCO 3 , 20 mmol/L; Glucose, 15 mmol/L) equilibrated with 95% O 2 + 5% CO 2 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 AE 0.5 mL min À1 throughout the perfusion period. In this condition, the average perfusion pressures were 77.5 AE 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 m _ VO 2 and the lactate and pyruvate concentrations.
During the perfusion period, the oxygen supply was modulated by adjusting the O 2 fraction of the equilibration gas using nitrogen gas, while 5% CO 2 concentration was maintain to keep pH in perfusate. This study set three levels of O 2 concentration: a 95.0% O 2 fraction, a 71.3% O 2 fraction, and a 47.5% O 2 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 m _ VO 2 followed the previous methods 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 lsec; 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% N 2 + 5% CO 2 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 (Δ[deoxy-Mb]) signal was evident. The final Δ[deoxy-Mb] signal intensity served as the normalization constant for 100% Mb deoxygenation.
The value of m _ VO 2 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 PO 2in and PO 2out are the arterial and venous oxygen tensions after adjusting for the vapor pressure of water. Inflow PO 2 and outflow PO 2 were measured continuously using two O 2 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 lmol mL À1 mmHg À1 at 37°C (Philip and Dorothy 1971). The m _ VO 2 at rest and during muscle contraction was calculated by using the values of PO 2in ÀPO 2out averaged over 15 sec in the steadystate conditions before and during muscle contraction.
The sampling rate for the NIRS data was 1 Hz. The other parameters (tension, perfusion pressure, O 2 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. , 2015. A simple moving average smoothed the Δ[deoxy-Mb] NIRS signals using a rolling average of 5 points, which corresponds to a 5 sec timeframe (Box et al. 1978). The Δ[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 S mb O 2 at rest could not be determined by NIRS, the value was assumed to be 90% based on previous studies reporting that the S mb O 2 at rest was greater than 90% (Chung et al. 2005). The %Δ [deoxy-Mb] plots were converted to S mb O 2 (%) plots using the following equation: S mb O 2 ¼ 100 À %D½deoxy À Mb S mb O 2 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 S mb O 2 signals, and s the time constant of S mb O 2 signal kinetics. MRT calculated by TD + s 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 P mb O 2 value (mmHg) at rest and steady state during muscle contraction was converted from the S mb O 2 value using the following equation: where P 50 is the partial oxygen pressure required to halfsaturate Mb. A P 50 of 2.4 mmHg was used for this equation, assuming a muscle temperature of 37°C (Schenkman et al. 1997 with P mb O 2 is in mmHg, and O 2 solubility in buffer is 0.00135 lmol mL À1 mmHg À1 at 37°C (Philip and Dorothy 1971).
The relationship of m _ VO 2 to conductance and O 2 gradient used the following equation: where m _ VO 2 is muscle oxygen consumption, k is constant, D is conductance, P cap O 2 is microvascular oxygen tension, P mb O 2 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 PO 2 and has set 30 mmHg as the reference the normoxic P cap O 2 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 AE SD. Statistical differences were examined using one-way paired measures analysis of variance (ANOVA) (factor: O 2 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 AE 1.3 lmol min À1 at 95% O 2 saturation to 12.0 AE 1.2 and 8.8 AE 0.9 lmol min À1 in the 71.3% and 47.5% O 2 saturation, respectively. The correspondence between the O 2 saturation and the measured O 2 concentration in the perfusate confirmed the appropriateness of the experimental conditions. As the O 2 supply volume decreased, the m _ VO 2 in resting muscle decreased gradually (Table 1). Even though S mb O 2, and P mb O 2 also decrease, the measurements could not detect a significant change in the estimated O 2 gradient as reflected in (P cap O 2 -P mb O 2 ), and did not reveal any significant alteration. Under all hypoxia conditions, the lactate to pyruvate ratio (L/P) showed no increase (Table 1).
Relative to the resting m _ VO 2 , muscle contracting at a similar tension (77-83 g) increased m _ VO 2 under all O 2 supply conditions. The representative time course of the change in twitch tension at each O 2 fraction condition was shown in Figure 1. However, the largest increase occurred at 95% O 2 , where the Δm _ VO 2 rose by 0.28 lmol g À1 min À1 . At 47.5% and 71.3% O 2 saturation, Δm _ VO 2 rose only by 0.11 and 0.16 lmol g À1 min À1 , respectively. Even though m _ VO 2 increased under hypoxia conditions, it increased much less than muscle under normoxic condition (Table 2).
In contrast, L/P increased with declining O 2 supply. L/P increased significantly higher at 47.5% O 2 (3.6 AE 3.4) than at 95.0% O 2 (1.8 AE 1.0). At 95.0% O 2 the muscle  Values are mean AE SD (n = 5 in each condition). m _ VO 2 , muscle oxygen consumption; Δm _ VO 2 , the net increase in m _ VO 2 due to muscle contraction; Outflow PO 2 : oxygen tension after perfusion of the hindlimb, Relative outflow PO 2 , relative value of outflow PO 2 based on the outflow PO 2 value at 95.0 %O 2 fraction; Estimated P cap O 2 , estimated microvasucular oxygen tension based on 30 mmHg of P cap O 2 at 95.0% O 2 fraction; S mb O 2 , intracellular O 2 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 contraction causes no significant change in the L/P ratio, consistent with previous observations . During muscle contraction, the steady S mb O 2 and P mb O 2 still decreased with decreasing oxygen supply. However, the S mb O 2 and P mb O 2 kinetics did not show a significant difference (Table 2). Figure 2 shows representative Δ[deoxy-Mb] kinetics during different levels of O 2 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% O 2 fraction condition, respectively. In protocol IV, the noncontracting muscle received a perfusate equilibrated with 95% N 2 + 5% CO 2 . Figure 3 shows the net increase in m _ VO 2 (Δm _ VO 2 ) relative to its resting value during muscle contraction in each O 2 fraction condition. The Δm _ VO 2 due to muscle contraction (0.11 AE 0.04, 0.16 AE 0.07, and 0.28 AE 0.05 lmol g À1 min À1 at 47.5%, 71.3%, and 95% O 2 fraction) decreased progressively with hypoxia. Under resting conditions, the measurements indicate P mb O 2 values of 3.1, 8.5, and 21.7 mmHg at 47.5, 71.3, and 95% O 2 . With muscle contraction, the P mb O 2 values decrease correspondingly to 0.4, 2.1, and 5.5 mmHg. Figure 4 shows then net decrease in P mb O 2 (ΔP mb O 2 ) due to muscle contraction (2.50 AE 0.81, 6.12 AE 3.52, and 16.10 AE 0.87 mmHg in 47.5, 71.3, and 95% O 2 fraction conditions, respectively). Even though P mb O 2 decreases under all   exercise conditions, the value changes the most at 95% O 2 . Figure 5 shows the relationship between the Δm _ VO 2 and the ΔP mb O 2 in each O 2 fraction condition. During muscle contraction, the decrease in oxygen supply caused a decrease in the ΔP mb O 2 , which led to a decrease in the Δm _ VO 2 . The linear relationship between Δm _ VO 2 and ΔP mb O 2 suggests that the O 2 gradient as reflected in (P cap O 2 -P mb O 2 ) has changed. Using an estimate P Cap O 2 , the analysis shows that indeed Δm _ VO 2 changes linearly with (P cap O 2 -P mb O 2 ) (Fig. 6).

Intracellular O 2 environment under hypoxic conditions
As oxygen delivery decreases, the P mb O 2 at rest decreased correspondingly from 21.7 to 8.5 to 3.1 mmHg. The m _ VO 2 decreases correspondingly from 0.55 to 0.43 to 0.34 lmol g À1 min À1 . It might appear that the decreasing O 2 supply leads consequently and directly to the decreasing m _ VO 2 . Yet in normoxic muscle the decreasing intracellular O 2 leads to an increasing m _ VO 2 , which implies a critical role for the O 2 gradient instead of the O 2 supply alone. An analysis of the O 2 gradient as reflected in (P cap O 2 -P mb O 2 ) shows that despite the decreasing intracellular O 2 , the O 2 gradient appears to have increased during hypoxia. The low m _ VO 2 in resting muscle most likely creates a shallow gradient, which obscures measurement accuracy. Nevertheless, the constant L/P ratio would argue that the O 2 supply and m _ VO 2 have not compromised oxidative metabolism, which would require glycolysis to compensate for any energy deficit. Consistent with a previous findings, the decreasing O 2 supply did not affect the low m _ VO 2 at rest (Shiota and Sugano 1986). However, when muscle begins to contract, m _ VO 2 rose and the P mb O 2 decreased further from the resting levels. At 47.5% O 2 , the P mb O 2 drops below the critical PO 2 level that limits oxidative phosphorylation in mitochondria (1.5 mmHg) (Kreutzer et al. 1992). L/P rises more at 47.5% O 2 than at 95.0% O 2 . However, the drop in P mb O 2 also expands the O 2 gradient under all O 2 saturation conditions during muscle contraction. But the widening O 2 gradient does not sufficiently enhance the O 2 flux to meet the cell's oxidative needs. Anaerobic glycolysis commences to supplement the energy demand, especially as the buffer PO 2 drops to 47%.
These findings indicate that even under hypoxia m _ VO 2 rises during contraction. Given the same O 2 saturation conditions, the rise in m _ VO 2 required an increase in DO 2 or (P cap O 2 -P mb O 2 ) as shown in equation (Richardson et al. 1995;Takakura et al. 2010). Even though the P mb O 2 decreases, the O 2 gradient has widened to increase the O 2 flux to balance out the O 2 supply and demand. A decreasing P mb O 2 with a rising m _ VO 2 agree with the Figure 5. Relationship between net decrease in intracellular oxygen tension (ΔP mb O 2 ) and net increase in muscle oxygen consumption (Δm _ VO 2 ) during muscle contraction. The ΔP mb O 2 and Δm _ VO 2 gradually decreased as the O 2 supply volume decreased. The relationship between the ΔP mb O 2 and the Δm _ VO 2 was represented with a line graph. Δm _ VO 2 : net increase in muscle oxygen consumption due to contraction. ΔP mb O 2 : net decrease in intracellular oxygen tension equilibrated with O 2 saturation myoglobin. Each data point represents a mean AE SD. n = 5 9 3 points. *P < 0.05 for Δm _ VO 2 parameter. † P < 0.05 for ΔP mb O 2 parameter. Figure 6. Relationship between delta change in muscle oxygen consumption (Δm _ VO 2 ) and estimated O 2 gradient between microvascular oxygen tension (P cap O 2 ) and intracellular oxygen tension (P mb O 2 ) under exercising condition. Delta change in m _ VO 2 due to muscle contraction increased linearly as a function of estimated O 2 gradient (P cap O 2 -P mb O 2 ). Regression line is based on mean values (m _ VO 2 = 0.020 9 (P cap O 2 -P mb O 2 ), R 2 = 0.99, n = 5 in each point). Each data point represents a mean AE SD. previously reported observations (Mol e et al. 1999;Takakura et al. 2010). In contrast, Richardson et al. (1995) reported that under normoxic or hypoxic condition, the P mb O 2 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 _ VO 2 rose to its maximum level (Richardson et al. 1995). In addition, the P mb O 2 reaches a low level (<5 mmHg) in the skeletal muscle, even when moderate levels of work were performed (Mol e et al. 1999;Chung et al. 2005;Takakura et al. 2010). These findings have led researchers to ignore the P mb O 2 variable from the formula for the calculation of the amount of O 2 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 P mb O 2 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 O 2 gradient can contribute to the Dm _ VO 2 during hypoxia. In addition, the P mb O 2 at the resting muscle did not reach a nadir as a result of the hypoxia. Instead, the P mb O 2 can decreased further during the muscle contraction even in the hypoxic conditions. Change in P mb O 2 from muscle contraction modulating change in m _ VO 2 under Hb-Free perfusion The study investigated whether the m _ VO 2 during muscle contraction in various oxygen concentration conditions depend on the changes in the P mb O 2 or the O 2 gradient. Because the oxygen delivery remained constant at any muscle contraction condition in the perfusion model at a constant flow, the decrease in P mb O 2 due to muscle contraction widen the O 2 gradient between the microvasculature and myocyte as reported in the our previous study . This change contributes the increase in m _ VO 2 due to muscle contraction. This interpretation is proven by the following equation; Because kDO 2 does not significantly contribute to the increase in m _ VO 2 due to muscle contraction in the constant-flow hindlimb perfusion model, the m _ VO 2 cannot increase unless the O 2 gradient expands by decrease in P mb O 2 in our previous studies (Takakura et al. , 2015. In the present study, the results have confirmed the hypothesis that the delta change in P mb O 2 and in the O 2 gradient contributes to delta increase in m _ VO 2 during muscle contraction. Our results show that under all oxygenation conditions, m _ VO 2 rises but P mb O 2 continues to decline by muscle contraction between 77% and 88% of maximal voluntary contraction (MVC). However, rise in the m _ VO 2 and O 2 gradient expansion under hypoxia conditions are much less than the corresponding changes under normoxic conditions. The observation then agree with the studies by Mol e et al. (1999) and Takakura et al. (2010), which indicate the progressive expansion of the O 2 gradient between the P cap O 2 and the P mb O 2 during muscle contraction contribute to the O 2 flux supporting the rising m _ VO 2 . No P mb O 2 plateau even under hypoxia condition seems apparent at MVC well above 40%.
Moreover, a changing m _ VO 2 in constant flow model also supports a P mb O 2 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 Δm _ VO 2 must have a contribution from P mb O 2 . Therefore, the mechanism behind the m _ VO 2 in hypoxic environments, includes then a contribution P mb O 2 (Mol e et al. 1999;Takakura et al. 2010).
The mechanism of the oxygen transport to the mitochondria in the myocytes is dependent on the Mbmediated oxygen transport or the dissolved O 2 flux. The contribution from the Mb-mediated oxygen transport increases with decreasing P mb O 2 . Below the equipoise diffusion PO 2 value (1.77 mmHg), 50% of the total oxygen transport to the mitochondria stems from the Mbmediated oxygen transport (Lin et al. 2007). The P mb O 2 during muscle contraction in the 47.5% O 2 fraction condition was 0.4 AE 0.3 mmHg. As this was below the equipoise diffusion PO 2 value, the Mb-mediated oxygen transport mechanism will dominate. In addition, any increase in Mb concentration will raise the equipoise PO 2 (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 (PO 2crit ). According to Hill (1965), PO 2crit at the maximum rate of oxygen consumption ( _ VO 2max ; in nmol mm À3 sec À1 ) is given by 2017 where CSA is the cross-sectional area of the cell (mm 2 ), D is the diffusion coefficient for oxygen in the muscle cell (mm 2 /sec), and a is the solubility of oxygen in muscle cell (mmol/L per mmHg). Da is known as Krogh's diffusion coefficient. Da seems to depend on fat content, extracellular space, and unknown factors (Jones and Kennedy 1986;Dutta and Popel 1995;Baranov et al. 2000). Indeed, Da 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) _ VO 2 is distributed homogeneously in the cells; (3) _ VO 2 by mitochondria is independent of local intracellular PO 2 ; and (4) myoglobin-facilitated oxygen diffusion is negligible, the role of Mb was not taken account of above Da. We have conducted the maximal twitch contraction under three O 2 fraction conditions to the same individual in the present perfusion model. Moreover, as the order of the three O 2 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, Da 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 O 2 diffusion to the mitochondria (Lin et al. 2007;Takakura et al. 2010Takakura et al. , 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 PO 2 value (1.77 mmHg). Also, we reported that decrease in P mb O 2 directly contribute the expansion of the O 2 gradient to lead the increased m _ VO 2 (Takakura et al. , 2015. As for a key factor regulating O 2 diffusion to intracellular mitochondria, the change in P mb O 2 might be one of candidates, because the change in P mb O 2 affects O 2 gradient, Mb-mediated O 2 transport and mitochondria respiratory.

Conclusion
This study investigated the effects of reduced oxygen supply on the P mb O 2 at rest and during muscle contraction, as well as the effect of the DP mb O 2 on the Dm _ VO 2 during muscle contraction. Our findings showed that the resting P mb O 2 decreases with the decrease in oxygen supply. However, the decrease in P mb O 2 expands the O 2 gradient, which then supports the rising Δm _ VO 2 during muscle contraction even under hypoxic conditions. The results suggest that the DP mb O 2 is a key determinant factor of the Dm _ VO 2 .