Endurance training facilitates myoglobin desaturation during muscle contraction in rat skeletal muscle

At onset of muscle contraction, myoglobin (Mb) immediately releases its bound O2 to the mitochondria. Accordingly, intracellular O2 tension (PmbO2) markedly declines in order to increase muscle O2 uptake (mO2). However, whether the change in PmbO2 during muscle contraction modulates mO2 and whether the O2 release rate from Mb increases in endurance-trained muscles remain unclear. The purpose of this study was, therefore, to determine the effect of endurance training on O2 saturation of Mb (SmbO2) and PmbO2 kinetics during muscle contraction. Male Wistar rats were subjected to a 4-week swimming training (Tr group; 6 days per week, 30 min × 4 sets per day) with a weight load of 2% body mass. After the training period, deoxygenated Mb kinetics during muscle contraction were measured using near-infrared spectroscopy under hemoglobin-free medium perfusion. In the Tr group, the mO2peak significantly increased by 32%. Although the PmbO2 during muscle contraction did not affect the increased mO2 in endurance-trained muscle, the O2 release rate from Mb increased because of the increased Mb concentration and faster decremental rate in SmbO2 at the maximal twitch tension. These results suggest that the Mb dynamics during muscle contraction are contributing factors to faster O2 kinetics in endurance-trained muscle.

At onset of muscle contraction, myoglobin (Mb) immediately releases its bound O 2 to the mitochondria. Accordingly, intracellular O 2 tension (P mb O 2 ) markedly declines in order to increase muscle O 2 uptake (mV . O 2 ). However, whether the change in P mb O 2 during muscle contraction modulates mV . O 2 and whether the O 2 release rate from Mb increases in endurance-trained muscles remain unclear. The purpose of this study was, therefore, to determine the effect of endurance training on O 2 saturation of Mb (S mb O 2 ) and P mb O 2 kinetics during muscle contraction. Male Wistar rats were subjected to a 4-week swimming training (Tr group; 6 days per week, 30 min 3 4 sets per day) with a weight load of 2% body mass. After the training period, deoxygenated Mb kinetics during muscle contraction were measured using near-infrared spectroscopy under hemoglobin-free medium perfusion. In the Tr group, the mV . O 2 peak significantly increased by 32%. Although the P mb O 2 during muscle contraction did not affect the increased mV . O 2 in endurance-trained muscle, the O 2 release rate from Mb increased because of the increased Mb concentration and faster decremental rate in S mb O 2 at the maximal twitch tension. These results suggest that the Mb dynamics during muscle contraction are contributing factors to faster V . O 2 kinetics in endurance-trained muscle.
R elative to control muscle, endurance-trained muscle increases O 2 consumption at the same level of maximal voluntary contraction (MVC) and increases maximal O 2 consumption, which is considered an indicator of improved aerobic exercise capacity. The increased O 2 consumption in the trained skeletal muscle depends on both O 2 utilization and vascular O 2 supply. Muscle O 2 utilization capacity is mainly determined by mitochondrial function and the quantity of mitochondria, whereas O 2 supply capacity to the mitochondria is determined by capillarization. Many studies have reported that endurance training upregulates mitochondrial function, and mitochondria number and volume [1][2][3] . It also increases capillarization [1][2][3] . However, the contribution of O 2 diffusion from the capillary to the mitochondria is still unknown, especially with respect to the intracellular factors involved in O 2 transport from the sarcolemma to the mitochondria.
Recent studies have shown that the O 2 gradient can contribute to the enhanced O 2 flux to meet the increased muscle O 2 demand during contraction 4 . The O 2 saturation of Mb (S mb O 2 ), which reflects the intracellular O 2 tension (P mb O 2 ), decreases as work intensity and muscle oxygen consumption (mV . O 2 ) increase. The decreasing P mb O 2 expands the O 2 gradient from the capillary to the muscle cell to increase the O 2 flux from the vasculature to the mitochondria. Whether the O 2 gradient contributes to the increased O 2 uptake in endurance-trained muscle remains uncertain. With the experimental model to investigate the intracellular O 2 dynamics 4 , we have hypothesized that the increase in O 2 consumption in endurance-trained skeletal muscle is accompanied with increased expansion of the O 2 gradient across the plasma membrane 4

Results
Descriptive data for muscle weight are presented in Table 1.
Although endurance training caused a significant reduction in body and muscle mass, the ratio of muscle mass to body mass differed slightly between the groups (with a difference in mean value of 0.1%). Table 2 shows the contractile and metabolic properties of the control and trained hind limb muscles. Although both groups showed no significant difference in mV . O 2 at rest, at maximal tension, the values of the peak mV . O 2 per gram per minute in the swimming training (Tr) group were significantly higher than those in the control (Con) group.
[Mb] and citrate synthase (CS) activities in the deep portion of gastrocnemius muscle significantly increased after endurance training, whereas the lactate-to-pyruvate ratio (L/P) decreased at peak maximal twitch tension. Table 3 Figure 1 shows the representative kinetics of S mb O 2 in each group. As for the S mb O 2 kinetic parameters, the steady-state value, amplitude (AP), and mean rate of change to 63% of the AP value ( 0.63 AP/ mean response time [MRT]) increased as work intensity increased in both groups, whereas MRT tended to accelerate in both groups. At maximal tension, the steady-state value and AP of S mb O 2 kinetic parameters in the Tr group were unchanged, but 0.63 AP/MRT of the kinetic parameters for S mb O 2 increased. The MRT also tended to be faster in the Tr group. At submaximal tension levels, the steadystate value, AP, and 0.63 AP/MRT in the Tr group were also unchanged, but the MRT of the kinetic parameters for S mb O 2 tended to be faster. The kinetic parameters for P mb O 2 showed that the steady-state value, AP, and 0.63 AP/MRT increased, and the MRT became faster in both groups as work intensity increased. When the kinetic parameters for P mb O 2 were compared at the same relative tension level between both groups, the relative temporal parameters for P mb O 2 kinetics in the trained muscle showed a tendency to accelerate to a higher level. In the present study, while the MRT was used to describe the overall dynamics of S mb O 2 and P mb O 2 fall following the onset of muscle contraction, 0.63 AP/MRT is the effective temporal parameter to show deoxygenation rate of Mb-O 2 per unit time in the initial phase of muscle contraction. The 0.63 AP/MRT would reflect the steep change in mitochondrial oxygen demand, because we previously reported that 0.63 AP/MRT increased in response to change in mitochondrial oxygen demand due to muscle contraction. Kindig et al. 12 also used AP/time constant in intracellular PO 2 kinetics during muscle contraction as an index of initial metabolic response. As for 0.63 AP/MRT parameter in the present study, while its value showed significant difference at 100% of the maximal twitch tension by endurance training, it did not differ at 50% and 75% of the maximal twitch tension between Con and Tr group. This result at submaximal tension level might be caused by non-significant difference in O 2 demand level during muscle contraction at the relative same intensity between groups. DmV . O 2 did not actually show the significant difference at the 50% and 75% of the maximal twitch tension between groups. On the other hand, at the maximal twitch tension level, 0.63 AP/MRT parameter and DmV . O 2 in the trained muscle showed significant difference compared with that in the control muscle. Figure 2 shows the relationship between muscle tension and the net increase in mV . O 2 during muscle contraction for both groups. While muscle tension and DmV . O 2 were significantly correlated in both groups, the mean individual slope in the Tr group (0.36 6 0.11 3 10 22 mmol/[g 2 ?min]) tended to be higher than that in the Con group (0.27 6 0.06 3 10 22 mmol/[g 2 ?min]; p 5 0.058). Figure 3 shows . O 2 in the trained muscle was higher than in the control muscle, suggesting that the trained muscle had more oxidative potential capacity compared with the control muscle. Figure 4 shows the O 2 release rate from Mb at same percent of MVC in the Tr and Con groups. The O 2 release rate from Mb increased progressively with the twitch tension level as follows: 1.1 6 0.3, 2.3 6 0.4, and 3.7 6 0.8 3 10 22 mmol/(g?min) at 50%, 75%, and 100% of maximal contraction in the Con group, respectively; and 1.1 6 0.5, 2.6 6 0.7, and 4.6 6 0.5 3 10 22 mmol/(g?min) at 50%, 75%, and 100% of maximal contraction in the Tr group, respectively. At maximal tension, the O 2 release rate from Mb showed a significant increase in the Tr group, suggesting more O 2 supply from Mb to the mitochondria at the onset of muscle contraction.  15 . This increase in mV . O 2 peak value without increase in O 2 delivery to the hind limb muscle would be caused by increased of both O 2 supply capacity to the mitochondria and O 2 utilization capacity such as capillary density, mitochondrial respiration capacity, and Mb function in the active muscle. At the equivalent muscle tension, the Tr group showed a slightly higher DmV . O 2 than the Con group ( Fig. 2). As reflected by a higher CS activity, the endurance-trained muscle had higher muscle oxidative potential. The increase in O 2 consumption and O 2 cost at a given work rate would imply a shift to more aerobic metabolism during muscle contraction.
The decrease in L/P at the maximal twitch tension also suggested a greater capacity to oxidize carbohydrate and a tightening in the coupling between ATP supply and demand 13,14 . This tight integration of ATP supply and demand is associated with less stimulation of glycolysis, resulting in a decrease in lactate production and a lower cytosolic redox state, and thus an improved coupling between pyruvate oxidation and glycolytic flux 13,15 . Collectively, the swimming endurance training in the present study enhanced muscle oxidative capacity, in agreement with evidence previous studies 1, 16,17 .
Relationship between P mb O 2 kinetics and muscle oxygen consumption.   . O 2 and mV . O 2 peak in the Tr group must also have a contribution from an increased capillarization and/or a greater DO 2 1 . In the extracellular segment, the angiogenesis of the muscle capillary in the hind limb muscles during training would enhance the supply of O 2 . An increase in capillarity would improve blood flow-to-V . O 2 relationships within the muscle, allowing for a greater O 2 extraction 1 . The change in capillarity can also affect DO 2 . A greater DO 2 after exercise training implies a decreased mean O 2 diffusion distance from the tissue capillaries to the muscle mitochondria 21,22 . In the intracellular segment, O 2 transport to the mitochondria depends upon Mb-mediated O 2 flux and free O 2 . The Mb-mediated O 2 flux in the trained muscle would show a greater value at a relatively higher     24 , which will presumably experience result in a smaller decrease in muscle phosphocreatine concentration, a smaller increase in lactate and proton (H 1 ) production, and a reduced degradation of muscle glycogen, compared with an individual with slow V . O 2 onkinetics [25][26][27] . Improvement in mitochondrial respiration capacity itself would largely contribute to this adaptation as a result of endurance training. However, no study has investigated the O 2 supply to the mitochondria at the intracellular level.
We previously found that Mb supplied O 2 immediately at the onset of muscle contraction and that the O 2 release rate from Mb increased linearly as the O 2 demand increased 4,28 . These facts suggest that Mb provides an immediate O 2 source for the sudden increase in mV . O 2 at the onset of muscle contraction. The present study reveals an increase in the O 2 release rate from Mb at the onset of muscle contraction at the maximal twitch tension after endurance training. Myocyte experiments have also suggested that a direct Mb-mediated oxygen delivery might contribute to mitochondrial respiration 29  The binding of O 2 to Mb and Hb certainly proceeds much faster than transport 30,31 , even though the rate-determining step depends on a much slower off rate. But, dismissing any contribution of Mb in regulating respiration in the cell (an inhomogeneous and compartmentalized system) based on just the steady-state rate determining step argument seems tenuous. If the blood delivers a sufficient O 2 supply, the cell would not need to withdraw O 2 from its Mb reservoir at the start of muscle contraction. But the cell does withdraw O 2 from Mb, as our data and all 1 H-NMR data show, and takes a finite amount of time to reach a new steady state 5,6 . Thermodynamics requires a demand to elicit the loss of O 2 from Mb, and both ATP utilization and respiration surge once contraction starts.  16,17,32,33 , the physiological significance of increased [Mb] has not been demonstrated in vivo. The present study also demonstrated that the deoxygenation rate of Mb became faster at the onset of muscle contraction after endurance training, suggesting a more efficient O 2 transport from Mb to the mitochondria at the transient phase.
In addition, the P mb O 2 response at the onset of muscle contraction showed the tendency to be faster after endurance training in the present study. Hirai et al. 20 reported that endurance training led to slower P cap O 2 kinetics during 1-Hz twitch contraction, indicating a relatively greater increase in muscle blood flow at the microvascular level than in O 2 diffusivity. This adaptation would be mainly caused by an increase in capillary density. Meanwhile, the intracellular O 2 environment was reported to adjust more effectively to the abrupt increase in oxygen demand at the onset of muscle contraction, before the microcirculatory O 2 environment has adapted 4 . In fact, the MRT of P mb O 2 kinetics became approximately 5 seconds faster on average by endurance training. The acceleration of P mb O 2 kinetics with slower P cap O 2 kinetics would imply a sharper expansion of O 2 gradient at the onset of muscle contraction, resulting in a more efficient O 2 transport from Mb to the mitochondria at the transient phase.
In the present study, we have performed additional statistical analyses on kinetics parameters to check the existence of type II error. As for MRT in P mb O 2 kinetics, the effect size was 0.052, and the statistical power was 0.109. This level of statistical power implies the existence of a type II error. However, based on our experimental content, increasing the sample size would not necessarily improve the accuracy or precision of our results. This might be one of limitations in this type of experiment. Actually, although significant difference was not recognized for kinetics parameters such as MRT between groups at the relative same tension level, the shorting of MRT by 5 sec on average at the maximal tension level in both of S mb O 2 and P mb O 2 kinetics suggests the possibility that swimming endurance training accelerates both kinetics during muscle contraction.
In summary, the results presented herein suggest that Mb plays an important role in the faster mV . O 2 response and increased mV . O 2 in trained skeletal muscles. However, how Mb-bound O 2 is supplied to the mitochondria at the onset of muscle contraction remains unclear. Recently, Yamada et al. 34 suggested the possibility that the presence of Mb in mitochondrial fractions indicates involvement in the immediate O 2 release from Mb at the onset of muscle contraction. If so, endurance training might impact Mb localization in the muscle cell. Further research is required to elucidate the mechanism of O 2 transport to the mitochondria within muscle cells and the causal relationship between cellular factors altering the O 2 off rate in Mb and mitochondrial respiration activity.

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 6 2uC 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-101636).
Five-week-old Wistar rats were randomly divided into the Con and 4 weeks Tr groups (n 5 9 in each group). The training protocol for the swimming group was as follows: On the first and second days, the rats swam for 1 h in two 30-min bouts separated by 5 min of rest. On the third and fourth days, the rats swam for 1.5 hours in three 30-min bouts separated by 5 min of rest. On and after the fifth day, the rats swam for 2 hours in four 30-min bouts separated by 5 min of rest. Except for the first bout of swimming training until the sixth day, a weight equal to 2% of the rats' body weight was tied to the bodies of the rats. The rats performed the above swimming protocol six days per week. During swimming exercise, the water temperature was kept at around 35uC. The tank's shape was square and its characteristics were 48 cm depth, 80 cm longitudinal and 60 cm width. All rats swam in that tank and an average surface area of at least 600 cm 2 /rat. Also, we kept monitoring to prevent the climbing, diving and bobbing of rat during swimming training. In cases where these behaviors were observed, they were dealt with immediately.
After 4 weeks (at 9-week of age), hindlimb perfusion was performed in each group (Con group: initial body weight (BW) at 5 weeks old; 143-168 g, final BW at 9 weeks old; 257-295 g, Tr group: initial BW; 140-176 g, final BW; 226-250 g). Preparation of isolated rat hindlimb and the perfusion apparatus are described in previous reports 4,28 . All surgical procedures were performed under pentobarbital sodium anesthesia (45 mg kg 21 intraperitoneal). The rats were killed by injecting 1 M KCl solution directly into the heart, followed by a surgical procedure, and an Hb-free Krebs-Henseleit buffer (NaCl, 118 mM; KCl, 5.9 mM; KH 2 PO 4 , 1.2 mM; MgSO 4 , 1.2 mM; CaCl 2 , 1.8 mM; NaHCO 3 , 20 mM; Glucose, 15 mM) equilibrated with 95%O 2 1 5%CO 2 at 37uC 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 22.0 6 0.0 ml min 21 in the Con group and 22.1 6 0.9 ml min 21 in the Tr group. In this condition, the average perfusion pressures were 78.6 6 7.7 mmHg in the Con group and 74.2 6 5.2 mmHg in the Tr group. Therefore, the perfusion resistance was unchanged throughout the perfusion period. In addition, no sign of oedema in the hindlimb was seen at the given flow rate. The effluent was collected from the inferior vena cava in order to measure mV . O 2 and the lactate and pyruvate concentrations.
Measurement Parameters. The twitch contraction protocol and measurement of Mb oxygenation and mV . O 2 followed the previous methods 4, 28 . 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 msec; duration, 1 msec) for 120 sec (120 twitch contractions). Target tension was controlled by changing the voltage of stimuli to obtain 50%, 75% and 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, even at the highest stimulation intensity.
An NIRS instrument (NIRO-300 1 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 1 5% CO 2 gas) was perfused for 30  O 2 due to muscle contraction by muscle tension. The L/P remains constant and shows no significant increase from the resting muscle value. Lactate levels can increase for a number of reasons, including lack of adequate O 2 delivery. Therefore, limited oxygen availability should lead to an increase in lactate level as anaerobic glycolysis starts. Indeed, the L/P increases during perfusion with anoxia buffer (95%N 2 ) increases. O 2 availability and delivery can sustain maximal contraction with no sign of fatigue.
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 4 . A simple moving average smoothed the D[deoxy-Mb] and D[oxy-Mb] NIRS signals using a rolling average of 5 points, which corresponds to a 5-sec timeframe 35 . The D[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% 5,36 .
The %D[deoxy-Mb] plots were converted to S mb O 2 (%) plots using the following equation: 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, USA): 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 t the time constant of S mb O 2 signal kinetics. MRT calculated by TD 1 t was used as an effective parameter of the response time for Mb deoxygenation at onset of muscle contraction. Mb Concentration and CS Activity in Buffer-Perfused Muscle Tissue. After buffer perfusion experiment, Mb concentration in muscle tissue was measured by a modified Reynafarje method 39 . CS activity, a mitochondrial enzyme and marker of muscle oxidative potential, was measured in whole muscle homogenates by using the spectrophotometric method of Srere 40 .
Statistical Analyses. All data are expressed as mean 6 SD. Statistical differences were examined using two-way unpaired measures analysis of variance (ANOVA) (tension level 3 training). A Turkey-Kramer post-hoc test was applied if the ANOVA indicated a significant difference. An unpaired t-test was used in comparing biochemical and physiological parameters between groups. Pearson's correlation www.nature.com/scientificreports coefficient was calculated when the relationship between two variables was evaluated. The level of significance was set at p , 0.05.