The purpose of this study was to use31P-magnetic resonance spectroscopy to examine the relationships among muscle PCr hydrolysis, intracellular H+ concentration accumulation, and muscle performance during incremental exercise during the inspiration of gas mixtures containing different fractions of inspired O2( ). We hypothesized that lower would result in a greater disruption of intracellular homeostasis at submaximal workloads and thereby initiate an earlier onset of fatigue. Six subjects performed plantar flexion exercise on three separate occasions with the only variable altered for each exercise bout being the (either 0.1, 0.21, or 1.00 O2 in balance N2). Work rate was increased (1-W increments starting at 0 W) every 2 min until exhaustion. Time to exhaustion (and thereby workload achieved) was significantly (P < 0.05) greater as was increased. Muscle phosphocreatine (PCr) concentration, Pi concentration, and pH at exhaustion were not significantly different among the three conditions. However, muscle PCr concentration and pH were significantly reduced at identical submaximal workloads (and thereby equivalent rates of respiration) above 4–5 W during the lowest condition compared with the other two conditions. These results demonstrate that exhaustion during all occurred when a particular intracellular environment was acheived and suggest that during the lowest condition, the greater PCr hydrolysis and intracellular acidosis at submaximal workloads may have contributed to the significantly earlier time to exhaustion.
- skeletal muscle
- oxygen uptake
- muscle bioenergetics
- adenosine 5′-triphosphate
- adenosine 5′-diphosphate
- inorganic phosphate
- mitochondrial respiration
- fraction of inspired oxygen
- magnetic resonance spectroscopy
the effect of breathing gas mixtures with varied fractions of inspired O2( ) on muscle respiration, metabolism, and fatigue during exercise has been extensively studied (2, 14, 15, 17, 19, 24). At any fixed workload up to near maximal, O2 uptake (V˙o 2) is identical for any (15, 19, 20,24). However, the subsequent fatigue development and the muscle maximal V˙o 2 can be significantly influenced by the (15, 22, 24). The effect of varied on muscle fatigue and performance is not only apparent when O2 availability to the muscle is altered at maximal work rates but also has been shown to influence muscle performance at submaximal workloads when the muscleV˙o 2 is not different among (1). However, the complete mechanisms by which muscle performance is affected by varied , both during submaximal and maximal work intensities, remain unclear. We have suggested previously (14-17) that differences in muscle oxygenation (ultimately resulting in differences in intracellular ; see Ref. 30), even at the same workload or V˙o 2, may result in differences in concentrations of some cellular metabolites, such as H+, lactate, phosphocreatine (PCr), and Pi (concentration denoted by brackets herein), that may affect cell homeostasis and thereby function. These changes in cell homeostasis during the breathing of different may be important factors in the subsequent development of fatigue, independent of any O2 limitation.
The purpose of the present research was to use31P-magnetic resonance spectroscopy (MRS) during plantar flexion incremental exercise in humans to test the hypotheses that1) breathing gas mixtures of different would significantly alter intramuscular [PCr] and pH at identical submaximal workloads and 2) these -induced alterations in cell metabolic state at submaximal workloads would lead to significantly different exhaustion time points (or maximal work rates) that would be associated with the attainment of a particular intracellular metabolic environment.
Six healthy subjects, three men and three women, aged 21–43 yr volunteered for this study and gave written informed consent. The study was approved by the Human Subjects Committee of the University of California, San Diego. Subjects were all healthy and active, ranging from recreational to well-trained athletes. The subjects refrained from strenuous exercise for 24 h before data collection.
Subjects were familiarized with plantar flexion exercise at a frequency of 1 contraction/s (keeping time with an electronic metronome) while lying supine in a superconducting 1.5-T magnet before the day of their experiment. On the experimental day, the subject performed three maximal exercise bouts, with 1 h between bouts. Each of the three exercise periods began with 2 min of unloaded contractions (at 1 contraction/s), followed by work rate increases of 1 W every 2 min until exhaustion. The wattage was increased by adding weight to be lifted by a rope and pulley system. Exhaustion was defined as the last work intensity at which the subject was able to fully complete the 2 min. The maximal work intensities achieved for the six subjects while they inspired room air were 8, 9, 10, 10, 10, and 12 W.
The only difference in the experimental protocol utilized for the three different exercise bouts was the inspired by the subject, being either 1.00, 0.21, or 0.10 O2 in balance N2. The order of the gases presented to each subject was varied so that all six of the possible orders of the three treatments were utilized. Subjects inspired the designated gas mixture for 8 min before the commencement of exercise, through the exercise period, and for the 6-min recovery period. Whereas four of the six subjects were not aware that the would be altered for each performance task, all subjects were blinded to both the inspired and the level of exercise intensity achieved for any given exercise period. Throughout each exercise bout subjects breathed through a low-resistance, two-way breathing valve (model 2700, Hans Rudolph, Kansas City, MO). Heart rate and arterial O2 saturation were monitored continuously throughout the experiment with a finger probe (Omni-Trak, In Vivo Research).
MRS data were acquired continuously for 2 min preexercise, for the entire exercise period, and for 6 min of recovery. MRS was performed by using a clinical 1.5-T General Electric Signa system (4.8 version) operating at 25.86 MHz for 31P.31P-MRS data were acquired with a transmit/receive surface coil (diameters 20 and 10 cm, respectively) placed under the calf at its maximum diameter. The centering of the leg over the coil was confirmed by T1-weighted1H localizing images obtained in the axial plane. Magnetic field homogeneity was optimized by shimming on the proton signal from tissue water. For31P-MRS the pulse power was adjusted so that ∼72% of the signal acquired was from tissue within 5 cm of the surface coil. The spectral width was 2,500 Hz, and data were acquired continuously through the exercise period with a single free induction decay (FID) generated every 4 s (repetition time = 4 s). As a result the data can be expressed with a time resolution of 4 s or summed as spectra representing an average over a defined time period.
Data were processed by using SAGE/IDL software on a Silicon Graphics Indigo workstation. Each FID consisted of 1,024 complex points and was processed with 5-Hz exponential line broadening before zero filling and Fourier transformation. All spectra were manually phased by using zero- and first-order phase corrections. Summing eight spectra provided sufficient signal-to-noise ratio to allow determination of ATP, PCr, and Pi levels at 32-s time intervals during the experiment.
All areas under the various spectral peaks were normalized to the previously measured value of β-ATP peak: 8.2 mM (35). Muscle intracellular pH was calculated from the chemical shift difference of the Pi peak relative to the PCr peak. ADP was calculated from the creatine kinase reaction Equation 1whereK eq is the equilibrium constant (1.66 × 109M−1) and total [creatine] was assumed to remain constant at 42 mM (35). The levels of PCr, Pi, and ATP determined from the areas under their respective spectral peaks were normalized (as a percentage of rest) to the average value obtained for the last 32 s (8 spectra) of rest for each subject.
The responses to the three were statistically assessed by repeated-measures ANOVA. Duncan’s multiple-range test was used to determine where differences occurred among conditions at the different work intensities. For all analyses, a 0.05 level of significance was used. All values are reported as means ± SE.
The estimate of resting arterial saturation for the three conditions was 70 ± 3, 98 ± 1, and 100 ± 1 (SE) % during 10, 21, and 100% O2 breathing, respectively. These arterial saturation values for the three different correspond to arterial values of ∼45, 100, and 600 Torr, respectively.
Time to exhaustion (Fig. 1), which we used as our index of muscle performance, was significantly different (P < 0.01) among each of the three : 17.7 ± 1.3, 21.0 ± 0.9, and 24.0 ± 1.8 min during 10, 21, and 100% O2 breathing, respectively. These times corresponded to work rates of 8.3 ± 0.5, 9.8 ± 0.5, and 11.5 ± 0.8 W, respectively.
Muscle [ATP] did not significantly change from resting values with increasing workloads and was never different among the three conditions.
The relationship between heart rate and work rate is illustrated in Fig. 2. Resting heart rates were significantly different (P < 0.01) among each of the three inspired gas mixtures. During exercise, the mean heart rate for the subjects during the low- condition was significantly greater than the other two conditions at all workloads.
Figure 3 illustrates the fall in the mean values of muscle [PCr] (relative to resting [PCr]) for each of the three different conditions as workload was increased. Resting muscle [PCr] was not significantly different among conditions. Muscle [PCr] fell from resting values systematically as workload increased in all three conditions. At work rates of 6 W and higher, [PCr] was significantly (P < 0.01) more depleted in the hypoxic treatment compared with the other two treatments. Even with the significantly greater work rates achieved as was increased, muscle [PCr] at exhaustion for the six subjects was not significantly different among the three treatments, being 41 ± 3, 44 ± 3, and 43 ± 4% of resting [PCr] during 10, 21, and 100% O2breathing, respectively.
Figure 4 illustrates the rise (relative to rest values) in measured [Pi] for each of the three different as workload was increased. Relative values of [Pi] were significantly greater (P < 0.01) at work rates above 5 W in the = 0.10 treatment compared with the other two treatments and was significantly greater in the = 0.21 vs. = 1.00 at 9 W. Relative muscle [Pi] at exhaustion for the six subjects was not significantly different among the three treatments, being 470 ± 60, 430 ± 50, and 440 ± 30% of resting [Pi] during 10, 21, and 100% O2breathing, respectively.
Figure 5 illustrates the changes in intracellular pH for each of the three different conditions as workload was increased. At work rates of 5 W and higher, pH was significantly less (P < 0.01) in the hypoxic treatment compared with the other two treatments. At the exhaustion time point for all six subjects (significantly greater workloads achieved as was increased), intracellular pH was not significantly different, being 6.84 ± 0.03, 6.90 ± 0.03, and 6.92 ± 0.02 during 10, 21, and 100% O2 breathing, respectively.
Figure 6 illustrates the relative increase above resting values in the calculated muscle [ADP]. Muscle [ADP] rose in a linear manner with workload, and there were no significant differences at any workload among the three conditions.
The present study demonstrates that, during incremental plantar flexion exercise in humans, muscle performance was significantly diminished at the lowest and significantly improved at the highest compared with normal. Muscle [PCr] and pH were significantly reduced at identical submaximal workloads during the lowest condition compared with the other two conditions when the work rate was above 40–50% of maximal. In addition, the intracellular metabolic state at exhaustion was similar among the three treatments, even though exhaustion occurred at significantly different time points (and thereby workloads).
Cellular O2 availabilty.
Under most work conditions, the rate of ATP production in the muscle cell is tightly coupled with the breakdown of ATP in the cytoplasm, thereby maintaining the required work output. Under normal steady-state conditions, aerobic metabolism provides the major source of ATP resynthesis. Although the role of O2 as a regulator of oxidative phosphorylation has often been considered only important when its concentration becomes rate limiting, it has been suggested that the [O2] available at the mitochondria, even when adequate for the required level of respiration, can modulate the levels of the other substrates in oxidative phosphorylation (14, 16, 31, 40, 41). Rumsey and colleagues (31) and Wilson and colleagues (40, 41), using isolated mitochondria and isolated cells, have demonstrated that oxidative phosphorylation is dependent on O2 at concentrations found in the physiological range and that there exists a wide range of O2 values that influence the metabolic state of the cell. It has also been demonstrated (14, 16) in isolated working muscle that the concentrations of several cellular metabolites (PCr, Pi, lactate, and ADP) can be altered at similar workloads and rates of respiration depending on the degree of oxygenation.
The availability of O2 for the mitochondria in working muscle will depend in part on the blood flow and O2 content of the blood perfusing the muscle. Blood flow to working muscle is typically increased (compared with normal) in hypoxemia and decreased in hyperoxemia (17). This offsets to some degree the differences in O2 content, and thereby total O2 delivery to the working muscle may be very similar among varied conditions. The result is that, as has been demonstrated previously (15, 20, 22, 29), the rate of V˙o 2 remains the same at identical submaximal workloads with breathing of varied . However, the gradient from capillary to mitochondrion is also an important factor that determines the total flux of O2 into a working cell. In the present study, the diminished arterial during the low- condition and the increased arterial in the high- condition likely resulted in an intracellular that was also different at any identical workload. Although this interpretation can become complicated during hyperoxemia, because the will fall rapidly (due to the shape of the O2 dissociation curve) on the first extraction of O2 in the capillary, Richardson et al. (30) have shown that, at similar workloads and rates of muscle respiration, intracellular myoglobin saturation (and thereby intracellular ) is significantly reduced during breathing of a low and increased in high . If indeed intracellular was different for the varied conditions in the present study, then it is possible that the greater PCr hydrolysis noted at submaximal work rates during hypoxia (compared with the other condition) was a metabolic readjustment to sustain a given rate of respiration caused by the differences in intracellular .
Although it is possible that the intracellular was different among the three conditions (see Ref.30) and may have influenced differences in metabolic state in the present study, other causes of the altered metabolic environment in the hypoxic condition need to be considered. The heart rate response (Fig.2) indicates a strong sympathetic response to the hypoxic treatment. Although PCr hydrolysis is not known to be affected by altered sympathetic stimulation or catecholamine levels in normal exercise, we cannot exclude the possibility of some neurohormonal-related influence on the altered metabolic state during submaximal work in hypoxia. It should also be noted that extrapolation of whole muscle results (as in the present study) to what is occurring in single fibers is complex and can be influenced by motor recruitment patterns. This is particularly true in incremental exercise when additional effort is achieved by additional recruitment of type I fibers until type II fibers are finally recruited as workload increases to maximal. The onset of substantial metabolic disturbances (see Figs. 3-5) in the present study may have been related to work rates at which type II fibers were progressively recruited. The motor unit recruitment pattern may have been different in the hypoxic condition, such that more fast-twitch units (type II) were recruited earlier in the incremental protocol (because type I fibers fatigued more quickly due to O2 deprivation), resulting in a more rapid PCr hydrolysis and metabolic acidosis. Sundberg (34) demonstrated a greater electromyogram during the same submaximal work in humans during modest reduction in blood flow (ischemia) and suggested that this was due to a greater total activation of muscle fibers to maintain the force output. With the present data, we cannot specifically determine whether a more rapid recruitment of type II fibers occurred during the submaximal workloads, but it is likely that at near-maximal levels this did occur (due to type I fibers becoming fatigued) and may have contributed to the earlier onset of exhaustion.
At the very lowest exercise intensities in the present study, proton consumption by PCr hydrolysis rose in a linear manner for the first three workloads and was not different among the three inspired (see Fig. 3). However, at 3–4 W during all three conditions, PCr depletion continued (Fig. 3), but the increase in pH (Fig. 5) began to level off as H+ consumption by the breakdown of PCr became offset by H+ accumulation from lactic acid production (33). From 3 W to exhaustion, pH fell to significantly lower levels in the low- condition compared with the other two inspired gases (see Fig. 5). These results suggest that glycolytic production of lactate during the low- condition began at an earlier workload and rate of respiration. Although lactate production has been historically viewed as resulting from an O2 limitation within working muscle (anaerobic glycolysis), it has become clear (7, 12, 21, 32, 36) that lactate production can occur in fully aerobic conditions as a result of slow activation of metabolic pathways or a simple mass action effect of pyruvate production. It is known that glycolysis is accelerated by increases in [Pi] (21), and it is possible that the increased muscle [H+] (and likely [lactate]) during the lowest condition at submaximal work rates in the present study, when cellular respiration was constant among , was due to the elevated Pi during this time period.
Control of cell respiration.
The signals responsible for the tight coupling between the rate of ATP demand by the actomysin and ion pump ATPases and ATP production by oxidative phosphorylation have been extensively studied (3-6, 11,23, 25, 26, 28), and it is clear that many factors interact to regulate respiration. Although ADP accumulation has been suggested (5) to be a key signal linking ATP demand to ATP resynthesis in the mitochondria, other investigators (6, 14, 16, 25, 28) have demonstrated that the level to which PCr is hydrolized is tightly coupled to the rate of respiration. In the present study, intracellular [PCr], [Pi], and [ADP] changed proportionally with workload (see Figs. 3, 4, and 6), as would be expected for a signal of oxidative phosphorylation. However, we have demonstrated previously (13, 14, 16) that tissue respiration could be dissociated from the various proposed regulators of tissue respiration during work, depending on the degree to which the tissue was oxygenated. At lower levels of arterial , greater changes in the proposed regulators were needed to achieve a givenV˙o 2 (13, 14, 16), suggesting that the sensitivity of mitochondrial respiration to the proposed regulators of respiration could be modified by the oxygenation state of the tissue.
In the present study the respiratory regulator that was least affected by as work intensity increased, and that demonstrated the best straight-line relationship to increases in work intensity (and thereby respiration), was the calculated rate of ADP accumulation (Fig. 6). Although that may implicate [ADP] as a stronger signal for respiration, the different intracellular that resulted from the three different arterial (as discussed previously) likely caused compensatory changes in the level of PCr to maintainV˙o 2 (13, 14,16). ADP may then have been held constant for each by the changes in [PCr] and [H+] caused by the altered O2 tension. Although the present study cannot address these issues directly, we have suggested previously (14) that the effect of differences in intracellular oxygenation on the respiratory regulators may be caused by mechanisms similar to what has been demonstrated with changes in the mitochondrial concentration of the cell: an altered mitochondrial sensitivity to the regulators of mitochondrial respiration (8, 9, 18). It is possible that, with changes in intracellular , there were subsequent changes in the quantity of functional mitochondria, equivalent to an altered mitochondrial content. A different concentration of regulator would thus be required to elicit a given rate of respiration.
An important finding in the present study was the significant increase in muscle performance, as identified by the time to exhaustion, as increased. This agrees with prior findings, in that along with the significant changes in maximal V˙o 2 during the breathing of altered (15, 19,22, 24, 29), muscle performance (as defined by the onset of fatigue) is enhanced at high (37) and diminished with low (15, 19). In addition, exercise performance at a submaximal workload (with similar rates of respiration) can be increased in hyperoxia and decreased in hypoxia (1). Although certainly a decreased or increased O2 availability for maximal mitochondrial respiration will affect the ability of the muscle to maintain the tight coupling between ATP turnover and ATP production, the present results demonstrate that hypoxia results in significant alterations (compared with the other condition) in muscle metabolic state before any potential O2 limitation.
Although it is likely that the altered muscle metabolic state (decreased [PCr] and increased [Pi] and [H+]) in submaximal work during the hypoxic condition was related to the significantly earlier onset of fatigue in that condition, the lack of significant changes in submaximal metabolic state (except for Pi at 1 workload) during the hyperoxic condition makes the improvement in muscle performance in that condition more difficult to interpret. It is possible that an elevated maximal V˙o 2 or other extracellular factors permitted a higher work rate to be achieved during hyperoxia. However, we (13) and others (24) have previously demonstrated that muscle [PCr] at identical, non-oxygen-limited submaximal workloads can be significantly greater during high arterial (hyperoxemic) conditions compared with normal. The factors that cause reduced tension development and muscle fatigue interact in a complex fashion (see Refs. 10 and 38), and accumulation of H+ and Pi have both been implicated in this process (27, 39). Interestingly, the significantly different exhaustion time points during the three different occurred when the same intracellular [H+] and [Pi] was achieved, suggesting that muscle performance was associated with the time point at which these potent inhibitors of force generation attained a specific level. Normalization of muscle [PCr], pH, and [Pi ] to 100% of the maximal work rate reached during each condition resulted in extremely tight relationships among that culminated in nonsignificant end points, as illustrated for [PCr] in Fig. 7. The similar changes in these normalized variables during all suggests that the variance in the time to exhaustion among the three was related to the different absolute rates of accumulation of these inhibitors of force generation. However, as noted previously, because there were little significant differences in metabolic state at identical submaximal work rates between normoxia and hyperoxia, this interpretation for the increased exercise performance in hyperoxia may not be satisfactory, and further work is needed to clarify the causes of this response.
This study demonstrated that during incremental plantar flexion exercise to exhaustion in humans, muscle performance was significantly improved as increased. Muscle [PCr] and pH were significantly decreased at identical submaximal workloads (above 40–50% of maximal) during the inspiration of the hypoxic compared with the other . A similar intracellular metabolic environment was attained at exhaustion among the varied , which occurred at significantly different workloads, suggesting that the greater disturbance of cellular homeostasis during submaximal work may have contributed to the earlier onset of fatigue in the lowest condition. Finally, the significantly altered intracellular environment during the lowest , at fixed rates of submaximal cellular respiration, support the hypothesis that the level of cellular oxygenation can modulate the concentrations of proposed respiratory regulators.
This research was supported by National Institutes of Health Grants AR-40155 and HL-17731. R. S. Richardson was funded by a fellowship from the Parker B. Francis Foundation during this research.
Address for reprint requests: M. C. Hogan, Dept. of Medicine 0623, Univ. of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0623 (E-mail:).
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- Copyright © 1999 the American Physiological Society