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ENVIRONMENTAL, EXERCISE AND RESPIRATORY PHYSIOLOGY
Department of Kinesiology, University of Waterloo, Ontario, Canada
Submitted 22 September 2004 ; accepted in final form 22 April 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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membrane excitability; Na+-K+-ATPase; peak aerobic power
Failure to generate repetitive action potentials at increasing frequency in the sarcolemma and t-tubules represents a plausible possibility for E-C failure during progressive exercise. The generation of repetitive action potentials is intimately dependent on being able to establish transmembrane gradients for Na+ and K+, given the flux of Na+ into and K+ out of the cell that occurs during excitation (11). Transmembrane gradients for Na+ and K+ depend on the active pumping of these cations by Na+-K+-ATPase (Na+-K+-pump), an integral membrane protein that uses the energy from ATP hydrolysis to counter transport of 3 Na+ and 2 K+ across the membrane (54). The catalytic activity of the Na+-K+-ATPase is a critical determinant of the rate of ATP hydrolysis and, consequently, the rate of the Na+ and K+ transport (41).
The maximal activity of the Na+-K+-ATPase depends on the amount of protein, the isoform composition, and acute regulatory stimulating factors (7). Even under optimal activating conditions, there is evidence that Na+-K+-ATPase activity may be insufficient to meet the demands for Na+-K+ transport imposed by muscle contractile activity, resulting in a loss of membrane excitability and fatigue (41). Repetitive contractile activity involving large muscle groups, as an example, particularly, if the demands for power are progressively increased, may be more problematic, given the alterations that occur in the intracellular environment. The accumulation of selected by-products of metabolism that occurs during progressive exercise, such as heat, hydrogen ions (H+), inorganic phosphate (Pi), and reactive oxygen species (ROS), (25, 52) could either individually or in combination inhibit the maximal activity of Na1-K+-ATPase similar to what has been shown for other cellular ATPases (13, 42, 57). The results of previous studies using heavy-cycle exercise support the possibility of a loss of membrane excitability, as indicated by disturbances in the properties of the compound action potential in the vastus muscles (2, 31).
Sustained repetitive contractions appear to cause inhibition of muscle Na+-K+-ATPase activity. Both our group (19) and others (22) have reported decreases in maximal Na+-K+-ATPase activity, as assessed in vitro by K+-stimulated 3-O-methylfluorescein phosphatase (3-O-MFPase), after exercise. As a consequence, it is possible that the Na+-K+-membrane transport and membrane excitability would be compromised in tasks in which large force levels are required. Moreover, if a similar task is performed under conditions where the muscle metabolic stress is exaggerated as during hypoxia (12, 48), the decrease in maximal Na+-K+-ATPase activity and the loss of membrane excitability may be even more pronounced. It is possible that a failure in membrane excitability secondary to reductions in maximal Na+-K+-ATPase activity may explain the lower mechanical PO and lower peak aerobic power observed in hypoxia compared with normoxia (46, 47). Support for this possibility comes from a recent study published by our group in which we have shown a greater reduction in maximal Na+-K+-ATPase activity during prolonged submaximal exercise when performed in hypoxia compared with normoxia (50). Although, we could find no evidence for a greater impairment in neuromuscular function during submaximal exercise in hypoxia, such may not be the case with progressive exercise. In contrast to submaximal exercise where force levels and motor unit firing rates are relatively low and 
O2 and muscle energy homeostasis are well protected (24, 50), the generation of increased forces needed to perform progressive exercise exaggerates the demands on membrane excitability and O2 requirements and, in the process, greatly elevates metabolic by-product accumulation in muscle (6, 49).
In this study, our objectives were to examine the effect of progressive cycle exercise to fatigue performed under both normoxic and hypoxic conditions on maximal Na+-K+-ATPase activity, neuromuscular fatigue, and membrane excitability. We have hypothesized that at fatigue in normoxia, a reduction in maximal Na+-K+-ATPase activity in the vastus lateralis would occur, accompanied by reductions in neuromuscular force-generating capacity and membrane excitability. Moreover, we have hypothesized that when the exercise is performed in hypoxia, the decrease in muscle maximal Na+-K+-ATPase activity, neuromuscular fatigue, and membrane excitability would be more pronounced.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Experimental design. To investigate our hypothesis, the experimental design used progressive cycle exercise to fatigue followed by measurement of muscle mechanical function and membrane excitability. Each volunteer was required to perform the cycle exercise to fatigue on two occasions, namely, while breathing room air (FIO2 = 0.21) and while breathing a normabaric hypoxic gas mixture (FIO2 = 0.14). We have used the terms normoxia (Norm) and hypoxia (Hypox) to describe the two conditions. The conditions were randomly assigned and separated by at least 4 wk. During the progressive exercise, measurement of respiratory gas exchange was made and tissue was sampled from the vastus lateralis for measurements of cellular properties. Assessment of muscle mechanical function and membrane excitability was performed before and after the cycle exercise.
The progressive exercise tests were replicated on a second occasion using a different group of participants. These tests, performed during both Norm and Hypox, in random order, were used for measurement of membrane excitability during the progressive exercise itself.
At least 2 wk before the first condition, each participant reported to the laboratory for an initial progressive cycle test to fatigue for measurement of peak aerobic power (O2 peak) and related functions. This test, administered in Norm, was used to calculate the tissue-sampling schedule. For both Norm and Hypox, matched tissue samples from the vastus lateralis muscles were obtained by the needle biopsy technique (5) at rest, before exercise (Pre), and at PO, equivalent to 50% O2 peak, as determined in Norm [50% O2 peak (Norm)]. These samples were obtained at the same absolute PO for both conditions. For Norm, additional tissue samples were obtained at 70% O2 peak and at fatigue [100% O2 peak (Norm)]. For Hypox, an additional tissue sample was obtained at fatigue [100% O2 peak (Hypox)]. The sample extracted at 70% O2 peak in Norm was labeled as 100% O2 peak (Hypox) for statistical comparisons. The power outputs at which these tissue samples were obtained was 214 ± 7 W and 250 ± 7 W for normoxia and hypoxia, respectively. Although, it would have been desirable to have matched tissue samples in Norm and Hypox at identical PO at which fatigue was observed in Hypox, this was not possible given the limited number of tissue samples that could safely be extracted and given the fact that the conditions were randomized.
To determine the effects of exercise and oxygen conditions on muscle mechanical properties and membrane excitability, isometric knee extension was used using a subset of the group (n = 7). These measurements were obtained 30–60 min before and within 4–5 min after the progressive tests while the participants were breathing room air. Mechanical characteristics were assessed using both voluntary contractions and contractions induced by electrical stimulation. Familiarization with the measurement protocols and standardization of conditions (see Mechanical function and membrane excitability protocols) were completed during a separate visit to the laboratory on the days before the performance of the progressive tests in Norm and Hypox.
The period after exercise represented the amount of time needed to instrument the subjects for the neuromuscular measurements. Previous research has demonstrated that alterations in muscle membrane excitability as measured by the amplitude of the muscle compound action potential (M wave) showed no recovery for and least 10 min after progressive exercise (31).
In the separate set of experiments, employing untrained males of similar age (21.5 ± 0.89 yr) and O2 peak (3.20 ± 0.17 l/min), we have recorded the M waves during the cycling itself. The properties of the M waves were assessed during Norm and Hypox at power outputs corresponding to the tissue sampling. No tissue samples were obtained, and no mechanical measurements were performed on these participants.
Progressive exercise protocol.
For the progressive cycle tests, the subjects sat in an upright position on an electrically braked cycle ergometer (Quinton 870) that had been calibrated on a daily basis. Calibration was accomplished using standardized weights. The exercise protocol consisted of a 4-min baseline period of cycling at 25 W followed by 15-W step increases in PO each minute until fatigue. Fatigue was defined as an inability to sustain pedal frequency at 
60 revolutions/min. Ventilation and gas exchange were monitored continuously beginning prior to exercise and until fatigue by previously described methods (29). The ventilatory volume and gas fraction signals were integrated to produce 30-s windows of E, O2, and CO2. Only the O2 values are presented in this paper. During the exercise tests, arterial oxygen saturation (Sp
) was monitored from the fingertip using oximetry (Ohmeda model 3700). The validity of this method has been previously established by our group (unpublished data) and by others (44). Measurements were obtained over a 15-s period, and the values recorded represented the average of three determinations. The finger was carefully cleaned with alcohol before attaching the probe.
For Norm, the four tissue samples were obtained from separate sites distributed between the two legs. For Hypox, only three samples were obtained. For Hypox, one sample was obtained from one leg and two from the other leg. All sampling sites were prepared during the preparatory period before exercise. As a consequence, during the exercise, only a brief interruption in cycling (15–20 s) was needed to rapidly secure the tissue samples. For the measurement of Na+-K+-ATPase activity, the tissue sample was frozen in liquid N2 and stored at –80°C.
Mechanical function and membrane excitability protocols. For muscle force and membrane excitability measurements, the subject sat upright in a straight-backed chair with hips and legs firmly secured by velcro straps, the knee at 90° to the thigh, and the arms folded across the chest. The variables measured included maximal voluntary contraction (MVC), the %activation (%ACT) using the interpolated twitch technique (3), and the forces generated during a twitch (Pt) obtained with supramaximal voltage, and during stimulation frequencies (Hz) of 10(P10), 20(P20), 30(P30), 50(P50), and 100(P100). The interpolated twitch, which is obtained by application of a single supramaximal stimulus during an MVC, is a measure of central or neural inhibition. For all of these frequencies, the maximal rate of force development (+dP/dtmax) and the maximal decline in force development (–dP/dtmax) were assessed. In addition, contraction time (CT) and one-half relaxation time (1/2 RT) were measured assessed for the twitch only. The potentiated twitch, which is a measure of the increased Pt that occurs as a consequence of a 10-s conditioning MVC applied just before measurement, was assessed in conjunction with Pt. The potentiated twitch force, which can be isolated to the muscle, is used to adjust the calculation of %ACT of muscle by the neural system. All of these procedures have been previously described in detail by our group (21, 55).
For measurement of force behavior, a 5-cm-wide plastic cuff placed around the lower right leg just proximal to the ankle malleoli was tightly attached to a linear variable differential transducer anchored at a level just below the malleoli. The linear variable differential transducer was amplified by a Daytronic carrier preamplifier at 1 kHz, converted to a digital signal and fed into a 12-bit analog-to-digital converter and then to an IBM computer for analyses. Calibration was provided before each test session with weights of known amounts. Twitches and tetani were elicited by stimulation of the quadriceps muscles using a Grass model S48 stimulator with an isolation unit. Two aluminium electrodes (8 x 13 cm) coated with warm electrode gel were used to deliver the electrical impulses to the quadriceps muscles. The ground electrode was placed centrally in the anterior aspect of the thigh just above the patella, whereas the active electrode was toward the hip on the proximal portion of the belly of the vastus lateralis. Each electrode was secured firmly with tensor bandage.
For twitch properties, a single supramaximal voltage (150 V) impulse of 50-µs duration was employed. For the measurement of the different impulse frequencies, the voltage selected was based on an 50% MVC response at 100 Hz. For the frequency measurements, pulse durations were 50 µs and train durations 1 s. The voltage and electrode placement were kept constant for both of the progressive tests. During a typical test session, subjects were first assessed for supramaximal twitch properties, followed by tetanic stimulations from low to high frequency and concluding with MVCs. All procedures for calculation of specific properties are as previously detailed (21).
Membrane excitability was measured in the vastus medialis using the compound mass action potential (M wave) obtained from the supramaximal twitch. The properties measured included the peak-to-peak amplitude (mV), duration (ms), and area (µV) (21). Amplitude was defined as the sum of the absolute values for maximum and minimum points of the biphasic (one positive and one negative deflection) M wave. Duration was defined as the time from baseline to baseline from the beginning to the end of the biphasic M wave, where the beginning is established as a positive deflection 2 SDs above baseline harmonic mean and the end as a return to baseline. The area was calculated as the integral of the absolute value of the entire M wave. The M-wave properties were based on the average obtained from two twitches. In addition to the M wave, raw electromyogram (EMG) signals for average integrated electromyogram (AEMG) signals were obtained from MVC contractions and were full-wave rectified, and the resulting signal was integrated over the duration of the contraction. This provides a measure of the combined neural and muscular EMG in response to maximal voluntary activation. The EMG signal (20- to 500-Hz bandwidth) was passed through an alternating current amplifier (National Instruments, AT-M10–16H Multifunction board). The gain was calibrated to optimize signal amplitude for analog-to-digital conversion and collected at 2,028 Hz. Custom-modified National Institutes of Allergy and Infectious Diseases software (National Instruments) was used to acquire EMG and force records and analyze raw data (Lab view 5–1 software routine). Additional details, including the calculation of specific properties, are as previously published from our laboratory (21). It should be noted that M-wave properties were collected from the vastus medialis, while tissue was sampled from the vastus lateralis. The possibility exists that different response patterns may occur between the two muscles.
In an additional group, M waves were recorded during the progressive cycling, according to the procedures of Jammes et al. (31). In this procedure, the M wave was obtained from the vastus medialis after stimulation of the vastus lateralis. Both the location of the stimulating and recording electrodes and the pulse characteristics were as described for the mechanical measurements. The M wave was collected from the right leg at the beginning of eight consecutive leg extensions. A signal generated from an electronic sensor was used to standardize the joint angle at which the twitch was induced (31).
Analytical procedures. The maximal activity of the Na+-K+-ATPase was assessed in whole muscle homogenates using the K+-stimulated 3-O-methylfluorescein phosphatase (3-O-MFPase) assay following the basic procedures of Huang and Askari (28) and Horgan and Kuypers (27) as modified by Fraser and McKenna (23). Briefly, tissue from frozen muscle samples was prepared in a homogenate (5% wt/vol) at 0°C in a buffer containing 250 mM sucrose, 2 mM EDTA, and 10 mM Tris (pH 7.40). Homogenization was performed at 0°C for 2 x 20 s, at 25,000 rpm (Polytron). Before analysis, the homogenate was frozen-thawed four times and then diluted 1:4 in cold homogenate buffer. The assay medium for the measurement of 3-O-MFPase activity contained 5 mM MgCl2, 1.25 mM EDTA, 100 mM Tris, and an 80 nM 3-O-methyl fluorescein standard (pH 7.40). The homogenate (30 µl) was incubated in a 2.5 ml of assay medium at 37°C for 5 min before the addition of 40 µl of 10 mM 3-O-MFP to initiate the reaction. After 60 s, 10 µl of 2.58 M KCl (final concentration 10 mM) was added to stimulate K+-dependent phosphatase activity, and the reaction was measured for a further 60 s. All assays were performed at 37°C, with continuous stirring, on a spectrofluorometer (Aminco Bowman AB2 SCM, Urbana, IL). Excitation wavelength was 475 nm, and emission wavelength was 515 nm, with 4-nm slit widths. The activity of 3-O-MFPase was determined by the difference in slope before and after the addition of KCl (23). The nonspecific activity represents the activity measured in the absence of K+. The nonspecific activity is thought to represent spontaneous hydrolysis and unspecific Mg2+-activated phosphatase activity of the homogenate. Maximal in vitro 3-O-MFPase activity was expressed relative to muscle protein content (pmol · min–1·mg protein–1). Protein content in the homogenate was determined by the method of Lowry as modified by Schacterle and Pollock (51). The activity was based on the average of three trials. To control for between-day variability in the assay, all samples for a given individual were analyzed during the same analytical session. The assay is a highly sensitive measurement of 3-O-MFPase, as indicated by the complete elimination of the slope with the addition of KCl by ouabain (4, 23).
Data analyses. Statistical analysis was performed on Statistica for Windows R.4.5 software (1993; Statsott A, Tulsa, OK). Both one- and two-way ANOVA procedures for repeated measures were used to analyze 3-O-MFPase changes with condition and with time. One-way ANOVA was used for each condition while two-way ANOVA was used to compare conditions using matched samples. For mechanical and membrane excitability, only two-way ANOVA procedures were employed. Post hoc analyses of main and interactive effects were performed using the Tukey test. The probability for statistical significance was set at P < 0.05. Throughout the test, all group values are represented as 0 ± SE.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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O2 and arterial O2 saturation.
In Norm, progressive increases in PO to fatigue resulted in progressive increases in O2 (l/min) (Fig. 1). A similar response was observed during progressive exercise in Hypox. The primary effect of Hypox was to reduce the peak mechanical power output and peak O2 that could be realized. In Norm compared with Hypox, peak PO was 292 ± 7.5 and 238 ± 7.0 W and O2 peak was 3.95 ± 0.18 vs. 3.20 ± 0.10 l/min. The reductions amounted to 18% and 19% for mechanical PO and O2 peak, respectively. No differences were observed between Norm and Hypox in O2 at any of the exercise intensities examined.
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(Fig. 2). Exercise in Hypox also resulted in a lower Sp compared with preexercise. The lower SaO2 was observed earlier in exercise and persisted throughout the progressive workload protocol until fatigue. In contrast, no reduction in Sp
from Pre regardless of the PO, was observed in Norm.
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12% in force was observed in Norm (Table 1). The reduction in MVC was not accompanied by reductions in %ACT as assessed by the interpolated twitch technique. No differences were observed between Norm and Hypox in MVC and %ACT. As with MVC, reductions in AEMG were observed with exercise, regardless of condition.
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O2 peak (Norm), 100% O2 peak (Hypox), and 100% O2 peak (Norm) (Fig. 4). In contrast, both the amplitude and the area of the M-wave were increased with Hypox. At both 50% O2 peak (Norm) and 100% O2 peak (Hypox), these properties were higher in Hypox compared with Norm.
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O2 peak. For both Norm and Hypox, the magnitude of the inactivation of Na+-K+-ATPase activity was not different, amounting to between 29 and 32%. Nonspecific activity was also observed to decrease with exercise. This was not unique to condition. As with the specific Na+-K+-ATPase activity, the decline was fully manifested by the first measurement point. For Norm, the nonspecific values (nmol·mg protein–1·h–1) were 935 ± 66, 771 ± 47, 719 ± 32 and 767 ± 34 for 0, 137, 250 and 300 W, respectively. For Hypox, the values were 779 ± 20, 646 ± 22 and 751 ± 42, for 0, 137 and 214 W, respectively.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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O2 peak that could be achieved, in general, neuromuscular performance and membrane excitability were not negatively affected compared with Norm. These findings suggest that the impairment in neuromuscular function, measured before and after the progressive exercise, is attributable to sites other than the sarcolemma. In this regard, our findings confirm our earlier research using a prolonged exercise protocol, namely that the neuromuscular deficit that occurs with Norm and Hypox cannot be explained by a failure in membrane excitability (50). The results of the current study indicate that although using a test protocol in hypoxia severely blunts power output, compromises O2 peak, disrupts energy homeostasis, and depresses Na+ and K+ membrane transport potential, membrane excitability does not appear to be limiting.
Muscular fatigue.
What is clear from our measurements is that the progressive exercise protocol resulted in a neuromuscular deficit in force-generating capacity. Measurements of MVC indicated that reductions of 92 N, or 12%. To isolate whether the fatigue was central or peripheral in nature, we have estimated the %ACT using the interpolated twitch technique (3). Our results indicate that the force loss was not central in nature since %ACT was near maximal and did not decline. However, some qualification is necessary. Because transcutaneous stimulation of the vastus lateralis involves activation of the small nerve endings, as demonstrated by the lack of a response with curare (a neuromuscular blocking agent), the failure to observe an increase in %ACT could also involve a failure in neuromuscular transmission (30). Accordingly, we can only conclude that the reduction in MVC could be due to the neuromuscular junction and/or the muscle itself (30). A failure in neuromuscular transmission is normally not thought to be rate limiting in repetitive exercise, regardless of the protocol employed (9). We have also found reductions in AEMG with exercise during the MVC. The reductions in AEMG could also be due to a reduction in neural drive, neural transmission, or membrane excitability (8). Because we have found no evidence for an inhibition of neural drive or a loss of membrane excitability, more peripheral site(s) in the muscle remain as the most likely source(s) for the reduction in MVC observed following cycling.
To examine the nature and magnitude of the loss in muscle force-generating potential, we have used a variety of stimulation conditions. One such condition involved the response to a single supramaximal twitch. Progressive exercise to fatigue resulted in a pronounced reduction in Pt. Because the reduction in Pt occurred in the absence of changes in the properties of the M wave, one or more sites could result in a reduction in [Ca2+]f and/or a depression in the sensitivity of the regulatory and contractile proteins to [Ca2+]f activation (37). Although changes in both of these properties have been documented with repetitive exercise (1, 18), a reduction in [Ca2+]f appears to be a major factor (10). The reductions in [Ca2+]f could be due to a failure in coupling between the t-tubule and CRC (53) or to direct alterations in the CRC (1). Reductions in SR Ca2+-release, mediated by apparent structural alterations in the CRC, have been observed to occur in skeletal muscle with repetitive stimulation as assessed in vitro (17).
The frequency-dependent nature of fatigue, induced by the progressive exercise protocol, was examined by brief submaximal stimulation of the muscle at frequencies ranging from 10 to 100 Hz. Reductions in force output of between 16% and 51% were observed across the frequencies examined. The reductions observed are also consistent with a depression in Ca2+ release (1). The fact that force is increased with increasing stimulation frequency after exercise, similar to preexercise, suggests that increasing activation can increase Ca2+ release and [Ca2+]f. However, with a given stimulation condition, Ca2+ release and, consequently, [Ca2+]f,is impaired (10).
To gain further insight into the contractile processes that might be altered with progressive exercise, we have also measured the rates of force development and relaxation. After exercise, the rate of twitch for both +dP/dtmax and –dP/dtmax decreased, while CT and 1/2 RT were unaltered. The decreases in +dP/dtmax and –dP/dtmax are suggestive of both a slower maximal rate of weak to strong binding and a slower maximal strong-to-weak binding and dissociation of cross bridges, respectively (39). At least for -dP/dtmax, reductions in SR Ca2+-uptake and Ca2+-ATPase activity occur after progressive exercise to fatigue (16). It should be emphasized that when the decrease in Pt was taken into account, neither +dP/dtmax nor –dP/dtmax were altered after exercise. This would suggest that the putative reductions in [Ca2+]f that occur with fatigue does not impair the maximal kinetics of force development and relaxation when adjusted for peak force (43).
Although force at different frequencies of stimulation after exercise was generally not differentially affected by Hypox, we found evidence that –dP/dtmax was not as disturbed. At 20 and 30 Hz, reductions in –dP/dtmax with Hypox were not as pronounced. This could suggest less of an impairment in actin-myosin dissociation and/or Ca2+ uptake in Hypox compared with Norm, during relaxation. At present, it is unclear whether these differences are due to Hypox per se on the shorter exercise time or PO realized in Hypox.
The reduction in the absolute maximal rate of relaxation could have important consequences to force levels, particularly at the lower frequency of stimulation where unfused tetani occurs. The effect of the slower rate of relaxation would be to attenuate the force loss, observed with exercise, given the higher force level that would persist between each impulse (20, 56).
M-wave properties. A particularly surprising finding was the lack of an impairment in the properties of the M wave, considering the approximate 32% reduction that we have observed in maximal Na+-K+-ATPase activity with progressive exercise to fatigue in Norm. Reductions in membrane excitability have been shown to parallel reductions in force in a variety of studies using repetitive contractions where the Na+-K+-ATPase activity has been partially inhibited by ouabain (41). However, in contrast to the previous studies where M-wave characteristics were measured concurrently with fatigue, initially, we assessed these properties only 4–5 min after exercise. It would be expected that some recovery in M-wave properties would occur during the period after exercise when the volunteers were being prepared for stimulation, as a result of recovery or partial recovery of selected intracellular metabolic by-products (40) and restoration of transmembrane Na+ and K+ gradients (41). To investigate whether or not significant recovery had occurred, we have performed a separate set of experiments measuring the M-wave properties during the progressive exercise task itself. During the progressive exercise in Norm, although a trend was evident, suggesting decreased levels of the M-wave properties, none of the changes were significant.
The failure to detect reductions in M-wave properties was not expected since Jammes et al. (31) have shown that the pronounced decline in vastus lateralis M-wave amplitude persists for at least 10 min in recovery. Interestingly, in the Jammes et al. study (31), the apparent disturbance in membrane excitation only occurred in untrained and not trained volunteers. Our study also employed untrained participants. However, in the our study, the volunteers were 20 years younger. It is possible that other factors such as differences in methodology may be involved in explaining the different results obtained between the two studies. Because Jammes et al. (31) also used progressive exercise, with the same general conditions—both with respect to time of exercise and the incremental power output—the differences in the exercise protocol would not appear to be important.
Na+-K+-ATPase. Contrary to our hypothesis, we found no additional inactivation of the Na+-K+-ATPase activity in Hypox at a comparable PO to Norm or when the progressive cycle exercise was performed to fatigue in Hypox. This was unexpected, as the exercise at comparable POs in Hypox compared with Norm results in a greater metabolic stress, including elevation of selected metabolic by-products such as Pi and H+ (36, 48) and conceivably in ROS accumulation (48). Previous studies have demonstrated a susceptibility of ion channel pumps, including the Na+-K+-ATPase, to damage caused by ROS (33). Although, it is known that exhaustive exercise causes ROS accumulation (45), it remains to be definitively established that progressive exercise in Hypox results in a more emphasized ROS accumulation. Unexpectedly, the reduction in Na+-K+-ATPase activity was observed early in the exercise protocol for both conditions. The inactivation that was observed would appear to involve an intracellular change related to the adjustment to exercise, possibly a rapid increase in ROS. It should be emphasized that our measures of Na+-K+-ATPase activity were performed under optimal in vitro conditions. In vivo, the catalytic activity of the Na+-K+-ATPase would be expected to be lower (32) given the accumulation of metabolic by-products in the intracellular environment with progressive exercise. In light of this, it is even more remarkable that the Na+-K+-ATPase activity that remains is sufficient to protect membrane excitability. At fatigue, no differences existed between Norm and Hypox in Na+-K+-ATPase activity. This finding was also unexpected given the longer exercise time at greater PO achieved in Norm compared with Hypox.
The possibility remains that the dissociation that we have observed between changes in Na+-K+-ATPase activity and membrane excitability reflects our sampling sites. Membrane excitability was measured in the vastus medialis muscle, while Na+-K+-ATPase activity was measured in the vastus lateralis. Previous studies, however, have reported generally similar M-wave responses between these muscles (34, 35).
O2 peak. The reduction in O2 peak observed in Hypox compared with Norm during the progressive exercise does not appear to be due to a failure in membrane excitability as a cause of the limited PO, which potentially could also limit oxidative phosphorylation. During the progressive exercise in Hypox, we have found increases in M-wave amplitude and area, not decreases as postulated. The increases in these properties have been observed earlier in a variety of exercise protocols and have been labeled pseudofacilitation (38). The increases have been attributed to hyperpolarization, secondary to activation of the electrogenic Na+-K+-ATPase (38). Interestingly, with hypoxemia, no evidence of pseudofacilitation during sustained contractile activity has been observed (15). Differences in muscle mass, type of muscle, and the task may be important in explaining the differences between the two studies.
In summary, the results of this study indicate that progressive cycle exercise to fatigue in Hypox compared with Norm results in a blunting of the peak mechanical PO and O2 peak. Progressive exercise to fatigue also results in an approximate 30% reduction in maximal Na+-K+-ATPase activity, regardless of condition. Mechanical function when measured after the exercise indicates a reduction in maximal force-generating capacity, possibly by disturbances at one or more sites in the muscle contractility. The intracellular processes involved in fatigue measured after exercise appear not to involve a failure in membrane excitability, since no alterations in the properties of the M wave occurred either in Norm or Hypox. A failure in the sarcolemma and t-tubule to conduct repetitive action potentials also does not remain as a viable site of fatigue during progressive exercise itself in the untrained. As such, a failure in membrane excitability cannot explain the O2 peak attained in Norm or the blunting in O2 peak observed in Hypox. It is possible that the intracellular site involved in muscle fatigue during Norm and Hypox is the same, the differences being that the Hypox stress precipitates the failure at a lower PO and O2 peak.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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 M. Amann, D. F. Pegelow, A. J. Jacques, and J. A. Dempsey Inspiratory muscle work in acute hypoxia influences locomotor muscle fatigue and exercise performance of healthy humans Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2007; 293(5): R2036 - R2045. [Abstract] [Full Text] [PDF]
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 H. J. Green, T. A. Duhamel, G. P. Holloway, J. W. Moule, J. Ouyang, D. Ranney, and A. R. Tupling Muscle Na+-K+-ATPase response during 16 h of heavy intermittent cycle exercise Am J Physiol Endocrinol Metab, August 1, 2007; 293(2): E523 - E530. [Abstract] [Full Text] [PDF]
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H. J. Green, T. A. Duhamel, K. P. Foley, J. Ouyang, I. C. Smith, and R. D. Stewart Glucose supplements increase human muscle in vitro Na+-K+-ATPase activity during prolonged exercise Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2007; 293(1): R354 - R362. [Abstract] [Full Text] [PDF]
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M. Amann, L. M. Romer, A. W. Subudhi, D. F. Pegelow, and J. A. Dempsey Severity of arterial hypoxaemia affects the relative contributions of peripheral muscle fatigue to exercise performance in healthy humans J. Physiol., May 15, 2007; 581(1): 389 - 403. [Abstract] [Full Text] [PDF]
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K. Katayama, M. Amann, D. F. Pegelow, A. J. Jacques, and J. A. Dempsey Effect of arterial oxygenation on quadriceps fatigability during isolated muscle exercise Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2007; 292(3): R1279 - R1286. [Abstract] [Full Text] [PDF]
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L. M. Romer, H. C. Haverkamp, M. Amann, A. T. Lovering, D. F. Pegelow, and J. A. Dempsey Effect of acute severe hypoxia on peripheral fatigue and endurance capacity in healthy humans Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R598 - R606. [Abstract] [Full Text] [PDF]
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M. Amann, M. W. Eldridge, A. T. Lovering, M. K. Stickland, D. F. Pegelow, and J. A. Dempsey Arterial oxygenation influences central motor output and exercise performance via effects on peripheral locomotor muscle fatigue in humans J. Physiol., September 15, 2006; 575(3): 937 - 952. [Abstract] [Full Text] [PDF]
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M. Amann, L. M. Romer, D. F. Pegelow, A. J. Jacques, C. J. Hess, and J. A. Dempsey Effects of arterial oxygen content on peripheral locomotor muscle fatigue J Appl Physiol, July 1, 2006; 101(1): 119 - 127. [Abstract] [Full Text] [PDF]
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Significantly different from 137 W (P < 0.05). #Significantly different from 214 W (P < 0.05). 
Significantly different from 250 W (P < 0.05).
Significantly different from 50% 