INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

A NUMBER OF INTRACELLULAR sites have been mechanistically linked to depressed force after activity. Accumulating evidence suggests that an impairment in excitation-contraction coupling can be a primary source of force failure with severe fatigue (49); however, other sites may contribute to varying degrees (19). More specifically, the role of the sarcolemma and t-tubular membranes in conducting repetitive action potentials has been identified as a possible contributor to fatigue in humans during voluntary exercise (20, 43). Failure at the level of the sarcolemma could occur because the reduction in the transmembrane gradients for Na⁺ and K⁺ during exercise reduces the ionic membrane potential. This would be reflected in decreases in the amplitude and area of the muscle compound action potential (M wave) and force (37).

Previous work studying the sarcolemma as a site in fatigue has relied primarily on properties of the electromyogram (EMG) and, subsequently, the M wave, as an indirect measure of sarcolemmal excitability. Reduced M-wave amplitude and/or area has been directly related to reduced force in experiments employing sustained muscle stimulation in both animals (2, 40) and humans (26). However, acute decreases in M-wave amplitude and area are not commonly observed during volitional exercise, especially if the fatigue is induced by high-intensity, short-duration (<5 min) activity (3, 9). This may be due to M-wave potentiating effects that counteract or supersede the effects of fatigue directly after these types of contraction schedules (34).

In contrast, contractions repeated for longer durations appear to induce greater M-wave depressions than brief, high-force, fast-fatiguing contractions (3, 18). In studies by Arnaud et al. (1) and Jammes et al. (27), the depression in M-wave amplitude persisted for a minimum of 15 min after bouts of supramaximal cycling and progressive cycling to fatigue, respectively. Persistent reductions in M-wave properties have also been observed by others (35) during recovery after sustained, intermittent contractile activity. Collectively, these findings suggest that reduced excitability may have a more significant role in fatigue induced by sustained exercise than previously believed. It is possible that acute alterations of membrane proteins may be the basis for sustained M-wave changes and, consequently, contribute to the long-lasting fatigue of postcontractile depression (PCD) that is commonly observed after sustained isometric exercise (45, 47).

The Na⁺-K⁺-ATPase is an integral membrane protein that maintains ionic gradients at the sarcolemma by pumping 3Na⁺ out and 2K⁺ into the cell during each cycle of the pump. The activity of the pump is increased in response to repetitive action potentials. This pump activity is also electrogenic in nature and, therefore, contributes to membrane potential and the maintenance of sarcolemmal excitability (11). Changes in M wave result primarily from a loss in muscle excitability, secondary to a loss in Na⁺ and K⁺ gradients (40). Consequently, the catalytic activity of the Na⁺-K⁺ pump to maintain ion gradients is believed to directly influence the magnitude of the M wave (24, 39). Although the Na⁺-K⁺-ATPase has a wide dynamic range of activity to maintain excitability in vivo (37), there is little evidence to indicate that exercise intrinsically alters the enzymatic activity of the Na⁺-K⁺-ATPase in skeletal muscle.

A recent study by Verburg et al. (48) supports this possibility. These investigators reported that, during sustained submaximal exercise, an increased loss of K⁺ from muscle was observed late in the exercise, which was attributed to an "insufficient activation of pumps." The "insufficient activation" could be due to an exercise volume-dependent inactivation of the Na⁺-K⁺-ATPase, resulting in a loss of enzymatic activity and an inability to maintain K⁺ homeostasis, membrane excitability, and, consequently, force. Evidence from cardiac muscle Na⁺-K⁺-ATPase indicates that the pump may be susceptible to damage by free radicals (29, 30), which are produced during contractile activity (44). Recently, our laboratory has demonstrated that moderate-intensity isometric contractions for 30 min in humans were associated with prolonged reductions in sarcoplasmic reticulum (SR) Ca²⁺- ATPase pump function in vitro, which could have contributed to PCD (47). PCD is a type of fatigue characterized by a depression in force across low and high frequencies of stimulation after exercise (47). It is possible, particularly with the high level of activation required to sustain this type of isometric exercise schedule, that the Na⁺-K⁺-ATPase may be similarly affected and result in reduced enzyme activity measured in vitro after exercise.

The purpose of this study was to determine whether Na⁺-K⁺-ATPase activity is affected by sustained, moderate-intensity, isometric exercise and to investigate the interrelationship among Na⁺-K⁺-ATPase activity, muscle excitability, and neuromuscular fatigue. We have hypothesized that Na⁺-K⁺-ATPase activity and muscle excitability will be reduced during exercise and will remain depressed during recovery, as was observed for the Ca²⁺-ATPase using a similar protocol.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subjects. Fourteen healthy, active volunteers [7 men, 7 women, age 23.4 ± 0.7 (SE) yr] participated in the study. Nine of these subjects volunteered for the biopsy component of the study. The study carried the approval of the Office of Human Research at the University of Waterloo, and all participants were fully informed of all experimental procedures and associated risks before written consent was obtained.

Experimental design. To investigate the relationship between Na⁺-K⁺ pump function and PCD, subjects completed a moderately intense isometric exercise protocol as previously described (47), with minor modifications. Subjects performed isometric single-leg extension at a target force of ~60% of maximum voluntary contraction (MVC) for 30 min using a 0.5 duty cycle (5-s contraction, 5-s rest) to induce PCD.

Muscle stimulation and force measurements. The experimental setup for measuring muscle contractile characteristics in isometric knee extension has been described previously (22, 47). Briefly, for all force measurements, both voluntary and involuntary, the participant sat upright in a straight-backed chair with hips and leg firmly secured, the knee at 90° to the thigh, and the arms folded across the chest. Twitches (Tw) and tetani were elicited by stimulation of the quadriceps muscles using a Grass model S48 stimulator with an isolation unit. 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. 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 into an IBM computer for analysis. Calibration was performed before each test session with weights of known amounts. Two aluminum chloride electrodes (8 × 13 cm) coated with warm electrode gel were used to deliver the electrical impulse to the quadriceps muscle. The ground electrode was placed centrally on 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 wrapped around the leg and over the top of the electrode to ensure good contact between the skin and the electrode.

EMG. The area required for muscle biopsies and positioning of stimulation electrodes precluded the use of the vastus lateralis for EMG recordings. Preliminary testing indicated that changes in vastus medialis EMG correlated with changes in vastus lateralis EMG at the specific joint angle used in this experiment (unpublished observations). EMG recordings were made with 10-mm-diameter Ag-AgCl (Meditrace 60) surface electrodes. Electrodes were placed over the belly of the vastus medialis (interelectrode distance was ~2 cm), with one ground electrode positioned on the lateral epicondyle of the tibia. The skin was shaved, abraded, and cleaned with alcohol. Electrode positioning was not altered during a single testing trial.

Na+-K⁺-ATPase activity. Activity of the Na⁺-K⁺-ATPase was assessed using the K⁺-stimulated 3-O-methylfluorescein phosphatase (3-O-MFPase) assay following the procedures of Fraser and McKenna (17), with minor modifications. In this assay, it is not the hydrolytic activity of the ATPase that is measured as such but the phosphatase activity. Measurement of phosphatase activity is based on the K⁺-dependence of the chromogenic substrate 3-O-methylfuorescein phosphate (3-O-MFP), which substitutes for the aspartylphosphate intermediate of the ATPase to represent the terminal step in ATP hydrolysis (25). In the measurement of 3-O-MFPase, the activity of the enzyme is based on the dephosphorylation of 3-O-MFP, which occurs during stimulation of the enzyme by K⁺. The change in fluorescence, which accompanies the hydrolysis of 3-O-methylfluorescein from 3-O-MFP and which is a measure of the 3-O-MFPase activity, is quantified by fluorescence spectrophotometry (excitation wavelength = 475 nm; emission wavelength = 515 nm, with 5-nm slit width). Briefly, tissue from the frozen muscle biopsy was homogenized (5% wt/vol) at 0°C for 2 × 20 s at 25,000 rpm (Polytron), in a buffer containing 250 mM sucrose, 2 mM EDTA, and 10 mM Tris (pH 7.40). Homogenates were freeze-thawed four times and diluted 1:4 in cold homogenate buffer. Approximately 30 µg of protein (~25-µl homogenate) were incubated for 4 min in medium containing 5 mM MgCl₂, 1.25 mM EDTA, and 100 mM Tris (pH 7.40). The K⁺-stimulated activity of the Na⁺-K⁺-ATPase was determined by the increase in activity after the addition of 10 mM KCl, at a substrate concentration of 160 µM 3-O-MFP. 3-O-MFPase activity was based on the difference in slope before and after the addition of KCl (F/min). We have demonstrated that the difference in slope is completely eliminated by [³H]ouabain, which is used to inhibit Na⁺-K⁺-ATPase activity (H. Green, unpublished observations). The Na⁺-K⁺-ATPase activity, which was based on an average of four trials for each sample, is expressed in nanomoles per milligram protein per hour. This value was obtained by dividing F for the sample by F for a standard solution of 3-O-MFP, multiplying by the concentration of the standard, and dividing by the amount of protein in the tissue sample assayed (25). Protein content of the homogenate was determined by the method of Lowry as modified by Schacterle and Pollock (42).

Na+-K⁺-ATPase content. The content of Na⁺-K⁺ pumps in muscle biopsy samples was determined using the assay developed by Nørgaard et al. (38) and employed previously in our laboratory (10). With the use of this procedure, two samples from each biopsy, weighing between 2 and 8 mg, are prewashed twice for 10-min periods in a Tris-sucrose buffer (10 mM Tris · HCl, 3 mM MgSO₄, 1 mM Tris-vanadate, and 250 mM sucrose) containing sodium metavanadate (NaVO₃) at 0°C. Samples were incubated in the Tris-sucrose buffer with [³H]oubain (1.8 µCi/ml) and unlabeled oubain (1 µM final concentration) for 2 × 60 min at 37°C. After the unbound oubain was removed by washing four times for 30 min in ice-cold buffer, the samples were blotted, weighed, placed in 1.5-ml Eppendorf tubes, and soaked in 1 ml 5% trichloroacetic acid for 16 h at room temperature, and then 0.5 ml of sample were counted for ³H activity in a scintillation mixture. [³H]oubain binding capacity was corrected (×1.05) for loss of specifically bound [³H]oubain during washout (38). No correction was made for nonspecific uptake and retention of ³H, which was estimated at <3% (H. Green, unpublished observations). The isotopic purity of the [³H]oubain was 99% as determined by the supplier (New England Nuclear-Du Pont Canada).

Data analysis. Statistical analysis was performed on Statistica for Windows R.4.5 software (1993; Statsoft A, Tulsa, OK). Descriptive statistics included means and SE. One-way ANOVA with repeated measures was used to analyze Na⁺-K⁺-ATPase properties over time. Correlational analysis by linear regression was used to relate M-wave area to Na⁺-K⁺-ATPase activity and Tw force. Two-factor ANOVAs were used to compare differences in force and EMG measures between the Con and Ex condition for each time point. Differences between Ex and Con legs were determined by paired comparison. Post hoc analysis of main effects was performed using the Tukey test. The probability level for statistical significance was accepted at P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Force measurement. The target force was maintained throughout most of the protocol, although some subjects were unable to maintain a target of ~60% of MVC in the last minutes of the exercise session (data not presented). All subjects completed 30 min of exercise, despite the neuromuscular fatigue.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Stimulated force in exercise (A) and control (B) vastus lateralis before (Pre), immediately after (PST0), 1 h after (PST1), and 4 h after (PST4) exercise. Values are means ± SE. * Significantly different from Pre (P < 0.05; n = 14). ** Symbols beside lines indicate only main effects, namely reduced force across all frequencies.

EMG. During the exercise, AEMG during repetitive contractions increased by 20% (P < 0.05) (results not presented). The AEMG at the beginning of the repetitive contractions was 52% of AEMG observed with the Pre MVC. The AEMG during MVC was reduced (P < 0.05) by 32 and 20% at PST0 and PST1, respectively (Table 1). There was no change in AEMG at MVC in the Con limb.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   M-wave amplitude (A), duration (B), and area (C) (see definitions in text), represented relative to Pre values in exercise (Ex) and control (Con) limbs at PST0, PST1, and PST4. M-wave properties were obtained from the vastus medialis. Values are means ± SE. * Significantly different from Pre (P < 0.05, n = 14).

Na+-K⁺-ATPase function. Na⁺-K⁺-ATPase activity, measured by 3-O-MFPase, was 81.2 ± 12.8 nmol · mg protein¹ · h¹ at Pre for Con. At PST0 in Ex, 3-O-MFPase was 35% less than at PST0 for Con (58.8 ± 9.8 vs. 90.6 ± 13.2 nmol · mg protein¹ · h¹; P < 0.05) (Fig. 3A). 3-O-MFPase activity was not different from Pre at any time point. [³H]oubain binding indicated that the Na⁺-K⁺-ATPase pump content quantified by this technique did not change over time (Fig. 3B). Linear regression indicated a low correlation between 3-O-MFPase and M-wave area (r = 0.38; P = 0.02; n = 45) (Fig. 4). In addition, 3-O-MFPase exhibited a correlation to Na⁺-K⁺-ATPase pump content in nonexercised samples (r = 0.59; P < 0.01; n = 16).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Na+-K+-ATPase activity indicated by 3-O-methylfluorescein phosphatase activity (3-O-MFPase) (A) and Na+-K+-ATPase content indicated by 3[H]ouabain binding (B) in response to exercise. Biopsies were taken from the vastus lateralis at Pre and PST0 in the Con leg and at PST0, PST1, and PST4 in the Ex leg. Values are means ± SE (n = 9). * Significantly different (P < 0.05).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Correlation between Na+-K+-ATPase activity, indicated by 3-O-MFPase and M-wave area.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In this study, we hypothesized that Na⁺-K⁺-ATPase activity and muscle excitability would be reduced during an isometric exercise protocol previously shown to be associated with a prolonged impairment in SR Ca²⁺ pump function (47). Using the 3-O-MFPase assay, we demonstrated a 38% difference in Na⁺-K⁺-ATPase activity between the Ex and Con limb directly after the contractile protocol. This change was also accompanied by depressions in M-wave amplitude and area. The fact that M-wave and Na⁺-K⁺-ATPase activity recovered by PST4 demonstrates the rapidly adaptable nature of the Na⁺-K⁺ pump in skeletal muscle. These results also suggest that, at least for the isometric task studied, compromised Na⁺-K⁺-ATPase activity may contribute to reduced sarcolemmal excitability and contribute to muscle fatigue. It should be emphasized that, although a strong trend was observed toward a decline in Na⁺-K⁺-ATPase measured at PST0 compared with Pre, statistical significance was not found. Using the contralateral limb as the control would appear appropriate because the nonexercised leg would be subjected to many of the vascular influences (hormonal, ionic) as the Ex leg.

Exercise protocol and fatigue. PCD is a type of fatigue characterized by a depression in force across low and high frequencies of stimulation after exercise (45, 47). The repetitive isometric exercise protocol employed in this experiment depressed force output across all muscle stimulation frequencies and in MVCs. Although some recovery was evident, fatigue persisted up to PST4. This protocol was previously observed to cause acute alterations of the Ca²⁺ pump, reducing maximal activity of the Ca²⁺-ATPase for at least PST1 (47). The authors suggested that Ca²⁺ cycling could be one of several sites of fatigue with this protocol. Previous investigations on the cause(s) of PCD identified a failure of neuromuscular transmission (8), although it was later demonstrated that muscle fibers retained the ability to generate normal action potentials during PCD (33). The results of this experiment indicate that, given the high level of activation required to sustain the voluntary isometric exercise schedule, impaired excitability of the sarcolemma could contribute to neuromuscular fatigue that was observed. However, definitive evidence that impaired sarcolemmal excitability is mechanistically linked to fatigue would depend on demonstrating impaired t-tubule excitability and coupling between the t-tubule and the SR Ca²⁺-release channel. During recovery, excitability was restored at a point at which force was still depressed. This observation supports other findings that indicate that other peripheral sites of fatigue are responsible for more prolonged reductions in force during the period after exercise [as reviewed by Fitts (16)]. The possibility that central fatigue, because of an impairment in neural drive, also occurred must be acknowledged. The reductions that were observed in MUA during the MVC and the reductions in AEMG observed both after exercise and in recovery are consistent with this notion.

Inactivation of the Na+-K⁺-ATPase. The capacity of the Na⁺-K⁺ pump to restore ionic gradients can be challenged by heavy contractile demands and resultant changes in the local environment (i.e., ionic and metabolite buildup, substrate depletion), and, therefore, appropriate regulation of Na⁺-K⁺-ATPase activity is essential for maintaining transport capacity and muscle excitability (37). Complex regulatory controls exist to ensure that Na⁺-K⁺ pump capacity is "activated" during exercise to maintain excitability. Intrinsic activity of the pump is increased over a 20-fold range by mechanisms associated with the action potential (14) and acute hormonal control (7). Pump capacity may also be increased by translocation of a specific pool of Na⁺-K⁺ pump subunits from intracellular sites to the muscle membrane (28). Despite a wealth of information published on the role of the Na⁺-K⁺-ATPase in muscle excitability and fatigue, the actual enzymatic capacity of the pump in skeletal muscle to meet the demands of exercise remains relatively unknown. Most experiments rely on indirect measures of excitability and pump function in response to a short-term fatiguing event. However, there has recently been evidence that Na⁺-K⁺ pump inactivation can occur during sustained exercise in humans. Verburg et al. (48) determined that, after a period of sustained submaximal exercise, increased loss of K⁺ from muscle occurred, which persisted in brief recovery. This observation suggests that a reduction in Na⁺-K⁺-ATPase activity occurred as the exercise progressed. This conclusion is supported by a study reported in abstract form that found direct reductions in Na⁺-K⁺-ATPase activity as measured by 3-O-MFPase during prolonged exercise (36). Our results offer further evidence that inactivation of the Na⁺-K⁺-ATPase can occur, at least with sustained isometric exercise.

EMG. M-wave amplitude and area assessed in the Ex limb were significantly lower than in the unexercised Con limb at PST0. Although the decrease in M-wave amplitude in the Ex leg of 12-14% was not significant on its own, similar but significant decreases have been shown with a long-duration voluntary fatigue protocol using the ankle plantarflexors and leg extensors (3) or first dorsal interosseus muscle (18). It has been proposed that the rundown of gradients for Na⁺ and K⁺ during intense exercise reduces the transmembrane ion potential and thereby reduce M-wave amplitude and force (37). A reduction in ion gradients with exercise can result from Na⁺ and K⁺ fluxes during successive action potentials, exceeding the ability for active transport mechanisms to maintain the gradients. Although complex regulatory controls exist, including the concentration of Na⁺ and specific ion channels in the sarcolemma, the Na⁺-K⁺-ATPase is the principal mechanism for maintaining excitability (43). The present results suggest that reduced M-wave amplitude after exercise may be due, at least in part, to intrinsic modifications of the Na⁺-K⁺-ATPase. According to this reasoning, modifications to the Na⁺-K⁺-ATPase should result in transmembrane disturbances in Na⁺ and K⁺. Impaired pump function would then indicate a compromised capability of the enzyme to maintain membrane excitability through its electrochemical contribution to membrane potential, resulting in depolarization, and reduced M wave.

Relationship of M wave and Na+-K⁺-ATPase. The depression of M wave after activity and subsequent return to Pre levels within 4 h could be explained, at least in part, by alteration of structure and subsequent repair, or activation of, Na⁺-K⁺-ATPase subunits in recovery. M-wave area is interpreted as a measure of the magnitude of the total current passage at the recording electrode, which is a function of the size of the individual action potentials and the number of activated fibers (39). Either factor may be affected by fatigue; however, the integrity of the Na⁺-K⁺-ATPase determines the ion gradients that these variables represent in a measure of M-wave area, making it suitable for correlation to Na⁺-K⁺-ATPase measured in vitro. A positive and significant correlation was observed between Na⁺-K⁺-ATPase activity and content (r = 0.59, P < 0.01, n = 16), which supports the hypothesis that the measure of activity is related to the Na⁺-K⁺- ATPase content. The capacity of these pumps is indicated by the measure of activity.

Conclusions. The results of this experiment indicate that the Na⁺-K⁺-ATPase activity can be altered by a single bout of heavy isometric exercise in humans. The association observed between Na⁺-K⁺-ATPase activity and measures of membrane excitability as obtained by the M wave support the contention that indirect measures of neuromuscular activity can reflect, at least in part, cellular processes measured through biochemical procedures in human muscle. The alteration in activity and the short time course for recovery emphasize the rapidly adaptive nature of the Na⁺-K⁺-ATPase and imply that reduced sarcolemmal excitability may contribute to neuromuscular fatigue in humans during the exercise. It is likely that other cellular processes contribute more to fatigue during the prolonged period of recovery. No doubt the role of Na⁺-K⁺-ATPase in a multifactorial fatigue process is dependent on the intensity, duration, and type of exercise performed.

Perspectives. Investigation of the skeletal muscle Na⁺-K⁺-ATPase indicates that it is highly adaptable to a wide variety of training protocols (21, 37). The rapid content changes observed for the Na⁺-K⁺-ATPase in response to training imply that acute events likely lead to the chronic adaptation. There are a number of intracellular signals that may predispose the enzyme to rapid adaptations, with one of them possibly being inactivation of the enzyme and the need to stimulate turnover and remodeling. It is reasonable to postulate that the stress of exercise induces structural modifications that reduce the activity of the enzyme and result in a need for compensation and adaptation. Heavy contractile activity that induces both high- and low-frequency fatigue challenges the capacity of the Na⁺-K⁺-ATPase to maintain transmembrane Na⁺ and K⁺ gradients and excitability. By mechanisms that are as yet uncertain, structural modifications appear to occur to the Na⁺-K⁺-ATPase, resulting in reduced activity. The fact that the Na⁺-K⁺-ATPase activity and EMG were restored within 4 h indicates that this modification has a rapid time course for recovery, as is observed in response to repeated training bouts for this key sarcolemmal protein. It must be emphasized, however, that the specific effect of alterations in Na⁺-K⁺- ATPase in modifying contractile activity needs to be established. Its role as a mechanism of fatigue may well depend on the characteristics of the task.