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Malleability of human skeletal muscle Na⁺-K⁺-ATPase pump with short-term training

Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1

Submitted 28 March 2003 ; accepted in final form 15 March 2004

	ABSTRACT

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To investigate the hypothesis that short-term submaximal training would result in changes in Na⁺-K⁺-ATPase content, activity, and isoform distribution in skeletal muscle, seven healthy, untrained men [peak aerobic power (peak oxygen consumption; O_{2 peak}) = 45.6 ml·kg^–1·min^–1 (SE 5.4)] cycled for 2 h/day at 60–65% O_{2 peak} for 6 days. Muscle tissue, sampled from the vastus lateralis before training (0 days) and after 3 and 6 days of training and analyzed for Na⁺-K⁺-ATPase content, as assessed by the vanadate facilitated [³H]ouabain-binding technique, was increased (P < 0.05) at 3 days (294 ± 8.6 pmol/g wet wt) and 6 days (308 ± 15 pmol/g wet wt) of training compared with 0 days (272 ± 9.7 pmol/g wet wt). Maximal Na⁺-K⁺-ATPase activity as evaluated by the 3-O-methylfluorescein phosphatase assay was increased (P < 0.05) by 6 days (53.4 ± 5.9 nmol·h^–1·mg protein^–1) but not by 3 days (35.9 ± 4.5 nmol·h^–1·mg protein^–1) compared with 0 days (37.8 ± 3.7 nmol·h^–1·mg protein^–1) of training. Relative isoform distribution, measured by Western blot techniques, indicated increases (P < 0.05) in ₂-content by 3 days and ₁-content by 6 days of training. These results indicate that prolonged aerobic exercise represents a potent stimulus for the rapid adaptation of Na⁺-K⁺-ATPase content, isoform, and activity characteristics.

submaximal exercise; sodium-potassium-adenosinetriphosphatase pump; content; activity; isoforms

The potentially beneficial effects of increases in Na⁺-K⁺ pump content with regular activity, however, remain unclear. Only one longitudinal study on senescent animals has apparently been made with measurements on the changes that occur in Na⁺-K⁺-ATPase activity and isoform expression (36). Although increases in maximal Na⁺-K⁺-ATPase activity would be expected to accompany increases in pump content, this may well depend on the adaptations that occur in specific isoforms. The Na⁺-K⁺-ATPase is a heterometric enzyme composed of an - and a -subunit. The -subunit is 112-kDa and contains the cation and ATP binding sites necessary for the catalytic and transport function of the enzyme. The -subunit is a 40- to 60-kDa glycosylated polypeptide that appears to regulate the assembly, expression, and function of the enzyme (3, 5, 21). In rat skeletal muscle, separate genes encode for three -subunits (₁, ₂, ₃) and three -subunits (₁, ₂, ₃) (1, 5, 28). In the rat adult locomotor muscles, the ₃- and ₃-isoforms have been reported to exist in only small amounts (1, 28). The expression of ₃ in skeletal muscles of the rat remain controversial, however, because two recent papers have reported its existence (23, 36). It is generally believed that expression of the subunits in rats is to some degree muscle fiber-type specific. In slow-twitch fibers, ₁₁, and ₂₁ predominate, whereas in fast-twitch, glycolytic-based (FG) fibers, ₁₂ and ₂₂ appear most prominent. Fast-twitch oxidative glycolytic (FOG) fibers appear to express all combinations (10). At present, the distribution of ₃ and ₃ in the different fiber types remains unclear. Species differences in muscle subunit isoform composition may also occur. In humans, as an example, no ₂-isoforms have been detected in muscle (28, 29).

On the basis of a variety of hormonal models that have been shown to result in altered Na⁺-K⁺-ATPase expression patterns in skeletal muscle (10), it has been suggested that the ₂-isoform is the highly regulated isoform, whereas the ₁ is not (41). Recent evidence obtained with the use of gene targeting in mice to selectively alter the expression of the -isoforms has revealed clear physiological differences (22). These differences in function have been attributed not to the unique characteristics of the enzyme itself but to the interaction with the Na⁺/Ca²⁺ exchanger (22). Another approach to determining the role of the subunit isoforms has been employed by Crambert et al. (8), who expressed nine different human Na⁺-K⁺-ATPase isoforms in Xenopus oocytes and investigated a variety of transport characteristics. A major finding was the dependence of the enzyme turnover rate on the -isoform (₁ highest). It has generally been acknowledged that the ₁-subunit plays a major role in maintaining basal pump activity, whereas the ₂-subunit can vary its catalytic activity over a wide range to maintain membrane excitability in the face of increased demands, as during exercise (5).

It is noteworthy that, in the only training study published to date using prolonged submaximal running in senescent rats, the ₂- and ₁-isoforms were increased and the ₂ was decreased, whereas no change occurred in the ₂ in muscles composed predominately of FG and FOG fibers (36). The ₁-isoforms increased in the FOG- but not in the FG-based muscles. Na⁺-K⁺-ATPase activity was observed to increase in both types of muscles. No measurements of total Na⁺-K⁺ pump content were made.

The purpose of this study was to determine the effect of a short period of prolonged exercise training in humans on Na⁺-K⁺-ATPase expression with specific regard to the content, isoform, and activity characteristics. We have hypothesized that increases in pump content observed with training would be accompanied by increases in the ₂- and ₁-isoforms and by increases in maximal Na⁺-K⁺-ATPase activity. Moreover, we propose that these adaptations would be observed during the first few days of training.

	METHODS

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Subjects

Seven untrained men were recruited for the study. All subjects were healthy (as determined by questionnaire), and none was engaged in physical exercise on a regular basis. Subject characteristics (mean ± SE) included an age of 20 ± 0.57 yr, a height of 180 ± 6.2 cm, and body mass of 84.4 ± 12.3 kg. Average peak aerobic power [peak oxygen consumption (O_{2 peak})] was 45.6 ± 5.4 ml·kg^–1·min^–1. Written consent was obtained from all volunteers as required after approval of the study by the Office of Research Ethics, at the University of Waterloo (Waterloo, ON, Canada).

Experimental Design

The training component of the study involved two segments of 3 days of prolonged submaximal cycle exercise. At least 2 wk before the initial training segment, all participants underwent a progressive cycle test to fatigue for measurement of O_{2 peak}. The workload-oxygen consumption relationships obtained from the progressive test were used to calculate a workload designed to produce 60–65% oxygen consumption that was used in testing and training. After the initial progressive test, all participants underwent a 15-min session of cycle exercise at the calculated workload. This session was used to confirm that the exercise intensity was appropriate for training or to make adjustments as necessary.

The supervised training program consisted of cycling for 2 h at 60–65% of O_{2 peak}. Beginning with the first session of training, all subjects were required to complete 2 h of exercise. Brief periods (5 min) of recovery were provided as required. After the first segment of training, all subjects were able to complete the 2 h of exercise without interruption. Exercise was conducted in normal room temperatures (20–21°C) and relative humidity (50–60%). Water was provided ad libitum throughout the exercise.

Before exercise and after 3 and 6 days of training, tissue was obtained from the vastus lateralis muscles by using the needle biopsy technique (4). Portions of the tissue obtained before exercise at 0, 3, and 6 days of training were used to measure the properties of the Na⁺-K⁺-ATPase. At all time points, the tissue was obtained between 24 and 36 h after the last exercise session.

Testing Protocols

The protocol used to measure O_{2 peak} was as previously published by our group (26) and involved 4 min of cycling at 25 W followed by step increases of 25 W each minute until the subject could no longer sustain a pedaling cadence of 50–60 rpm. All exercise testing was conducted on an electronic cycle (model 870, Quinton) that was calibrated daily and with the use of an open-circuit gas-exchange analytic system as previously described (26). The values used for O_{2 peak} were the highest values obtained over a 30-s period.

Analytic Procedures

Preparation of tissue homogenates.   Whole muscle homogenates were employed for both the measurement of Na⁺-K⁺-ATPase activity and the isoform subunit protein analyses of the enzyme. Briefly, tissue from the frozen biopsy samples was homogenized (5% wt/vol) at 0–4°C for 2 x 20 s at 25,000 rpm (Polytron) in a buffer containing 250 mM sucrose, 2 mM EDTA, and 10 mM Tris (pH 7.40).

Na⁺-K⁺-ATPase isoform determination.   Both (₁, ₂)- and (₁)-isoforms of the Na⁺-K⁺-ATPase were resolved by using electrophoresis on 7.5% SDS-polyacrylamide gels (Mini-Protean II, Bio-Rad) as described by Laemmli (32). Detection of isoforms was performed in duplicate ( only) using two different aliquots for each sample and two different gels. For analyses of -subunit isoforms, the amounts of protein employed was 40 µg and for the -subunit isoform the amount was 50 µg. Gels were run both for the glycosylated and deglycosylated -subunits. Only the deglycosylated results are reported in this paper. The shift in the bands before and after deglycosylations were as expected (36). Deglycosylation of the -subunit was accomplished with N-glycosidase F (Boehringer Mannheim, Indianapolis, IN) overnight at room temperature before electrophoresis. After SDS-PAGE, gels were electrophoretically transferred to polyvinylidene difluoride membrane (Bio-Rad) by placing the gel in transfer buffer (25 mM Tris, 192 mM glycine, and 20% wt/vol methanol) and applying a high voltage (20 V) for 45 min (Trans-Blot Cell, Bio-Rad). To detect each of the isoforms, the nonspecific binding sites were blocked with 5 or 7.5% BSA in Tris-buffered saline (pH 7.5) for 2 h at room temperature before incubation with primary polyclonal antibodies. The primary antibodies included ₁, ₂, and ₁ (Upstate Biotechnology, Lake Placid, NY). Immunoblotting was performed for 2 h at room temperature with the antibodies diluted in 5% BSA (₁, 1:1,000; ₂, 1:1,000; ₁, 1:1,000). Bound antibodies were detected with goat anti-rabbit IgG1.

An enhanced chemiluminescence procedure was used to assess antibody content (Amersham-ECL-RPN2106P1). Blots were developed in Kodak GBX developing solution and fixed in Kodak GBX fixer after exposure to photographic film (Kodak Hyperfilm-ECL). Blots were exposed to multiple films to ensure that signals were within the linear range of the film. Relative isoform protein levels were determined by scanning densitometry (Scion Image software). The linearity of the blot signal and the amount of protein applied were determined in pilot work.

For any particular isoform, samples from each muscle homogenate were added to each lane. For each individual, a total of three samples (0, 3, and 6 days) were applied on an individual gel, and each gel was run in duplicate. The samples from two to three volunteers were run on an individual gel. For each isoform, two sets of gels were run in parallel, each gel with a known protein content (1–2 µg) of brain standard (rat) for relative control. For each sample, data were calculated first as a relative percentage of brain standard and then with the samples obtained at 3 and 6 days of training as a relative percentage of 0 days. The intra-assay variability, as determined by the coefficient of variation was 3.6 and 9.4% for the ₁- and ₂-isoforms, respectively.

Na⁺-K⁺-ATPase activity.   The K⁺-stimulated 3-O-methylfluorescein phosphatase assay (3-O-MFPase), using the basic procedures of Huang and Askari (25) but with higher substrate concentrations (2, 15), was used to estimate Na⁺-K⁺-ATPase activity. The homogenate (25 µl) was freeze-thawed for 4 cycles and diluted 1:4 in cold homogenate buffer before incubation at 37°C for 4 min in a medium containing 5 mM MgC1₂, 1.25 mM EDTA, 1.25 mM EGTA, 5 mM NaN₃, and 100 mM Tris (pH 7.40). The K⁺-stimulated activity was determined by the increase in activity after the addition of 10 mM KC1 and at a substrate concentration of 160 µM 3-O-MFP. 3-O-MFPase activity was determined by the difference in the linear signal slope by using fluorescence spectroscopy (13, 14). Actual 3-O-MFPase was determined by the difference in slope before and after the addition of KCl. The 3-O-MFPase activity was based on the average of three trials. Protein content of the homogenate was determined by the method of Lowry as modified by Schacterle and Pollock (38). Average intra-assay variability for 3-O-MFPase activity was 8.5%.

Na⁺-K⁺-ATPase content.   To measure Na⁺-K⁺-ATPase content, the [³H]ouabain-binding procedure was employed as previously developed (37) and as employed in our laboratory (13). For this determination, two samples from each biopsy, weighing between 2 and 8 mg, were used. Briefly, samples were incubated in a Tris-sucrose buffer (10 mM Tris·HCl, 3 mM MgSO₄, 1 mM Tris-vanadate, and 250 mM sucrose) with [³H]ouabain (1.8 µC/ml) and unlabeled ouabain (1 µM final concentration) for 2 x 60 min at 37°C. After the samples were washed (4 x 30 min in ice-cold buffer), blotted, and weighed, they were soaked in 1 ml of 5% trichloroacetic acid for 16 h at room temperature, and then an aliquot (0.5 ml of sample) was counted for ³H radioactivity in a scintillation mixture. [³H]ouabain-binding capacity was corrected (x1.05) for loss of specifically bound ³H during washout (13). The [³H]ouabain-binding protocol has been shown to detect a major part of the functional Na⁺-K⁺ pumps present in intact muscle (34). The values are presented as picomoles per gram of wet weight. For this assay, interassay variability was 11%.

	RESULTS

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Na⁺-K⁺-ATPase content, as determined by [³H]ouabain binding, increased by 24 pmol/g wet wt or 9% during the first 3 days of training (Fig. 1). No further increases were observed with an additional 3 days of training.

View larger version (11K): [in this window] [in a new window]   Fig. 1. Training-induced changes in muscle Na+-K+-ATPase content as determined by the [3H]ouabain-binding technique. Values are means ± SE for 7 subjects. Training days are indicated by 0, 3, and 6. *Significantly different from 0 days, P < 0.05.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Effects of short-term training on maximal Na+-K+-ATPase activity as determined by 3-O-methylfluorescein phosphatase. Values are means ± SE for 7 subjects. Training days are indicated by 0, 3, and 6. *Significantly different from 0 and 3 days, P < 0.05.

View larger version (11K): [in this window] [in a new window]   Fig. 3. Effects of short-term training on relative changes in Na+-K+-ATPase isoform distribution. A: 1-isoform. B: 2-isoform. C: 1-isoform. Values are means ± SE for 7 subjects. Note that relative values were first calculated against a standard and that the training effects for 3 and 6 days were calculated against 0 days, which was set to 100%. Training days are indicated by 0, 3, and 6. *Significantly different from 0 days, P < 0.05.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Representative immunoblots of the 1-, 2-, and 1-isoforms in human vastus lateralis muscle before (0) and after 3 (3) and 6 (6) days of training. The standard (STD) used was the rat brain. Primary polyclonal antibodies were used to identify each of the isoforms. Deglycosylation of the -subunits was performed before electrophoresis.

	DISCUSSION

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The increase in Na⁺-K⁺-ATPase content was not unexpected given our laboratory's earlier work where our group has demonstrated a rapid upregulation in this enzyme with both CLFS (17) and submaximal exercise training (6). Others have also confirmed the potent nature of CLFS for inducing a large and early increase in Na⁺-K⁺-ATPase content (24). This study adds to the growing evidence that the Na⁺-K⁺-ATPase pump is highly adaptable and closely dependent on the chronic contractile history of the muscle cell (9, 18, 30, 35).

At this time, it is unclear what the nature of the molecular events are leading to the increased levels of Na⁺-K⁺ pump content or the nature of the signaling mechanisms regulating altered expression. Because prolonged exercise results in extensive alterations in a wide range of hormones associated with the autonomic nervous system (norepinephrine, epinephrine), fuel homeostasis (insulin), and fluid and electrolyte balance (aldosterone, vasopressin) (16) and because these hormones can, in general, alter Na⁺-K⁺-ATPase expression (7, 10), several signaling possibilities exist. However, on the basis of previous work using the one-legged training model (20) and CLFS (17, 24) where changes in Na⁺-K⁺-ATPase content are confined to the active muscle, it is clear that the signal must be local in nature and dependent on the contractile activity.

Changes in one or both of the two cations Na⁺ and K⁺, which are regulated by the Na⁺-K⁺-ATPase via transmembrane transport, represent potential candidates for the changes in adaptations that we have observed (7, 10). Prolonged exercise is known to result in an increase in plasma K⁺ concentrations (42) and ostensibly in intracellular Na⁺ levels (39), changes that could be secondary to inhibition of the Na⁺-K⁺-ATPase activity (11, 13). Both the extracellular K⁺ and intracellular Na⁺ levels can effect changes in the long-term regulation of the Na⁺-K⁺-ATPase (10, 33). In the case of K⁺, the experimental models employed have only utilized K⁺ deficiency (33). In such cases, early and pronounced reductions in muscle pump content are observed (40). It is not clear what the effect of chronic hyperkalemia is. In contrast, elevations in intracellular Na⁺ levels promote increases in pump content (10). Interestingly, the effects of increased intracellular Na⁺ -subunit isoform expression in the C₂C₁₂ skeletal muscle cell only indicates an increase in the ₂-isoform (31). In our training study, although there is a suggestion that ₁-isoform increased, only the ₂-isoform abundance was significantly increased.

It is apparent from our training study that the increase in Na⁺-K⁺-ATPase content, as determined by the vanadate-facilitated [³H]ouabain-binding technique, must be explained primarily by the relative increases in subunit abundance (₂ and possibly ₁) because both of these properties increased significantly by 3 days of training and because the [³H]ouabain is specific for the -catalytic subunit (8). It has been shown that [³H]ouabain binds to the nucleotide site of the enzyme (37), and an increase in the number of binding sites would be expected with increased Na⁺-K⁺-ATPase content, as we have observed. Moreover, unlike the rodent where the binding affinity of ouabain for the ₁-isoform is low, all human -isoforms have a high affinity for binding ouabain (8).

Although ouabain binds specifically to the -subunit, it should be emphasized that the functional properties of the enzyme depend on -heterodimer (3, 21). As a consequence, changes in the -subunit with training would appear to play a vital role. The role of the specific -subunit isoforms in regulating Na⁺-K⁺ pump activity in muscle is uncertain.

It is known that rat muscles express the - and -isoforms in a tissue-specific manner that appears to depend on the fiber-type composition. Accordingly, we (11) and others (33) have shown that muscles composed of primarily slow, type I fibers mainly express ₁₂ and ₂₂, whereas fast, type II isoform expression depends on the oxidative potential. Muscles of FG fiber predominance mainly express ₁₂ and ₂₂, whereas those with FOG fiber potential express all four combinations, namely ₁₁, ₂₁, ₁₂, and ₂₂ (27, 33).

Surprisingly, few studies have characterized human muscle Na⁺-K⁺-ATPase isoform distribution, in general, and by fiber type. Although, it is known that ₁,- ₂-, and ₁-isoforms are expressed in human skeletal muscle, in our experience, ₂ content, if detectable, is very low, at least in the vastus lateralis muscle (unpublished observations). In both vastus lateralis (29) and human soleus muscle (28), ₂ has not been detected. It is for this reason why we have not probed for training effects on the ₂-isoform content. In addition, ₃ has been detected in rat muscle (1, 23, 36), but its existence in human muscle has been uncertain. In recent pilot work, using a monoclonal antibody specific for ₃, we have been able to detect ₃ in human muscle. In general, the changes that we have observed are generally consistent with the effects of chronic administration of glucocorticoids (41).

The increases that we have observed in the - and -subunit isoforms are consistent with what has been reported to occur with endurance training in senescent rats (36). In the rat study, increases in muscle ₁,- ₂-,and ₁-isoforms have been found after 13–14 wk of training. These changes are similar to what we have reported by 6 days of training in humans.

It is of interest that our findings suggest that increases in Na⁺-K⁺-ATPase activity depend not solely on increases in pump and ₂-isoform abundance per se but also an increase in the ₁-isoform (and possibly other -isoforms). This suggests that the ₂-isoform heterodimer is responsible for the increase in enzyme activity observed. The -subunit plays an important role in modulating the catalytic activity of the enzyme (21). A dissociation between changes in pump and -isoform levels and pump activity is not an uncommon finding in studies investigating the long-term regulation of the enzyme. Glucocorticoids (41) and K⁺ deprivation (33) represent two models where discordant responses are observed between activity and protein responses. It is possible that specific -isoform changes observed with training could account for the increased enzyme activity. Increases in the ₂- and ₃-isoforms with training, however, cannot explain increases in activity because these isoforms are either decreased (₃) or remain unchanged (₂) (36). However, Crambert et al. (8) have observed that, when the human Na⁺-K⁺-ATPase isoforms are expressed in Xenopus oocytes, the enzyme turnover rates depended primarily on the -isoform with ₁ displaying the highest rate.

It is emphasized that our results indicate a net increase in protein as the primary effect to explain the change in Na⁺-K⁺-ATPase content. These changes observed could be mediated by alterations in synthetic and/or degradation rates. Depending on the model employed and the tissue examined, alteration in protein levels could occur as a result of transcription, transcript stability, translation, and protein stability (10). Extensive changes have been shown in pump content and Na⁺-K⁺-ATPase properties in the absence of changes in the mRNA levels for the - and -isoforms (40). Determination of the molecular mechanisms involved in the training-induced alterations in Na⁺-K⁺-ATPase expression awaits further research.

The results of this study serve to reemphasize that the profound effect of abrupt increases in contractile activity is not only the alteration of extensive protein components and processes in the muscle cell but also the mediatation of rapid changes. In earlier work, our laboratory has demonstrated that metabolic adaptations, typical of the trained active muscle, occur within the first 3 days of exercise with the use of the same model as employed in this study (19). The metabolic adaptations occurred in untrained groups with both low and high O_{2 peak} values. Interestingly, these adaptations were not accompanied by increases in O_{2 peak} after the full 6 days of training. Moreover, when both groups were combined into a single group (previously subdivided into high and low O_{2 peak}), our laboratory has found that citrate synthase activity was increased (P < 0.05) (5.71 ± 0.29, 6.42 ± 0.37 and 7.18 ± 0.37 mol·kg protein^–1·min^–1 before and after 3 and 6 days of training, respectively). It is tempting to speculate that the change in Na⁺-K⁺-ATPase, oxidative potential, and metabolic behavior are all coupled.

In summary, this appears to be the first study investigating training-induced adaptations in the Na⁺-K⁺-ATPase at the protein, isoform, and activity levels. Our results demonstrate as little as 3 days of prolonged exercise are capable of inducing adaptations in pump content. Our results also indicate that increases in pump content are not sufficient to increase activity but rather that activity increases appear to depend on the expression of the -isoform, possibly the ₁ as suggested in this study. The role of the specific -isoforms in modulating activity remains unclear, given the percent increase observed for both ₁ and ₂. Future studies with expanded numbers and more frequent time course measurements during the training should be able to identify the significance of specific isoforms in regulating the adaptations in the catalytic behavior of the enzyme. Such studies should also assess the presence and significance of all isoforms in skeletal muscle.

	GRANTS

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The authors gratefully acknowledge the financial support of the Natural Sciences and Engineering Research Council (Canada) and the Heart and Stroke Foundation (Canada) for this project.

	FOOTNOTES

 

Address for reprint requests and other correspondence: H. J. Green, Dept. of Kinesiology, University of Waterloo, Waterloo, ON, Canada N2L 3G1 (E-mail: green{at}healthy.uwaterloo.ca).

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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