The Na+-K+-ATPase enzyme is vital in skeletal muscle function. We investigated the effects of acute high-intensity interval exercise, before and following high-intensity training (HIT), on muscle Na+-K+-ATPase maximal activity, content, and isoform mRNA expression and protein abundance. Twelve endurance-trained athletes were tested at baseline, pretrain, and after 3 wk of HIT (posttrain), which comprised seven sessions of 8 × 5-min interval cycling at 80% peak power output. Vastus lateralis muscle was biopsied at rest (baseline) and both at rest and immediately postexercise during the first (pretrain) and seventh (posttrain) training sessions. Muscle was analyzed for Na+-K+-ATPase maximal activity (3-O-MFPase), content ([3H]ouabain binding), isoform mRNA expression (RT-PCR), and protein abundance (Western blotting). All baseline-to-pretrain measures were stable. Pretrain, acute exercise decreased 3-O-MFPase activity [12.7% (SD 5.1), P < 0.05], increased α1, α2, and α3 mRNA expression (1.4-, 2.8-, and 3.4-fold, respectively, P < 0.05) with unchanged β-isoform mRNA or protein abundance of any isoform. In resting muscle, HIT increased (P < 0.05) 3-O-MFPase activity by 5.5% (SD 2.9), and α3 and β3 mRNA expression by 3.0- and 0.5-fold, respectively, with unchanged Na+-K+-ATPase content or isoform protein abundance. Posttrain, the acute exercise induced decline in 3-O-MFPase activity and increase in α1 and α3 mRNA each persisted (P < 0.05); the postexercise 3-O-MFPase activity was also higher after HIT (P < 0.05). Thus HIT augmented Na+-K+-ATPase maximal activity despite unchanged total content and isoform protein abundance. Elevated Na+-K+-ATPase activity postexercise may contribute to reduced fatigue after training. The Na+-K+-ATPase mRNA response to interval exercise of increased α- but not β-mRNA was largely preserved posttrain, suggesting a functional role of α mRNA upregulation.
- [3H]ouabain binding
- muscle fatigue
in skeletal muscle, the Na+-K+-ATPase enzyme (Na+-K+ pump) regulates transsarcolemmal [Na+] and [K+] gradients and thus is critical for the maintenance of membrane excitability and contractility (4, 35, 39). It is therefore not surprising that the muscle Na+-K+-ATPase content is upregulated by training, with, for example, 8–16% increases after 7 wk of high-intensity sprint training in untrained participants (16, 26). Similar increases have been found after endurance training, in untrained and moderately trained participants (12, 13, 24), as well as in already well-trained participants after 3–5 mo of intensified training (8, 27). Corresponding increases in maximal Na+-K+-ATPase activity with training would be expected. However, little is known about training and Na+-K+-ATPase activity, with two inconsistent reports of a disproportionately large increase in activity with training (13), and no difference between trained and untrained individuals (10). Elevated muscle Na+-K+-ATPase content and maximal activity could enable higher Na+-K+-ATPase activity during exercise (and recovery), and thereby be important for preserving muscle function and enhancing performance in already well-trained athletes. We therefore investigated whether a short period of high-intensity training (HIT), commonly undertaken by endurance cyclists, would correspondingly enhance both muscle Na+-K+-ATPase content and maximal activity.
Human skeletal muscle expresses three Na+-K+-ATPase isoforms of both the catalytic α-subunit (α1, α2, α3) and the regulatory β-subunit (β1, β2, and β3) at each of mRNA and protein levels (31). Training-induced muscle Na+-K+-ATPase upregulation must reflect an increased abundance of one or more of these α isoforms, but training effects on isoform mRNA and protein expression are far from clear. Only one study has examined training effects on Na+-K+-ATPase mRNA expression in human muscle (36). HIT in untrained participants for 5.5 wk did not alter α1, α2, or β1 mRNA expression in resting muscle (36). This is surprising, given the acute increase in α-isoform mRNA with intense exercise in humans (31), acute stimulation of isolated rat muscle (28), and the Na+-K+-ATPase upregulation seen with training (8, 12, 13, 16, 24, 26, 27). It was also not reported whether any Na+-K+-ATPase upregulation occurred with training in that study (36). Four studies have investigated training effects on Na+-K+-ATPase isoform protein abundance in human muscle (7, 12, 34). Endurance, intermittent and strength training each induced an increase in α2 abundance, but responses in α1 and β1 proteins were inconsistent (7, 12, 34). None of these studies probed for possible effects on α3, β2, and β3 isoforms (7, 12, 34, 36). Importantly, only untrained participants were used (7, 12, 34); thus it is unclear whether upregulation of Na+-K+-ATPase isoforms also occurs with training in well-trained athletes. No studies have investigated the effects of HIT on muscle Na+-K+-ATPase in well-trained athletes, and none have investigated HIT effects on α3, β2, and β3 isoform mRNA or protein expression. The present study is the first to comprehensively investigate training effects on human muscle Na+-K+-ATPase by exploring adaptations in each of content, maximal activity, isoform mRNA expression, and protein abundance. We tested the hypotheses that HIT in well-trained athletes would induce corresponding increases in both content and maximal activity, together with increased mRNA expression and protein abundance of each of the Na+-K+-ATPase α1, α2, and α3 isoforms.
Few studies have investigated training effects on the immediate muscle Na+-K+-ATPase responses to acute exercise. In untrained participants, these immediate responses include an acutely depressed maximal activity (9, 10, 22, 29, 40, 42) and elevations in mRNA expression of several isoforms, although discrepancies exist in the isoform responses (30, 31, 36, 37). We have previously found moderate correlations between depressed maximal activity and increased α-isoform mRNA with intense (40) but not prolonged exercise (30). Thus the significance of our earlier finding (40) is unclear. In well-trained athletes, a decline in maximal Na+-K+-ATPase activity with acute exercise is evident (1, 2), but interestingly HIT blunted the α1 mRNA increase with acute exercise, at least in untrained participants (36). Well-trained athletes may have attenuated mRNA expression in response to acute exercise, due to a blunted activation of signaling intermediates with years of training (47). However, no study has examined the effects of training on Na+-K+-ATPase isoform mRNA or protein expression responses to acute exercise in well-trained athletes. Furthermore, it is unknown whether training dissociates the acutely increased α-isoform mRNA response with acute exercise from the depressed Na+-K+-ATPase activity. We therefore tested the hypothesis that HIT in well-trained athletes would not attenuate the acute depression in maximal Na+-K+-ATPase activity with high-intensity interval exercise but would blunt the increased Na+-K+-ATPase isoform mRNA expression, thus dissociating these events.
Twelve well-trained male cyclists/triathletes gave written consent to participate in this study, which conformed to the Declaration of Helsinki and was approved by the Human Research Ethics Committees of Victoria University and of RMIT University. All subjects refrained from vigorous exercise, caffeine and alcohol consumption, and consumed a controlled diet for 24 h before each of the exercise tests. This paper forms part of a larger study that also investigated muscle metabolism responses to HIT; hence details of subjects, and of some test methodologies, have already been presented elsewhere (3). The physical characteristics of the subjects are (mean and SD) age 31 yr (SD 3); height 177 cm (SD 6); body mass (kg) at baseline 75.7 (SD 4.5), pretraining 75.3 (SD 2.1), and posttraining 74.1 (SD 2.8).
Subjects were training on a daily basis, cycling at a low to moderate intensity of >350 km/wk, and had not performed any interval training for at least 6 wk. In the current study, subjects acted as their own controls. To achieve this, each subject underwent a resting muscle biopsy and performed a peak power output (PPO) test (baseline). Subjects then maintained their normal endurance training for a further 4 wk before undergoing pretraining testing (pretrain), followed by the HIT program and posttraining testing (posttrain). HIT sessions replaced a portion of their normal endurance training. An untrained control group could not be included in this study, as untrained subjects are unable to ride for >15 min at the same relative intensity sustained by well-trained subjects (47). Furthermore, untrained subjects may also have altered mechanisms facilitating gene transcription (47). Each test was performed on an electromagnetically braked ergometer (Lode, Groningen, The Netherlands).
Subjects completed an incremental exercise test at each of baseline, pretraining, and posttraining. Each test commenced with subjects cycling at a workrate equivalent to lactate threshold, as previously detailed (3), followed by 25-W increments each 150 s until voluntary exhaustion, defined as the inability to maintain pedal cadence above 60 rpm. The incremental exercise PPO [PPO = Wf + (Ti/150) × 25, where Wf is final workrate with 150 s completed, Ti is time at incomplete workrate, 150 is time spent at each workrate, and 25 is the size of the step in workrate] was used to determine the subsequently described high-intensity interval training session. Peak O2 uptake (V̇o2 peak) was also determined during this incremental test, as previously detailed (3).
High-Intensity Interval Exercise
Each high-intensity interval exercise session was comprised of a warm-up at 58% PPO for 20-min followed by eight, 5-min intervals performed at a workrate corresponding to 80% PPO, equivalent to ∼85% V̇o2 peak (45); each interspersed with 1 min of recovery cycling at 100 W, equivalent to ∼1.3 W/kg.
High-Intensity Interval Training
The above interval exercise session was repeated on a further six occasions in a 21-day period, constituting high intensity interval training (HIT), with 3, 2, and 2 sessions completed in weeks 1, 2, and 3, respectively. The PPO was redetermined after the third session to enable adjustment of workloads for sessions 5 and 6. This HIT regime improves performance in well-trained subjects (45). The workrates used for sessions 1 (pretrain) and 7 (posttrain) were identical. The use of matched high-intensity interval exercise bouts pre- and posttraining enabled greater precision in detecting possible training effects on the exercise-induced changes in Na+-K+-ATPase mRNA and maximal activity, as previously shown for muscle metabolites and plasma ion regulation (16).
Muscle Biopsy Sampling and Analyses
An initial resting muscle biopsy was taken at baseline, with further biopsies taken at rest (rest) and immediately postexercise (exercise), during the pretrain and posttrain testing sessions. The biopsy was taken from the vastus lateralis muscle under local anaesthesia (Xylocaine, 1%). The resting muscle biopsy sample was taken ∼15 min before the beginning of the first (pretrain) and seventh (posttrain) HIT session. Each of pretrain and posttrain occurred within 24 h of a standardized low-intensity cycling training session to minimize the possibility of interference from a different training stimulus. The posttrain session occurred 2 or 3 days post the sixth HIT training session. The postexercise sample was taken immediately after cessation of the final exercise bout, with the subject lying supported on the cycle ergometer. Serial biopsies were taken from separate incisions in the same leg, with the exercise sample taken from an incision ∼1.5 cm proximal to the rest sample. Muscle samples were removed, rapidly frozen in liquid N2, and stored at −80°C for subsequent analysis of Na+-K+-ATPase isoform mRNA and crude homogenate protein expression, content, and maximal activity. Muscle was also analyzed for AMPK response to exercise, as reported elsewhere (3), which restricted our ability to complete all analyses for each subject at each time point. Consequently, the sample size for each Na+-K+-ATPase analysis was as follows: isoform mRNA, n = 12; isoform protein expression, n = 5; Na+-K+-ATPase content, n = 7; and maximal activity, n = 7.
Real-Time RT-PCR Measurement of mRNA
Na+-K+-ATPase isoform mRNA expression was measured as previously described (31). Total RNA was extracted from 5–10 mg of muscle using the FastRNA reagents (BIO 101, Vista, CA) (32). The resulting RNA pellet was dissolved in EDTA-treated water, and total RNA concentration was determined spectrophotometrically at 260 nm. RNA (1 μg) was transcribed into cDNA using the Promega AMV Reverse Transcription Kit (Promega, Madison, WI), and the resulting cDNA was stored at −20°C for subsequent analysis.
Real-time PCR (GeneAmp 5700 Sequence Detection System) was run for 1 cycle (50°C for 2 min, 95°C for 10 min) and 50 cycles (95°C for 15 s, 60°C for 60 s). Primer sequences were designed for the Na+-K+-ATPase α1, α2, α3, β1, β2, and β3 genes from published sequences (31). The Na+-K+-ATPase α4 gene was also probed for but was undetected by RT-PCR (30). All samples were run in triplicate, and measurements included a no-template control as well as a human skeletal muscle sample endogenous control. Cyclophilin mRNA expression was unchanged with exercise [pretrain rest, 1.89 × 10−8 (SD 0.92 × 10−8); pretrain ex, 1.75 × 10−8 (SD 1.14 × 10−8; P < 0.77] and training [pretrain rest, 1.89 × 10−8 (SD 0.92 × 10−8; posttrain rest, 1.79 × 10−8 (SD 0.94 × 10−8; P < 0.80] and was therefore used as a control (housekeeping gene) to account for any variations in the amount of input RNA and the efficiency of reverse transcription. Gene expression was quantified using a cycle threshold (CT) method, whereby the relative expression of the genes compared with pretrain resting sample was made using the expression, 2−··CT, in which the expression of each gene and that of the housekeeping gene were both log transformed and the resulting expression of the target genes was normalized to that of the housekeeping gene cyclophilin (30).
Immunoblotting methods were as previously described (30). Muscle samples (20–30 mg) were diluted 1:40 with extraction buffer (25 mM Tris·HCl, pH 6.8, 1% SDS, 5 mM EGTA, 50 mM NaF, 1 mM sodium vanadate, 10% glycerol, 17.4 μg/ml PMSF, 10 μg/ml leupeptin, and 1 μg/ml aprotinin) and homogenized on ice for 15 s at a speed rating of 4 (Polytron PT1200, Kinematica, Luzern, Switzerland). A portion of each homogenate was heated for 10 min at 90°C and analyzed for total protein content (BCA Assay Kit, Pierce, Rockford, IL) with BSA as the standard. Stabilizing buffer (0.75 M Tris·HCl, pH 6.8, 25% glycerol, 25 mM DTT, 5% SDS, and 5% 2-mercaptoethanol) was then added to the remaining homogenate in a 1:5 dilution, and each sample was subsequently frozen at −80°C for immunoblotting.
SDS-PAGE (10% separating gel, 5% stacking gel) was performed, and gels were loaded with 20 (β1) or 70 μg (α1, α2, α3, β2, β3) of protein. Following electrophoresis (20 min at 100 V and 90 min at 150 V), the protein was transferred (90 min, 100 V) to a 0.45-μm nitrocellulose membrane and blocked for 2 h with blocking buffer [5% nonfat milk in TBS-Tween (TBST)]. Membranes were incubated overnight at 4°C in primary antibodies diluted in blocking buffer containing 0.1% NaN3. Membranes were washed in 0.05% TBST and incubated for 1 h in horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse immunoglobulins or goat anti-rabbit immunoglobulins) diluted 1:10,000 in TBST buffer. Following three washes in 0.05% TBST, membranes were dried and treated with chemiluminescent substrate (Pierce SuperSignal, West Pico, IL). The signal was captured and imaged (Kodak Digital Science Image Station 400CF, Eastman Kodak). Positive control samples included rat brain and kidney homogenates, and these were run on each gel to assess the reactivity of the probe.
Blots were probed with antibodies specific to each isoform. These were for α1: monoclonal α6F (developed by D. Fambrough and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA); α2: polyclonal anti-HERED (kindly donated by T. Pressley, Texas Tech University); α3: monoclonal MA3-915 (Affinity Bioreagents, Golden, CO); β1: monoclonal MA3–930 (Affinity Bioreagents); β2: polyclonal 610915 (Transduction Laboratories, Lexington, KY); and β3: polyclonal 610993 (Transduction Laboratories).
[3H]ouabain Binding Sites
Na+-K+-ATPase content was determined in quadruplicate using the vanadate-facilitated [3H]ouabain binding site content (38). Muscle samples were cut into 2- to 5-mg pieces and washed for 2 × 10 min in 37°C vanadate buffer containing (in mM) 250 sucrose, 10 Tris, 3 MgSO4, and 1 NaVO4 (pH 7.2–7.4). This was to thaw the samples and to maintain lower Na+ and K+ concentrations to minimize interference with vanadate-facilitated [3H]ouabain binding. Muscle samples were then incubated for 120 min at 37°C in the above buffer with the addition of [3H]ouabain (10–6 M, 2.0 mCi/ml). After incubation, muscle samples were washed for 4 × 30 min in ice-cold vanadate buffer to remove any unbound [3H]ouabain, blotted on filter paper, and weighed before being soaked overnight in vials containing 0.5 ml of 5% trichloroacetic acid (TCA) and 0.1 mM ouabain. The following morning, 2.5 ml of scintillation cocktail (Opti-Fluor, Packard) was added before liquid scintillation counting of the [3H]-activity. The content of [3H]ouabain binding sites was calculated on the basis of the sample wet weight and the specific activity of the incubation medium and samples and expressed as pmol/g wet wt. The resting muscle [3H]ouabain binding site content was contrasted between baseline, pre, and post conditions. The resting muscle [3H]ouabain binding site content intra-assay coefficient of variation (CV) was 11.0% (n = 20). The [3H]ouabain binding site content was also expressed relative to muscle protein content, which was measured spectrophotometrically (23). Postexercise [3H]ouabain binding was not analyzed due to tissue limitations since this is not changed by brief exercise (2, 22).
Maximal 3-O-MFPase Activity
Approximately 5 mg of muscle was thawed, quickly blotted on filter paper, weighed, then homogenized (5% wt/vol) on ice for 2 × 20 s, 20,000 rpm (Omni 1000, Omni International) in an homogenate buffer containing (in mM) 250 sucrose, 2 EDTA, and 10 Tris (pH 7.40). The homogenate maximal in vitro Na+-K+-ATPase activity was determined in triplicate using the K+-stimulated 3-O-methylfluorescein phosphatase (3-O-MFPase) activity assay (10, 11). The muscle 3-O-MFPase activity intra-assay CV was 2.7% (n = 34). The 3-O-MFPase activity was also expressed relative to the muscle homogenate total protein content.
All data are means (SD). To establish stability of performance and resting muscle measures before training, differences between baseline and pretrain were analyzed with a paired t-test. Resting muscle [3H]ouabain binding (pretrain, posttrain) was also analyzed with a paired t-test. A two-way ANOVA with repeated measures for time (rest, exercise) and day (pretrain, posttrain) was used to test for main and interaction effects for muscle 3-O-MFPase activity. Post hoc analyses used the Student-Newman-Kuels test. For isoform mRNA and protein expression, differences between rest and Ex, for both pretrain and posttrain, were separately analyzed by calculation of the change score for isoform mRNA and protein expression using a paired t-test, as uneven numbers for these trials precluded use of ANOVA. Significance was accepted at P < 0.05. In addition and as a method to partially circumvent the likelihood of a Type II error as a consequence of our small sample size, the effect size was calculated for selected results that did not achieve significance. The effect size [ES = (mean1 − mean2)/SD] was calculated for isoform mRNA and protein expression, and the pooled SD was calculated when the SDs were unequal (6). Cohen's (6) conventions for effect size were adopted for interpretation, where ES of 0, 0.2, 0.5, and 0.8 are considered as trivial, small, moderate, and large, respectively. Furthermore, a moderate to large effect size likely represents a functional effect of an intervention.
Stability from Baseline to Pretrain
Before commencing the HIT intervention, these well-trained athletes had very stable exercise PPO [baseline 363 (SD 35) and pretrain 366 W (SD 30), n = 12, CV 1.2%, P = 0.32] and V̇o2 peak [baseline 4.98 (SD 0.63) and pretrain 4.96 l/min (SD 0.56), n = 5, CV 2.2%, P = 0.31].
Neither maximal 3-O-MFPase activity [baseline 284 (SD 7); pretrain 282 (SD 6) nmol·min−1·g wet wt−1, n = 7, CV 1.0%, P = 0.21] nor [3H]ouabain binding [baseline 350 (SD 36); pretrain 367 (SD 28) pmol/g wet wt, n = 8, CV 11%, P = 0.39] differed significantly from baseline to pretrain. Similarly, there were no significant differences from baseline to pretrain for any Na+-K+-ATPase isoform mRNA [all expressed relative to baseline; α1, 1.25 (SD 0.27), P = 0.37; α2, 0.65 (SD 0.40), P = 0.97; α3, 1.01 (SD 0.22), P = 0.75; β1, 1.20 (SD 0.13), P = 0.37; β2, 2.16 (SD 0.66), P = 0.61; and β3, 1.01 (SD 0.26), P = 0.30 arbitrary units, n = 12, CVs 15.7, 1.3, 15.7, 7.9, 30.2, and 35.1% respectively, not significant] or Na+-K+-ATPase crude homogenate protein expression [α1, 0.86 (SD 0.13), P = 0.84; α2, 0.80 (SD 0.10), P = 0.18; α3, 1.38 (SD 0.29), P = 0.95; β1, 1.17 (SD 0.24), P = 0.36; β2, 1.03 (SD 0.19), P = 0.25; and β3, 1.11 (SD 0.25), P = 0.30 arbitrary units, n = 11, CV 10.8, 15.4, 22.4, 11.1, 2.4, and 7.4% respectively, not significant].
Acute High-Intensity Interval Exercise Effects on Muscle Na+-K+-ATPase
Maximal 3-O-MFPase activity.
The muscle maximal in vitro 3-O-MFPase activity expressed per gram wet weight (nmol·min−1·g wet wt−1) declined by 12.5% (SD 5.1) immediately after acute high-intensity interval exercise (P = 0.0002, Fig. 1). A similar reduction in 3-O-MFPase activity of 12.3% (SD 5.9) was observed when expressed per gram of homogenate protein (P = 0.0001, data not shown).
Na+-K+-ATPase isoform mRNA expression.
A single bout of high-intensity interval exercise immediately elevated Na+-K+-ATPase α1, α2, and α3 mRNA expression by 2.0- (P = 0.03), 2.4- (P = 0.01), and 4.0-fold (P = 0.04), respectively (Fig. 2) but had no significant effect on β1 (P = 0.11, ES = 1.6), β2 (P = 0.87, ES = 1.7), or β3 (P = 0.14, ES = 1.7) mRNA expression (Fig. 2).
Na+-K+-ATPase isoform protein abundance.
Acute high-intensity interval exercise had no significant effect on the protein abundance of any of the Na+-K+-ATPase α1 (P = 0.18), α2 (P = 0.77), α3 (P = 0.96), β1 (P = 0.71), β2 (P = 0.18), or β3 isoforms (P = 0.47, Table 1).
Training Effects on Performance
HIT increased PPO by 3% (SD 3) in these already well-trained athletes [pretrain 366 (SD 30); posttrain 379 W (SD 39), P = 0.04, Fig. 3] with maintenance of V̇o2 peak [pretrain 4.96 (SD 0.56); posttrain 5.08 l/min (SD 0.62), P = 0.22].
Training Effects on Muscle Na+-K+-ATPase Content and Activity
[3H]ouabain binding site content.
The muscle [3H]ouabain binding site content was not significantly altered after training [pretrain 355 (SD 80), posttrain 337 (SD 124) pmol/g wet wt, P = 0.40, Fig. 4].
HIT increased maximal 3-O-MFPase activity in resting muscle by 5.4% (SD 2.9) and in muscle sampled postexercise by 5.6% (SD 3.4) (P = 0.03, Fig. 1). This training effect was also present when maximal 3-O-MFPase activity was expressed per gram of protein for resting [pretrain 1,653 (SD 93); posttrain 1,748 (SD 67), P = 0.03] and postexercise muscle [pretrain 1,443 (SD 129); posttrain 1,536 (SD 148) nmol·min−1·g protein−1, P = 0.03].
Training Effects on Muscle Na+-K+-ATPase mRNA and Protein Expression
HIT elevated resting muscle Na+-K+-ATPase α3 (P = 0.02) and β3 (P = 0.03) mRNA by 4.6- and 2.5-fold, respectively, but had no significant effect (α1 P = 0.31, α2 P = 0.37, β1 P = 0.40, β2 P = 0.41) on any other isoform mRNA expression (Fig. 2). HIT did not significantly alter resting muscle protein abundance for any Na+-K+-ATPase isoform (α1 P = 0.44, α2 P = 0.94, α3 P = 0.82, β1 P = 0.77, β2 P = 0.33, and β3 P = 0.60, Table 1).
Adaptation to acute exercise.
After 3 wk of HIT, the upregulatory effects of high-intensity, intermittent exercise on α1 (P = 0.013) and α3 mRNA expression (P = 0.008) persisted, but α2 was unchanged (P = 0.77, Fig. 2). After HIT, acute exercise also immediately increased β2 mRNA expression by 2.7-fold (P = 0.023), with no significant change in β1 (P = 0.06, ES = 0.2) or β3 (P = 0.28) mRNA expression (Fig. 2). After HIT, acute exercise did not significantly alter muscle protein abundance for any Na+-K+-ATPase isoform (Table 1). There was no significant difference between pretrain and posttrain in the exercise effect on the protein expression of any of the α1 (P = 0.62), α2 (P = 0.51), α3 (P = 0.75), β1 (P = 0.11), β2 (P = 0.24), or β3 (P = 0.36) isoforms (Table 1). The effect size for the exercise effect on isoform protein abundance was greater for α2 (ES = 0.1, 0.3), β1 (ES = 0.1, 0.6), and β2 (ES = 0.5, 0.9), suggesting some possible changes with training, but the same for α1 (ES = 0.4, 0.4), α3 (ES = 0.6, 0.6), or β3 (ES = 0.2, 0.3) for pretrain and posttrain, respectively (Table 2).
Correlations Between Acute Exercise-Induced Changes in mRNA Expression and 3-O-MFPase Activity
The changes in Na+-K+-ATPase isoform mRNA expression with acute exercise were not significantly correlated to either the absolute (data not shown) or relative changes in 3-O-MFPase activity at either pretrain (α1, r = 0.20; α2, r = 0.41; α3, r = −0.18; β1, r = −0.11; β2, r = 0.27; and β3, r = 0.38, n = 7, not significant), or posttrain (α1, r = 0.11; α2, r = 0.12; α3, r = 0.41; β1, r = 0.06; β2, r = 0.04; and β3, r = −0.09, n = 7, not significant).
This study demonstrated Na+-K+-ATPase adaptability in skeletal muscle in already well-trained athletes with 3 wk of HIT. HIT increased maximal activity at rest and immediately following acute exercise, despite unchanged total content or isoform abundance. Furthermore, HIT increased α3 and β3 isoform mRNA expression in resting muscle, and the immediate increases with acute exercise in α1 and α3 mRNA expression were preserved. No significant correlations were detected between the decline in maximal Na+-K+-ATPase activity and elevations in α-isoform mRNA expression with acute exercise.
HIT Increased Maximal Na+-K+-ATPase Activity but not Content or Isoform Protein Abundance
An important finding was that HIT increased maximal Na+-K+-ATPase activity by ∼6% in already well-trained athletes, in the absence of any change in Na+-K+-ATPase total content ([3H]ouabain binding site) or in α-isoform protein abundance. This unchanged Na+-K+-ATPase content contrasts the well-known upregulatory effects of training in untrained (12, 13, 17, 26) or moderately trained participants (24). Insufficient exercise stimulus is unlikely to explain this, since [3H]ouabain binding was increased after only 3 days of training in previously untrained participants (12). One possible explanation is that these highly endurance trained participants may have elevated Na+-K+-ATPase content compared with untrained individuals (10, 21). The only other study to find increased Na+-K+-ATPase content with intense exercise training in well-trained endurance athletes used 5-mo intensified endurance training (8), a much greater increase in training load than used here. Any subtle changes in Na+-K+-ATPase content with short-term HIT could therefore go undetected with the [3H]ouabain binding site assay. However, undetected increases in Na+-K+-ATPase seems unlikely since HIT had no effect on Na+-K+-ATPase α-isoform protein abundance. This finding contrasts reports of an early α2 protein upregulation after only 3 days of endurance training in untrained participants (12), most likely reflecting the different training status. Interestingly, 6 days of training were required for an increase in Na+-K+-ATPase maximal activity, indicating a short-term dissociation between content and maximal activity (12). Here, the opposite effect occurred, with an increase in maximal Na+-K+-ATPase activity, despite unchanged Na+-K+-ATPase content. Given the almost identical resting muscle Na+-K+-ATPase content and maximal activity from baseline to pretrain, it seems likely that other mechanisms may be responsible for the increased maximal Na+-K+-ATPase activity observed with HIT. This requires further investigation but might occur through changes in membrane composition, protein phosphorylation, and/or changes in other associated regulatory proteins.
HIT Increased the End-Exercise Maximal Na+-K+-ATPase Activity
In these well-trained athletes, HIT increased maximal 3-O-MFPase activity but did not attenuate the exercise-induced acute depression in maximal 3-O-MFPase activity. Consequently, a higher maximal activity was found end-exercise after HIT. This suggests that an elevated muscle Na+-K+-ATPase activity might be an important functional adaptation with training. The decrease in maximal Na+-K+-ATPase activity with high-intensity interval exercise was qualitatively similar to that previously reported after a range of different exercise modalities and in subjects from widely different training backgrounds (2, 10, 22, 30, 42). This finding strengthens evidence for the decline in maximal Na+-K+-ATPase activity as reproducible and as potentially an important acute exercise response, consistent with our contention that this occurs as a necessary component of muscle fatigue, possibly as a muscle-protective strategy (2, 10).
Passive cellular Na+ influx (19, 43) and K+ efflux (14, 15, 19, 20, 43, 44) during muscle contraction exceed the rate of Na+-K+-ATPase-mediated Na+/K+ transport (5), causing membrane potential depolarisation (43), slow inactivation of voltage-dependent Na+ channels (41), which may contribute to a decline in muscle excitability (9), therefore accelerating muscle fatigue (4). Depressed maximal Na+-K+-ATPase activity could theoretically further exacerbate these transmembranous Na+ and K+ fluxes during muscle contraction, and training might be anticipated to alleviate this decline with exercise. The higher end-exercise Na+-K+-ATPase activity was in the context of stable performance and Na+-K+-ATPase measures from baseline to pretrain in these athletes. The 3% performance gain with short-term HIT represents an important gain in these athletes where an improvement of even 0.5% may improve sporting outcomes (18).
HIT Increases Resting Muscle Na+-K+-ATPase α3 and β3 mRNA Expression
A novel finding was that 3 wk of HIT upregulated α3 and β3 mRNA expression in resting muscle in well-trained athletes. The lack of change in α1, α2, and β1 isoform mRNA expression is consistent with an earlier finding, albeit in previously untrained participants (36). This lack of change might also reflect our low statistical power for HIT on the α2, β1, and β2 isoforms (Table 3). We cannot discern whether the increased α3 and β3 mRNA expression reflects increased mRNA transcription, reduced mRNA degradation, and/or enhanced mRNA stability (25). It is possible that the detected preferential upregulation of α3 and β3 mRNA expression is a consequence of their lower expression, relative to the more abundant α1 and α2 isoforms and β1 mRNA (37). It is difficult to assess the functional importance of this increase, since α3 and β3 mRNA expression have previously been found to be very low in human muscle (30, 37) and indeed are very low with our analyses since 35–38 PCR cycles were required for amplification of the primers for these isoforms. Furthermore, no α3 or β3 protein upregulation occurred with training.
Na+-K+-ATPase mRNA and Protein Responses to Acute Exercise Persist After Training
Before training, acute, intense interval-exercise immediately increased mRNA expression for each of the α1, α2, and α3 isoforms. This is consistent with immediately upregulated α1–3 isoform mRNA expression with fatiguing knee-extensor contractions in untrained individuals (31, 40) and also upregulation in isolated rat EDL muscle after electrical stimulation (28). These results differ from prolonged exercise, where only α3 isoform mRNA expression was immediately upregulated, with a delayed rise in α1 mRNA expression at 24 h postexercise (30). Differing results were obtained in two studies from the same laboratory, whereby α1 mRNA expression was increased but there was no detectable change in α2 and β1 mRNA after high-intensity single-leg kicking in one study (36), possibly due to the low observed statistical power; but α1, α2, β1, and β3 isoform mRNA expression were all increased after similar exercise in another (37). In rats, α1 and β2 mRNA expression was elevated in slow-twitch and fast-twitch muscle, respectively, following 1 h of treadmill running exercise (46). Although no immediate significant increase in mRNA expression was found for any of the three β-isoforms in this study before HIT, there was a tendency toward increased β1 and β2 mRNA with a large effect size for exercise present for each β isoform. We previously found in untrained individuals that fatiguing knee-extensor contractions upregulated mRNA expression of the three Na+-K+-ATPase β isoforms (31), but prolonged exercise upregulated only β2 mRNA expression (30). Our inability to detect changes in β1, β2, or β3 isoform mRNA expression with exercise may have been influenced by the lack of additional postexercise biopsy sampling time points or that isoform mRNA expression may be fiber-type dependent (46). The cycling in this study may not have recruited as many fast-twitch fibers (33) as with fatiguing leg kicking, or this response may have been blunted in these well endurance trained athletes (31, 47).
The increased Na+-K+-ATPase α-isoform mRNA with acute exercise persisted after 3 wk of HIT, in contrast to a recent report that HIT blunted the acute-exercise upregulation of α1 mRNA but consistent with the unaltered α2 mRNA expression response (36). These differences may be explained by the different training regimes and previously untrained participants used in their study. Our findings suggest that, even in well-trained athletes, the muscle Na+-K+-ATPase mRNA response to altered training can be maintained for at least 3 wk. It is unknown whether continuing this training regime for a greater time period would have eventually blunted this acute exercise response. Posttraining, this exercise effect size was large for α1 mRNA expression only, and moderate for the remaining isoforms.
There were no statistically different changes in protein abundance following acute exercise either pre- or post-HIT. It is likely that this represents a type II error due to the small sample size, as well as the greater variability evident in the Western blots for samples posttraining. Indeed, the effect size using Cohen's conventions (6) for exercise pre-HIT was moderate for α1 and large for α3, β2, and β3 protein abundance. Post-HIT, the effect size for exercise was small for α1 and α3 and moderate for α2 and β2. Collectively, these results suggest that changes in protein abundance might be possible with acute exercise and with this training in already well-trained athletes. Care must be taken when interpreting these Na+-K+-ATPase isoform mRNA and protein expression results, given the variability of results, the low number of observations for some measures, and the lack of additional postexercise biopsy sampling times to detect delayed responses.
No Relationships Between Depressed Na+-K+-ATPase Activity and Increased mRNA
Finally, we were unable to find any significant relationships between the acute depression in maximal Na+-K+-ATPase activity with acute exercise and the elevation in mRNA of any isoform. This contrasts findings in untrained or moderately trained participants (40) but is consistent with prolonged exercise, where no relationships were found (30). It seems unlikely that our lack of recovery biopsy analyses precluded us from detecting significant relationships, since relationships were previously detected in muscle samples taken immediately postexercise (40).
In conclusion, even in highly trained endurance athletes, short-term intensified training leads to adaptations in skeletal muscle Na+-K+-ATPase maximal activity and isoform mRNA expression and enhanced exercise performance. The Na+-K+-ATPase maximal activity in resting muscle was increased after training, independently of any change in total content or α-isoform protein abundance, pointing to as yet undefined independent regulatory mechanism(s) in skeletal muscle. Furthermore, a higher maximal Na+-K+-ATPase activity in exercised muscle after HIT is consistent with a possible functional role for Na+-K+-ATPase upregulation in minimizing muscular fatigue. Acute high-intensity interval exercise in well-trained athletes preferentially upregulated Na+-K+-ATPase α-isoform mRNA expression, and these responses persisted after training for the α1 and α3 isoforms but were not significantly correlated with the acutely depressed maximal Na+-K+-ATPase activity. These findings question a possible role of depressed activity in the immediate Na+-K+-ATPase isoform mRNA upregulation that occurs with exercise. HIT increased α3 and β3 isoform mRNA expression, pointing to a possible adaptive role in α3 and β3 isoforms to intensified training in already well-trained athletes.
This study was partially funded by an Australian Research Council Grant C00002552 and by the National Health and Medical Research Council of Australia Grant 256603.
We thank our participants for generous involvement in this lengthy and demanding study. We also thank Drs. Benedict Canny and David Newman for conducting some muscle biopsy sampling.
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.
- Copyright © 2007 the American Physiological Society