in skeletal muscle, Na⁺-K⁺-ATPase activity maintains the transmembrane Na⁺ and K⁺ concentration gradients necessary for repeated action potential generation. The Na⁺-K⁺-ATPase comprises a catalytic α-subunit and a glycosylated β-subunit, with different genes encoding for four α-isoforms (α₁–α₄) and three β-isoforms (β₁–β₃). Human skeletal muscle has recently been shown to express mRNA for each of the α₁- to α₃- and β₁- to β₃-isoforms (24, 29) and also for the α₄-isoform (29). Rat skeletal muscle has also been reported to express mRNA for each of the α₁- to α₃- and β₁- and β₂-isoforms (31, 43), whereas the mRNA for the α₄- and β₃-isoforms do not appear to have been probed.

Only ∼6 min of intense exercise elevated the mRNA expression of the α₁- to α₃- and β₁- to β₃-isoforms in human muscle (24), whereas more prolonged exercise elevated the mRNA expression of the α₁-, α₃-, and β₂-isoforms in human muscle (23) and the mRNA expression of the α₁- and β₂-isoforms in rat muscle (43). Whether electrical stimulation exerts similar effects is unknown and was therefore investigated here. It was hypothesized that three bouts of high-frequency electrical stimulation of isolated rat extensor digitorum longus (EDL) muscle would increase the mRNA expression of one or several of the Na⁺-K⁺-ATPase α₁- to α₃- and β₁- to β₃-isoforms.

The intracellular signals involved in the regulation in the mRNA expression of the Na⁺-K⁺-ATPase isoforms in skeletal muscle have not been identified. Because acute exercise increases the mRNA expression of the Na⁺-K⁺-ATPase isoforms in mammalian muscle (24, 43), it is likely that one or several of the transmembrane ionic fluxes and subsequent intracellular ionic concentration changes, which occur with exercise, may be involved in the signaling pathways inducing mRNA expression of the Na⁺-K⁺-ATPase isoforms.

Repeated muscle contractions induce an elevation in intracellular Na⁺ content in human (40) and rat muscle (20). However, because this increases Na⁺-K⁺-ATPase activity (26), this elevation in intracellular Na⁺ content is transient. It is possible that this transient rise in intracellular Na⁺ content is involved in increasing the mRNA expression of the Na⁺-K⁺-ATPase isoforms in skeletal muscle. In rat kidney cells, increasing the intracellular Na⁺ content and/or Na⁺ influx with ouabain, an inhibitor of Na⁺-K⁺-ATPase, induced an ∼100% increase in the mRNA expression of both the α₁- and β₁-isoforms (36). Furthermore, in chicken skeletal muscle cells, veratridine, an activator of the voltage-gated Na⁺-channels, induced a 70 and 150% increase in the mRNA expression of the α- and β-subunit isoforms, respectively (42). Monensin, a specific Na⁺ ionophore, has also been used to elevate the intracellular Na⁺-to-K⁺ ratio in rat skeletal muscle (8), but the effects of monensin on the mRNA expression of the Na⁺-K⁺-ATPase isoforms are unknown. Interestingly, in rat hindlimb muscle, an increase in intracellular Na⁺ content induced by dietary K⁺ deficiency reduced the mRNA expression of the α₂-isoform by 35%, with no significant effect on the mRNA expression of either of the α₁- or β₁-isoforms (1). Despite the above-mentioned exception, it was therefore hypothesized that elevated intracellular Na⁺ content and/or Na⁺ influx, induced by either ouabain, veratridine, or monensin, would increase the mRNA expression of one or several of the Na⁺-K⁺-ATPase α₁- to α₃- and β₁- to β₃-isoforms in rat skeletal muscle.

Repeated muscle contractions induce a reduction in intracellular K⁺ concentration and a subsequent increase in muscle extracellular K⁺ concentration ([K⁺]_o), leading to membrane depolarization (38). During intense exercise in humans, muscle [K⁺]_o can reach as high as ∼13 mM (41). In isolated rat EDL muscle, a [K⁺]_o of 13 mM induced a membrane depolarization of 26 mV (13). No study has elevated muscle [K⁺]_o to investigate the mRNA expression of the Na⁺-K⁺-ATPase isoforms. However, in rat liver cells, low [K⁺]_o (0.25–0.65 mM) induced a 250% increase in the mRNA expression of the α-subunit isoform (35); in canine kidney cells, low [K⁺]_o induced a 200% increase in the mRNA expression of both the α- and β-subunit isoforms (3). Such low [K⁺]_o would lead to membrane depolarization (10). It was therefore hypothesized that membrane depolarization induced by high [K⁺]_o, to replicate repeated muscle contractions, would increase the mRNA expression of one or several of the Na⁺-K⁺-ATPase α₁- to α₃- and β₁- to β₃-isoforms in rat skeletal muscle.

Baseline cytosolic Ca²⁺ concentration increases during repeated muscle contractions in isolated mammalian muscle (44). In rat kidney cells, elevating the intracellular Ca²⁺ concentration from 0.1 to 4.0 μM induced a 300% increase in the mRNA expression of both the α₁- and β₁-isoforms (37). Whether increased intracellular Ca²⁺ content exerts similar effects on the mRNA expression of the other Na⁺-K⁺-ATPase isoforms is unknown. The Ca²⁺ ionophore A-23187 has been shown to elevate both muscle Ca²⁺ content and ⁴⁵Ca uptake in rat EDL muscle (11), as well as the intracellular free Ca²⁺ content in cultured myotubes (16). On the basis of the findings by Rayson (37), it was hypothesized that elevated intracellular Ca²⁺ content and/or Ca²⁺ influx would increase the mRNA expression of one or several of the Na⁺-K⁺-ATPase α₁- to α₃- and β₁- to β₃-isoforms in rat skeletal muscle.

The effects of electrical stimulation on the mRNA expression of the Na⁺-K⁺-ATPase isoforms was studied here, with the aim to also identify the ionic factors modulating this expression in isolated rat EDL muscle. We show that electrical stimulation increased the mRNA expression of the Na⁺-K⁺-ATPase α-isoforms but not of the β-isoforms in isolated rat EDL muscle. Furthermore, the mRNA expression of the Na⁺-K⁺-ATPase isoforms appears to involve the cellular changes associated with membrane depolarization and increased intracellular Ca²⁺ content and/or Ca²⁺ influx but not of increased intracellular Na⁺ content.

METHODS

Animals and preparation of muscles.

Experiments were carried out with 4-wk-old female and male Wistar rats weighing ∼60–70 g. Rats of this age were used because the relatively small size of their EDL muscles (∼25 mg) minimizes the diffusional barriers to substrates, ions, and oxygen to the cell surface. The animals were fed ad libitum and were maintained in a temperature-controlled environment (21°C) with constant day length (12 h). The animals were killed by cervical dislocation, followed by decapitation, with intact EDL muscles, a predominantly fast-twitch fiber muscle (2), dissected out as previously described (27). All handling and use of animals complied with Danish animal welfare regulations.

Muscles were equilibrated for 30 min at 30°C in standard Krebs-Ringer bicarbonate buffer (KR) (pH 7.4) containing the following (in mM): 122.1 NaCl, 25.1 NaHCO₃, 2.8 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 1.3 CaCl₂, and 5.0 d-glucose; this was bubbled continuously with a mixture of 95% O₂-5% CO₂. In buffer with 13.0 mM [K⁺]_o, an equivalent amount of Na⁺ was omitted to maintain isosmolarity.

Effect of electrical stimulation on intracellular Na⁺ and K⁺ contents.

Intact muscles were mounted at resting length on electrodes for isometric contractions and equilibrated for 30 min in KR at 30°C. Muscles were then either rested or exposed to field stimulation across the central region through platinum electrodes, using either one, two, or three stimulation bouts, each comprising 10 s of continuous 60-Hz stimulation (0.2 ms, 12 V), given at 10-min intervals. We measured force (mN) using force displacement transducers, which were recorded with a chart recorder and/or digitally on a computer. Muscles exposed to two stimulation bouts were allowed to rest for 10 min after the second stimulation bout to represent the intracellular Na⁺ and K⁺ contents immediately before the third stimulation bout. Muscles were then washed for 4 × 15 min in ice-cold Na⁺-free Tris sucrose buffer (pH 7.45, containing the following in mM: 263.5 sucrose, 4.7 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 1.3 CaCl₂) to remove all extracellular Na⁺ (8). Muscles were blotted, tendons were removed, muscle wet weight was determined, and the muscles were soaked overnight in 0.3 M trichloroacetic acid (TCA) to give complete extraction of ions from the tissue (5). The Na⁺ content in the TCA extract was measured by flame photometry (FLM3; Radiometer, Copenhagen, Denmark) with lithium as internal standard. Values for Na⁺ content were then multiplied by 1.59 to correct for the loss of intracellular Na⁺ during the ice-cold washout (see Fig. 1, as described below). In contrast, the loss of K⁺ during the washout was minimal (8).

Effect of washout in ice-cold Na⁺-free Tris-sucrose buffer on intracellular Na⁺ content.

Control experiments were performed to determine the loss of intracellular Na⁺ during the 4 × 15 min (60 min) washout in ice-cold Na⁺-free Tris sucrose buffer in the EDL muscle, as this has previously only been determined in the soleus muscle (8). Intact muscles were mounted at resting length on electrodes for isometric contractions and equilibrated for 30 min in KR, after which muscles were transferred to ice-cold Na⁺-free Tris-sucrose buffer for washout for 30, 60, 90, 120, 150, or 180 min. Every 15 min during washout, muscles were transferred to new tubes and were kept agitated by continuous bubbling with air. After washout, muscles were blotted, tendons were cut off, muscle wet weight was determined, and the muscles were soaked overnight in 0.3 M TCA. The Na⁺ content in the TCA extract was measured by flame photometry, as described above. Figure 1 demonstrates that, after an initial 30 min of washout, which allows for removal of extracellular Na⁺ from the tissue, the muscle Na⁺ content showed an exponential decline with washout duration. This relationship can be tightly fitted by a linear regression line in the semi-logarithmic plot (r² > 0.99) and is assumed to represent the washout of intracellular Na⁺ (33). To determine the intracellular Na⁺ content of the muscles at time 0 (t = 0; prewashout), the regression line was used to calculate a correction factor. The correction factor was calculated by dividing the intercept value of the regression line (t = 0) with the regression line value at 60 min of washout. From this, all values for intracellular Na⁺ contents determined after a 60-min (4 × 15 min) washout were corrected by multiplying by a factor of 1.59.

Effects of electrical stimulation on the mRNA expression of the Na⁺-K⁺-ATPase isoforms.

Muscles were mounted for isometric contractions in thermostated chambers containing standard KR and were adjusted to optimal length for force production. After a standard equilibration of 30 min in KR, muscles were either rested or exposed to three stimulation bouts, each comprising 10 s of continuous 60-Hz stimulation (0.2 ms, 12 V) given at 10-min intervals. After the final stimulation bout, muscles were either immediately removed or allowed to recover for a further 3 h, because previous studies (24, 34) have demonstrated increased mRNA expression in the 2- to 4-h period after exercise. After removal, muscles were blotted, tendons were removed, and muscles were immediately frozen in liquid N₂ for analyses of the mRNA expression of the Na⁺-K⁺-ATPase isoforms.

Effects of muscle stretching on the mRNA expression of the Na⁺-K⁺-ATPase isoforms.

Control experiments were performed to show that any increase in mRNA expression of the Na⁺-K⁺-ATPase isoforms with electrical stimulation was not due to an artifact of the experimental design, specifically from stretching of muscles to optimal force-generating length. No difference was found between control muscles and those stretched passively for 30 min to produce a force of 10 or 50 mN, which is within the range required to reach optimal force-generating length, for the mRNA expression of any isoform (unpublished observations).

Effects of ouabain, veratridine, and monensin on intracellular Na⁺ content.

Muscles were placed in polyethylene baskets; after a standard equilibration of 30 min in standard KR, muscles were incubated for either 120 min with ouabain (1.0 mM), for 30 min with veratridine (0.1 mM), or for 30 min with monensin (0.1 mM). Muscles were then allowed to recover for a further 3 h in standard KR, before being washed for 4 × 15 min in ice-cold Na⁺-free Tris-sucrose buffer to remove all extracellular Na⁺. Muscles were then blotted, tendons were removed, and muscle wet weight was determined. Muscles were soaked overnight in 0.3 M TCA, and the Na⁺ content in the TCA extract was measured by flame photometry, as described above. Values were then multiplied by 1.59 to correct for the loss of intracellular Na⁺ during the ice-cold washout.

Effects of interventions utilized to induce increased intracellular Na⁺ and Ca²⁺ contents and of membrane depolarization on the mRNA expression of the Na⁺-K⁺-ATPase isoforms.

For each of the following interventions, muscles were placed in polyethylene baskets and, after equilibration for 30 min in standard KR, were incubated in the appropriate buffer for the indicated duration. All muscles were then allowed to recover for a further 3 h in standard KR (4 mM K⁺), after which they were blotted, tendons were removed, and the muscle was frozen in liquid N₂ for measurement of the mRNA expression of the Na⁺-K⁺-ATPase isoforms. Control muscles were incubated for durations matching their respective experimental muscles in standard KR.

Increased intracellular Na⁺ content and/or Na⁺ influx induced by ouabain, veratridine, and monensin.

Matching groups of muscles were used to determine the mRNA expression of the Na⁺-K⁺-ATPase isoforms after ouabain (120 min, 1.0 mM), veratridine (30 min, 0.1 mM), or monensin (30 min, 0.1 mM) exposure and compared to those used to determine the changes in intracellular Na⁺ content arising from these interventions.

Membrane depolarization induced by high [K⁺]_o.

Membrane depolarization was induced by incubating muscles for 60 min in KR containing 13 mM [K⁺]_o (13).

Increased intracellular Ca²⁺ content and/or Ca²⁺ influx induced by A-23187.

Increased intracellular Ca²⁺ content and/or Ca²⁺ influx was induced by incubating muscles for 30 min in KR containing the Ca²⁺ ionophore A-23187 (0.02 mM) (11, 16).

Measurement of the mRNA expression of the Na⁺-K⁺-ATPase isoforms.

Total RNA was extracted from ∼10 mg of muscle using the FastRNA reagents (BIO 101, Vista, CA), with methods previously described (25). The resulting RNA pellet was dissolved in EDTA-treated water, and total RNA concentration was determined spectrophotometrically at 260 nm. The ratio of absorbance at 260 and 280 nm (260/280) was 1.95 (SD 0.33), and the concentration of yielded RNA was not significantly different between control and test muscles (P = 0.152). RNA (1 μg) was transcribed into cDNA using the Promega AMV reverse transcription kit (Promega, Madison, WI), with oligo(dT) primers, with the resulting cDNA stored at −20°C for further 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 40 cycles (95°C for 15 s, 60°C for 60 s). Primer sequences were designed for the rat Na⁺-K⁺-ATPase α₁–α₄ and β₁–β₃ genes from published sequences (Table 1). However, mRNA expression of the Na⁺-K⁺-ATPase α₄-isoform could not be detected in all muscle samples by RT-PCR. The sizes of the PCR fragments amplified with each primer (126–268 bp) are included in Table 1 and are within the size range for close to 100% PCR efficiency, thereby validating this method. All samples were run in triplicate, and measurements included a no-template control (no cDNA), as well as a rat skeletal muscle sample endogenous control. Primer sequences for the commonly used housekeeping gene cyclophilin (Cyc) (24, 25) were also designed from published sequences (Table 1), and Cyc mRNA was used as a control to account for any variations in the amount of input RNA and the efficiency of reverse transcription. The mRNA expression of Cyc was not significantly altered with any intervention (electrical stimulation, P = 0.690; ouabain, veratridine, and monensin, P = 0.976; 13 mM [K⁺]_o, P = 0.133; A-23187, P = 0.761), when expressed in the logarithmic form of 2^−CT and using the statistical analyses described below. Gene expression was quantified from fluorescence emission using a cycle threshold (C_T) method, whereby the relative expression of the genes compared with control sample was made with the expression, 2^−ΔΔCT, in which the expression of each gene was normalized for input cDNA with the housekeeping gene Cyc. As an estimate of the relative basal mRNA expression of the Na⁺-K⁺-ATPase isoforms, a ΔC_T value was calculated by subtracting the basal CYC C_T from the basal isoform C_T, with the relative basal isoform mRNA expression then calculated with the expression 2^−ΔCT. The order of relative basal mRNA expression of the Na⁺-K⁺-ATPase isoforms was (highest to lowest) β₁ > α₂ > β₂ > β₃ > α₃ > α₁. This order compares roughly to that found in human vastus lateralis muscle (β₁ > α₂ > α₁ > β₃ > β₂ > α₃) (29), with the main difference being that the mRNA expression of the α₁-isoform in that study was higher than that of the β₃- and α₃-isoforms. This contrast may reflect species differences or variations in fiber-type composition between the predominantly fast-twitch EDL muscle and the vastus lateralis muscle of mixed fiber-type composition (7), because, in the predominantly slow-twitch soleus muscle, we have also estimated the relative basal mRNA expression of the α₁-isoform to be higher than that of the β₃- and α₃-isoforms (Murphy, Macdonald, McKenna, and Clausen, unpublished observations). The intra-assay coefficient of variation for each target gene was <13.0% for 2^−CT (Table 2), which is within those previously reported (25).

Chemicals.

All chemicals were of analytical grade. Monensin, veratridine, ouabain, A-23187, and d-glucose were purchased from Sigma (St. Louis, MO).

Statistical analysis.

All data are presented as means (SD). Statistical differences between two groups in the relative mRNA expression were analyzed with an independent-sample Student's t-test. The statistical difference in the relative mRNA expression or intracellular Na⁺ and K⁺ contents between three or more groups of muscles was analyzed by using a one-way ANOVA. Differences were located with a Student-Newman-Keuls post hoc test. Significance was accepted at P < 0.05. Power analyses were performed with Power and Precision software (Biostat).

RESULTS

Effect of electrical stimulation on intracellular Na⁺ and K⁺ contents and on the mRNA expression of the Na⁺-K⁺-ATPase isoforms.

A representative trace of the force responses to the electrical stimulation paradigm is shown in Fig. 2. During the three stimulation bouts, each at 10 s of stimulation at 60 Hz, force decreased by 27.0% (SD 8.1), 17.3% (SD 7.0), and 15.1% (SD 9.4), respectively (n = 6). At 3 h after the final 10-s stimulation bout, peak tetanic force was 49.2% (SD 7.8) of initial peak tetanic force (n = 6).

The first 10-s stimulation bout immediately increased intracellular Na⁺ content by 32.2% (P = 0.003) and reduced intracellular K⁺ content by 7.9% (P = 0.049; Fig. 3). Intracellular Na⁺ content was 28.9% (P = 0.016) lower in muscles exposed to two stimulation bouts followed by a 10-min recovery than in resting muscles (Fig. 3). The third 10-s stimulation bout, given at 20 min after the first 10-s stimulation bout, increased intracellular Na⁺ content by 50.7% (P = 0.001) and reduced intracellular K⁺ content by 4.4% (P = 0.030), compared with before the third stimulation bout (Fig. 3).

Immediately after the third stimulation bout, there was no significant increase in the mRNA expression of any of the α₁-, α_2-, or α₃-isoforms (Fig. 4). At 3 h after stimulation, these were, however, increased by 223% (P = 0.011), 621% (P = 0.001), and 892% (P = 0.001), respectively (Fig. 4). The mRNA expression of the α₂- and α₃-isoforms at 3 h poststimulation was also higher than that immediately after electrical stimulation, by 132% (P = 0.020) and 161% (P = 0.004), respectively, whereas there was no significant difference in the mRNA expression of the α₁-isoform between these two time points (P = 0.231; Fig. 4). However, electrical stimulation had no significant effect on the mRNA expression of any of the β₁- to β₃-isoforms, either immediately or at 3 h after stimulation (Fig. 4).

Effects of ouabain, veratridine, and monensin on intracellular Na⁺ content and on the mRNA expression of the Na⁺-K⁺-ATPase isoforms.

Ouabain, veratridine, and monensin elevated intracellular Na⁺ content by 769% (P = 0.001), 724% (P = 0.001), and 598% (P = 0.001), respectively (Fig. 5), as measured after the cessation of exposure to these agents, the 3-h recovery in standard KR, and the ice-cold 4 × 15 min washout.

Ouabain exposure followed by a 3-h recovery had no significant effect on the mRNA expression of any of the α₁-, α₂-, α₃-, or β₁-isoforms but reduced the mRNA expression of the β₂- and β₃-isoforms, by 76% (P = 0.044) and 92% (P = 0.009), respectively (Fig. 6). Neither veratridine nor monensin exposure, followed by a 3-h recovery, had any significant effect on the mRNA expression of any of the α₁-, α₃-, β₁-, or β₂-isoforms, but both reduced the mRNA expression of the β₃-isoform, by 87% (P = 0.013) and 90% (P = 0.011), respectively, whereas veratridine also reduced the mRNA expression of the α₂-isoform by 69% (P = 0.001; Fig. 6).

Effect of 13 mM [K⁺]_o on the mRNA expression of the Na⁺-K⁺-ATPase isoforms.

Exposure to 13 mM [K⁺]_o followed by a 3-h recovery increased the mRNA expression of the α₁-isoform by 160% (P = 0.021) and tended to increase the mRNA expression of the β₃-isoform (P = 0.055; Fig. 7). There was no significant effect of 13 mM [K⁺]_o on the mRNA expression of any of the Na⁺-K⁺-ATPase α₂-, α₃-, β₁-, or β₂-isoforms (Fig. 7).

Effect of A-23187 on the mRNA expression of the Na⁺-K⁺-ATPase isoforms.

A-23187 exposure followed by a 3-h recovery increased the mRNA expression of the α₃-isoform by 123% (P = 0.035) and, in contrast, reduced the mRNA expression of the β₁-isoform by 76% (P = 0.001; Fig. 8). There was no significant effect of A-23187 on the mRNA expression of any of the α₁-, α₂-, β₂-, or β₃-isoforms (Fig. 8).

DISCUSSION

This study investigated the effects of high-frequency electrical stimulation on the mRNA expression of the Na⁺-K⁺-ATPase isoforms in isolated rat EDL muscle. Factors modulating this expression were also explored, using interventions designed to induce either increased intracellular Na⁺ or Ca²⁺ content or membrane depolarization. The first main finding was that three bouts of high-frequency electrical stimulation followed by 3 h of recovery induced subunit-specific Na⁺-K⁺-ATPase mRNA expression, with the mRNA expression of each of the catalytic α₁-, α₂-, and α₃-isoforms increased with stimulation. The second main finding was that ouabain, veratridine, and monensin each markedly increased intracellular Na⁺ content but, surprisingly, did not increase the mRNA expression of any isoform. In fact, each of these interventions reduced the mRNA expression of the β₃-isoform and ouabain and veratridine also reduced the mRNA expression of the β₂- and α₂-isoforms, respectively. In contrast, 13 mM [K⁺]_o, which induces a 26-mV membrane depolarization (13), increased the mRNA expression of the α₁-isoform, whereas A-23187, which elevates intracellular Ca²⁺ content (17) and Ca²⁺ influx (11), increased the mRNA expression of the α₃-isoform but reduced the mRNA expression of the β₁-isoform. Thus the mRNA expression of the six Na⁺-K⁺-ATPase isoforms expressed in isolated rat EDL muscle appears to be regulated by different intracellular stimuli.

Three bouts of high-frequency electrical stimulation specifically increased the mRNA expression of the α-isoforms.

The effects of three bouts of high-frequency electrical stimulation on the mRNA expression of the Na⁺-K⁺-ATPase isoforms clearly differs between the α- and β-subunits. These findings are in contrast to the previously reported increase in the mRNA expression of the β₂-isoform but not of the α₁-isoform in fast-twitch muscles after 1 h of treadmill running in rats (43). Furthermore, in human vastus lateralis muscle, ∼6 min of intense exercise elevated the mRNA expression of all six Na⁺-K⁺-ATPase isoforms (24) whereas ∼55 min of submaximal exercise elevated the mRNA expression of the α₁-, α₃-, and β₂-isoforms (23). Thus the changes in the mRNA expression of the Na⁺-K⁺-ATPase isoforms induced with whole body exercise are not necessarily matched with those induced with in vitro electrical stimulation in isolated rat skeletal muscle. This difference may suggest that the mRNA expression of the Na⁺-K⁺-ATPase isoforms in skeletal muscle may involve both systemic (e.g., hormonal) and local factors. However, a recent study in humans demonstrated that, despite the concentrations of epinephrine and norepinephrine being significantly higher after exercise involving both arms and legs compared with that involving only legs, there was no difference in the mRNA expression of any of the α₁-, α₂-, β₁-, β₂-, and β₃-isoforms between the two exercise regimens (29). Thus it appears likely that local factors rather than systemic effects are involved in the mRNA expression of the Na⁺-K⁺-ATPase isoforms in skeletal muscle.

The upregulatory effect of electrical stimulation on the mRNA expression of the α-isoforms was not evident until 3 h after stimulation. This is consistent with the higher mRNA expression of genes regulating energy metabolism in the 2- to 4-h period after exercise (34), indicating that the mechanisms involved in increasing mRNA expression (i.e., accelerated transcription, attenuated mRNA degradation, or a combination of both) require several hours to induce a detectable increase in mRNA expression of the Na⁺-K⁺-ATPase isoforms. Despite an apparent increase, the lack of significant change in the mRNA expression of any of the α₁-, α₂-, and α₃-isoforms immediately after the final stimulation bout may reflect a type II error (14); however, the statistical power values of 0.72, 0.67, and 0.75, respectively, suggest that this is unlikely. Importantly, we also found that the elevations in mRNA expression of the α-isoforms with electrical stimulation were not due to an artifact of the experimental design, specifically due to the stretching of muscles to optimal force-generating length.

A significant increase in the mRNA expression of the α-isoforms, but not of the β-isoforms, with three bouts of 10 s of electrical stimulation at 60 Hz was also observed in an experiment involving 90 s of 60-Hz electrical stimulation followed by 3 h of recovery in isolated rat EDL muscle (Murphy et al., unpublished observation). Thus α-subunit-specific Na⁺-K⁺-ATPase mRNA expression may be an obligatory response to high-frequency electrical stimulation in isolated rat EDL muscle. This response may reflect the overabundance (5.5-fold for mRNA; 1.4- to 3.3-fold for protein) of the β-subunit in mammalian skeletal muscle (18, 29). Thus only an increased expression of the α-subunit may be required for the formation of additional αβ-heterodimers. This response may also reflect the catalytic nature of the α-isoforms, predisposing these isoforms to tight regulation imposed by changes associated with altered ion fluxes. As previously discussed, such changes were thought to include elevations in intracellular Na⁺ content (36, 42) and intracellular Ca²⁺ concentration (37) and also membrane depolarization (3, 35). Indeed, as evidenced with the first and third stimulation bouts, the electrical stimulation protocol used in the present study significantly elevated intracellular Na⁺ content and reduced intracellular K⁺ content, which would lead to membrane depolarization (21). The observed undershoot in intracellular Na⁺ content in muscles exposed to two stimulation bouts followed by a 10-min recovery, compared with that in resting muscles, reflects excitation-induced activation of the Na⁺-K⁺-ATPase (26). This finding suggests that the first 10-s stimulation bout was sufficient to increase Na⁺-K⁺-ATPase activity for at least 20 min. If the excitation-induced increase in intracellular Na⁺ content is involved in triggering the increase in Na⁺-K⁺-ATPase mRNA expression, it is surprising that such a short-lasting event is a sufficient signal. Although not measured in the present study, it is well-documented that there is an elevation in baseline cytosolic Ca²⁺ concentration with repeated muscle contractions in isolated mammalian muscle (44).

Ouabain, veratridine, and monensin increased intracellular Na⁺ content but did not increase the mRNA expression of any of the Na⁺-K⁺-ATPase isoforms.

Despite a clear increase in intracellular Na⁺ content with each of ouabain (120 min, 1.0 mM), veratridine (30 min, 0.1 mM), and monensin (30 min, 0.1 mM), there was no significant increase in the mRNA expression of any of the Na⁺-K⁺-ATPase isoforms. These findings are in contrast to those with cultured rat kidney cells, where 45–60 min of incubation with ouabain (0.1 mM) induced a ∼100% increase in the mRNA expression of both the α₁- and β₁-isoforms (36). Furthermore, in cultured chicken skeletal muscle cells, 5–30 h of exposure to veratridine (0.01 mM) induced a 70 and 150% increase in the mRNA expression of the α- and β-subunit isoforms, respectively (42). It is unlikely that the incubation periods used in the present study were insufficient because large elevations (598–769%) in intracellular Na⁺ content were induced. Furthermore, the interventions used to increase intracellular Na⁺ content would have induced membrane depolarization in isolated skeletal muscle (6) in the present study and also in cell cultures (4) used in the previous studies (36, 42). As such, differences between this and previous studies (36, 42) regarding the effect of elevated intracellular Na⁺ content on the mRNA expression of the Na⁺-K⁺-ATPase isoforms probably reflect differences in the preparations (isolated whole muscle vs. cultured cells) and species and tissues used (rat skeletal muscle vs. chicken skeletal muscle and rat kidney). In fact, ouabain reduced the mRNA expression of the β₂- and β₃-isoforms, veratridine reduced the mRNA expression of the α₂- and β₃-isoforms, and monensin also reduced the mRNA expression of the β₃-isoform. It therefore is probable that mRNA expression of the α₂-, β₂-, and β₃-isoforms may be negatively related to intracellular Na⁺ content. On the other hand, because intermittent electrical stimulation was also shown to increase intracellular Na⁺ content, as well as the mRNA expression of the Na⁺-K⁺-ATPase α-subunit isoforms, the lack of elevation in mRNA expression of any of the Na⁺-K⁺-ATPase isoforms with ouabain, veratridine, or monensin may have been because of the sustained increase in intracellular Na⁺ content. Thus increases in intracellular Na⁺ content may need to be intermittent in nature to elevate the mRNA expression of the Na⁺-K⁺-ATPase isoforms in isolated skeletal muscle.

It should be noted that, in skeletal muscle from rats, mice, and guinea pigs, an increase in intracellular Na⁺ induced by dietary K⁺ deficiency leads to a marked downregulation of the content of Na⁺-K⁺-ATPase enzymes, measured as [³H]ouabain binding capacity (30) or as α₂-isoform protein abundance (1). This would suggest that elevated intracellular Na⁺ under some conditions elicits downregulation of Na⁺-K⁺-ATPase mRNA expression. Indeed, Azuma et al. (1) showed that, in skeletal muscle from K⁺-deficient rats, mRNA expression of the α₂-isoform was reduced by 35%. In the present study, Na⁺ loading with veratridine decreased mRNA expression of the α₂-isoform by 69%, whereas Na⁺ loading with ouabain induced a nonsignificant 47% reduction in mRNA expression of the α₂-isoform. These changes are in keeping with the downregulation seen in K⁺-deficient rats. The physiological significance of this relationship and the cellular changes involved in reducing the mRNA expression of the β₂- and β₃-isoforms are unclear and require further investigation.

Furthermore, the lack of increase in mRNA expression of the Na⁺-K⁺-ATPase isoforms with interventions utilized to increase intracellular Na⁺ content is unlikely to be due to a reduction in intracellular Ca²⁺ content. In rat soleus muscle, ouabain (120 min, 1.0 mM) increased intracellular Na⁺ content, induced a small increase in ⁴⁵Ca uptake, and had no effect on muscle Ca²⁺ content (12). Additionally, in rat EDL muscle, veratridine (15 min, 0.1 mM) increased ⁴⁵Ca uptake by 139% (12). Thus, if there were any changes in intracellular Ca²⁺ content, it would have been elevated rather than reduced, with the interventions used here to increase intracellular Na⁺ content.

13 mM [K⁺]_o increased mRNA expression of the α₁-isoform.

The elevation of [K⁺]_o to levels mimicking those occurring in contracting muscle during exercise (41) induced an increase in the mRNA expression of the α₁-isoform and a tendency (P = 0.055) toward an increased mRNA expression of the β₃-isoform but had no significant effect on the mRNA expression of any of the α₂-, α₃-, β₁-, or β₂-isoforms. In rat liver cells, low [K⁺]_o (0.65 mM) was shown to increase the mRNA expression of the α-subunit isoform by 250% (35), whereas in canine kidney cells, an even lower [K⁺]_o (0.25 mM) induced a 200% increase in the mRNA expression of both the α- and β-subunit isoforms (3). Because such low [K⁺]_o would have actually depolarized the muscle (10) and the 13 mM [K⁺]_o used in the present study would have also depolarized the muscle (13), these results suggest that membrane depolarization may increase the mRNA expression of the Na⁺-K⁺-ATPase isoforms in both cultured cells and isolated muscles.

The effects of membrane depolarization on the mRNA expression of the Na⁺-K⁺-ATPase isoforms is unlikely to be due to any increase in intracellular Ca²⁺ content. In skinned fibers from rat EDL muscle, membrane depolarization induced via reduction of intracellular K⁺ concentration decreased sarcoplasmic reticulum Ca²⁺ release, as indicated by a reduction in twitch force (28), whereas in isolated rat EDL muscles, 30-min incubation in 20 mM [K⁺]_o had no significant effect on ⁴⁵Ca uptake (9). It therefore appears that intracellular Ca²⁺ content was more likely to have been reduced, rather than elevated, with the membrane depolarization induced here with 13 mM [K⁺]_o.

A-23187 increased mRNA expression of the α₃-isoform but reduced mRNA expression of the β₁-isoform.

The Ca²⁺ ionophore A-23187 (0.02 mM) was used to induce an elevation in intracellular Ca²⁺ content and/or Ca²⁺ influx. Indeed, in isolated rat EDL muscle, only 15 min of incubation with 0.02 mM A-23187 significantly increased ⁴⁵Ca uptake by 323% (11). Additionally, in cultured rabbit myocytes, only 10 min of incubation with 0.4 μM A-23187 was sufficient to increase intracellular free Ca²⁺ content by 200–900% (16). In the present study, A-23187 induced complex changes in the mRNA expression of the Na⁺-K⁺-ATPase isoforms, by increasing the mRNA expression of the α₃-isoform, reducing the mRNA expression of the β₁-isoform, and having no significant effect on the mRNA expression of any of α₁-, α₂-, β₂-_, or β₃-isoforms. These findings are in contrast to the 300% elevations in the mRNA expression of both the α₁- and β₁-isoforms found in rat kidney cells after 1-h incubation in solution containing 1.0 μM Ca²⁺ compared with that containing 0.1 μM Ca²⁺ (37). In that study, the author confirmed that the increase in extracellular Ca²⁺ (0.1–1.0 μM) also induced an increase in intracellular Ca²⁺ (0.1–4.0 μM). However, the validity of those results is uncertain because the extracellular Ca²⁺ concentrations used in that study are nonphysiologically low, with the normal resting extracellular Ca²⁺ concentration being 1.0–1.5 mM, as utilized here.

The physiological significance of an elevation in the mRNA expression of the α₃-isoform and a reduction in the mRNA expression of the β₁-isoform in response to increased intracellular Ca²⁺ content and/or Ca²⁺ influx is unclear. Nonetheless, because the relative mRNA expression of the α₃-isoform in skeletal muscle is likely to be low (29), the quantitative importance of an increase in the mRNA expression of the α₃-isoform is uncertain.

Importantly, the effects of A-23187 on the mRNA expression of the Na⁺-K⁺-ATPase isoforms were unlikely to be due to any A-23187-induced contracture because no increase in baseline tension was previously found with application of A-23187 (11). Although the mechanisms responsible for the elevation in mRNA expression of the α₃-isoform and the reduction in mRNA expression of the β₁-isoform with A-23187 are unknown, they may involve altered rates of transcription and degradation, respectively. In rat kidney cells, the elevation in mRNA expression of the α₁-isoform with an increase in extracellular Ca²⁺ concentration could almost completely be accounted for by an acceleration in the transcription rate of the α₁-isoform (37). On the other hand, the effect of an increase in extracellular Ca²⁺ concentration on the mRNA expression of the β₁-isoform was thought to be mediated by an altered degradation rate of the β₁-isoform. However, in that study, an increase in extracellular Ca²⁺ concentration was thought to attenuate the degradation rate of the β₁-isoform.

Perspectives

The functional significance of an increase in mRNA expression of the Na⁺-K⁺-ATPase isoforms with three bouts of 10-s electrical stimulation is uncertain. In isolated rat soleus and EDL muscles, protein expression of the Na⁺-K⁺-ATPase α₂-isoform was unchanged with up to 240 min of electrical stimulation (22). In human vastus lateralis muscle, [³H]ouabain binding was not changed after 72 min of exercise (19) but was increased by 13% with ∼10 h of running (32). It therefore appears that a time course discrepancy exists between the mRNA and protein expression of the Na⁺-K⁺-ATPase. Thus mRNA expression of the Na⁺-K⁺-ATPase may be elevated with only 30 s of repeated muscle contractions, whereas the protein expression of the functional Na⁺-K⁺-ATPase may only be elevated after several hours of repeated muscle contractions.

The mRNA expression of the Na⁺-K⁺-ATPase isoforms appears to be regulated by different stimuli in rat skeletal muscle. In the EDL, a muscle comprising predominantly fast-twitch fibers, the present study suggests that membrane depolarization and elevated intracellular Ca²⁺ content and/or Ca²⁺ influx may induce increased mRNA expression of the α₁- and α₃-isoforms, respectively. Thus other factors not explored in this study may be responsible for inducing the increased mRNA expression of the other Na⁺-K⁺-ATPase isoforms observed after electrical stimulation and exercise. This may include increased reactive oxygen species (ROS) since repeated muscle contractions increase ROS in both rat and human muscle (15, 39). Furthermore, there is accumulating evidence that increased ROS may act as a second messenger in signaling pathways involved in the mRNA expression of the cardiac Na⁺-K⁺-ATPase (45). Further work is required to investigate the role of increased ROS in the mRNA expression of the Na⁺-K⁺-ATPase isoforms in skeletal muscle.

In conclusion, the effects of three bouts of high-frequency electrical stimulation on the mRNA expression of the Na⁺-K⁺-ATPase isoforms in rat EDL muscle were subunit specific, with increases in mRNA expression of the α-isoforms but not of the β-isoforms. Furthermore, mRNA expression of the Na⁺-K⁺-ATPase isoforms appears to be regulated by different stimuli, including the cellular changes associated with high [K⁺]_o such as membrane depolarization, as well as with A-23187 such as elevated intracellular Ca²⁺ content and/or Ca²⁺ influx. Surprisingly, there was no increase in the mRNA expression of any of the Na⁺-K⁺-ATPase isoforms with interventions used to elevate intracellular Na⁺ content but rather a decreased mRNA expression of several isoforms. Thus a surprising diversity of signals appears to be involved in upregulating the mRNA expression of the different Na⁺-K⁺-ATPase isoforms. However, this is consistent with the expression of multiple isoforms, with presumably a corresponding diversity in function.

GRANTS

This study was supported by grants from the National Health and Medical Research Council of Australia, The Foundation for Young Australians, and The Lundbeck Foundation.

FOOTNOTES

• 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.