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ENVIRONMENTAL, EXERCISE AND RESPIRATORY PHYSIOLOGY

Ionic mechanisms of excitation-induced regulation of Na⁺-K⁺-ATPase mRNA expression in isolated rat EDL muscle

¹Muscle, Ions and Exercise Group, School of Human Movement, Recreation and Performance, Centre for Ageing, Rehabilitation and Sport Science, Victoria University of Technology, Melbourne, Australia; and ²Institute of Physiology and Biophysics, University of Aarhus, Århus, Denmark

Submitted 4 October 2005 ; accepted in final form 14 December 2005

	ABSTRACT

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This study investigated the effects of electrical stimulation on Na⁺-K⁺-ATPase isoform mRNA, with the aim to identify factors modulating Na⁺-K⁺-ATPase mRNA in isolated rat extensor digitorum longus (EDL) muscle. Interventions designed to mimic exercise-induced increases in intracellular Na⁺ and Ca²⁺ contents and membrane depolarization were examined. Muscles were mounted on force transducers and stimulated with 60-Hz 10-s pulse trains producing tetanic contractions three times at 10-min intervals. Ouabain (1.0 mM, 120 min), veratridine (0.1 mM, 30 min), and monensin (0.1 mM, 30 min) were used to increase intracellular Na⁺ content. High extracellular K⁺ (13 mM, 60 min) and the Ca²⁺ ionophore A-23187 (0.02 mM, 30 min) were used to induce membrane depolarization and elevated intracellular Ca²⁺ content, respectively. Muscles were analyzed for Na⁺-K⁺-ATPase ₁–₃ and ₁–₃ mRNA (real-time RT-PCR). Electrical stimulation had no immediate effect on Na⁺-K⁺-ATPase mRNA; however at 3 h after stimulation, it increased ₁, ₂, and ₃ mRNA by 223, 621, and 892%, respectively (P = 0.010), without changing mRNA. Ouabain, veratridine, and monensin increased intracellular Na⁺ content by 769, 724, and 598%, respectively (P = 0.001) but did not increase mRNA of any isoform. High intracellular K⁺ concentration elevated ₁ mRNA by 160% (P = 0.021), whereas A-23187 elevated ₃ mRNA by 123% (P = 0.035) but reduced ₁ mRNA by 76% (P = 0.001). In conclusion, electrical stimulation induced subunit-specific increases in Na⁺-K⁺-ATPase mRNA in isolated rat EDL muscle. Furthermore, Na⁺-K⁺-ATPase mRNA appears to be regulated by different stimuli, including cellular changes associated with membrane depolarization and increased intracellular Ca²⁺ content but not increased intracellular Na⁺ content.

gene expression; Na⁺-K⁺ pump; skeletal muscle

Only 6 min of intense exercise elevated the mRNA expression of the ₁- to ₃- and ₁- to ₃-isoforms in human muscle (24), whereas more prolonged exercise elevated the mRNA expression of the ₁-, ₃-, and ₂-isoforms in human muscle (23) and the mRNA expression of the ₁- and ₂-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 ₁- to ₃- and ₁- to ₃-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 ₁- and ₁-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 - and -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 ₂-isoform by 35%, with no significant effect on the mRNA expression of either of the ₁- or ₁-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 ₁- to ₃- and ₁- to ₃-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 -subunit isoform (35); in canine kidney cells, low [K⁺]_o induced a 200% increase in the mRNA expression of both the - and -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 ₁- to ₃- and ₁- to ₃-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 ₁- and ₁-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 ₁- to ₃- and ₁- to ₃-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 -isoforms but not of the -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

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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 x 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).

View larger version (8K): [in this window] [in a new window]   Fig. 1. Time course of muscle Na+ content during washout in ice-cold Na+-free Tris-sucrose buffer in rat extensor digitorum longus (EDL) muscle. Muscles were mounted for isometric contractions and equilibrated for 30 min at 30°C in standard Krebs-Ringer (KR). Muscles were then transferred to ice-cold Na+-free Tris-sucrose buffer for washout for durations ranging from 30 to 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, and tendons were removed, weighed, and taken for flame photometric analysis of Na+ contents. Data are means and SD; n = 4; r2 of regression line > 0.99. The correction factor for intracellular Na+ content was obtained by dividing the intercept value of the regression line (time = 0) with the regression line value at 60 min of washout (correction factor = 1.59). Na+ content of the muscles at the end of the 60-min washout was corrected for the loss of intracellular Na+ by multiplying with this factor. Note that the muscle Na+ contents are presented on a log scale.

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 x 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 ₁–₄ and ₁–₃ genes from published sequences (Table 1). However, mRNA expression of the Na⁺-K⁺-ATPase ₄-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^–CT, 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 C_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^–CT. The order of relative basal mRNA expression of the Na⁺-K⁺-ATPase isoforms was (highest to lowest) ₁ > ₂ > ₂ > ₃ > ₃ > ₁. This order compares roughly to that found in human vastus lateralis muscle (₁ > ₂ > ₁ > ₃ > ₂ > ₃) (29), with the main difference being that the mRNA expression of the ₁-isoform in that study was higher than that of the ₃- and ₃-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 ₁-isoform to be higher than that of the ₃- and ₃-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).

View this table: [in this window] [in a new window]   Table 1. Rat Na+-K+-ATPase genes 1–4 and 1–3, and Cyc primer sequences used for mRNA analyses

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

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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).

View larger version (8K): [in this window] [in a new window]   Fig. 2. Representative trace of the force responses to the electrical stimulation paradigm. Muscles were mounted for isometric contractions and equilibrated for 30 min in standard KR. Muscles were then stimulated for 0.5 s at 60 Hz and allowed to rest for 30 min before being stimulated for 10 s at 60 Hz, at 10-min intervals, for 3 stimulation bouts. After the third stimulation bout, muscles were allowed to recover for a further 180 min and were then stimulated for 0.5 s at 60 Hz.

View larger version (10K): [in this window] [in a new window]   Fig. 3. Effects of 3 bouts of high-frequency electrical stimulation (stim) on intracellular Na+ (A) and K+ contents (B) in rat EDL muscle. Muscles were mounted for isometric contractions, equilibrated for 30 min in standard KR, and either allowed to rest for 10 s or stimulated for 10 s at 60 Hz, at 10-min intervals, for either 1 (1 x 10 s), 2 (2 x 10 s), or 3 stimulation bouts (3 x 10 s). Muscles stimulated for 2 stimulation bouts were then allowed to rest for a further 10 min to represent the intracellular Na+ and K+ contents immediately before the third stimulation bout. All muscles were then washed for 4 x 15 min in ice-cold Na+-free Tris-sucrose buffer and blotted; tendons were then removed, weighed, and taken for flame photometric analysis of Na+ and K+ contents. Values for Na+ content were multiplied by 1.59 to correct for the loss of intracellular Na+ during the washout. Bars denote 10-s stimulation bouts. Data are means () and SD; n = 4–10. *P < 0.05 vs. rest, P < 0.05 vs. 1 x 10 s, P < 0.05 vs. 2 x 10 s plus 10-min recovery.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Effects of 3 bouts of high-frequency electrical stimulation and subsequent 3-h recovery on mRNA expression of the Na+-K+-ATPase 1- to 3- and 1- to 3-isoforms in rat EDL muscle. Isoform mRNA expression is expressed relative to control (Con) (1.0). Muscles were mounted for isometric contractions, equilibrated for 30 min in standard KR, and stimulated electrically for 10 s at 60 Hz at 10-min intervals. Muscles were then either immediately removed (Stim) or allowed to recover in standard KR for a further 3 h (Stim+3h). Data are means and SD; n = 8 except Stim where n = 6. *P < 0.02, greater than Con; P < 0.02, greater than Stim.

View larger version (13K): [in this window] [in a new window]   Fig. 5. Effects of ouabain, veratridine, and monensin on intracellular Na+ content in rat EDL muscle. Muscles were placed in polyethylene baskets, equilibrated for 30 min in standard KR, and then incubated without (solid bars) or with ouabain (120 min, 1.0 mM), veratridine (30 min, 0.1 mM), or monensin (30 min, 0.1 mM). They were then allowed to recover in standard KR for a further 3 h (hatched bars). Muscles were then washed for 4 x 15 min in ice-cold Na+-free Tris-sucrose buffer and blotted; tendons were then removed, weighed, and taken for flame photometric analysis of Na+ content. Values for Na+ content were multiplied by 1.59 to correct for the loss of intracellular Na+ during the washout. Data are means and SD; n = 13 for Con, n = 4–7 for ouabain, and n = 4–6 for veratridine and monensin. *P = 0.001, greater than Con.

View larger version (13K): [in this window] [in a new window]   Fig. 6. Effects of ouabain, veratridine, and monensin on mRNA expression of the Na+-K+-ATPase 1- to 3- and 1- to 3-isoforms in rat EDL muscle. Isoform mRNA expression is expressed relative to control (1.0). Muscles were placed in polyethylene baskets, equilibrated for 30 min in standard KR, and then incubated with ouabain (120 min, 1 mM), veratridine (30 min, 0.1 mM), or monensin (30 min, 0.1 mM). They were then allowed to recover in standard KR for a further 3 h. Data are means and SD; n = 6. *P < 0.05, lower than Con.

View larger version (14K): [in this window] [in a new window]   Fig. 7. Effects of 13 mM extracellular K+ concentration ([K+]o) on mRNA expression of the Na+-K+-ATPase 1- to 3- and 1- to 3-isoforms in rat EDL muscle. Isoform mRNA expression is expressed relative to control (1). Muscles were placed in polyethylene baskets, equilibrated for 30 min in standard KR, and then incubated for 60 min at 13 mM [K+]o, before being allowed to recover in normal KR (4 mM K+) for a further 3 h. Data are means and SD; n = 12. *P < 0.03, greater than Con; #P < 0.06, greater than Con.

View larger version (14K): [in this window] [in a new window]   Fig. 8. Effects of A-23187 on mRNA expression of the Na+-K+-ATPase 1- to 3- and 1- to 3-isoforms in rat EDL muscle. Isoform mRNA expression is expressed relative to control (1). Muscles were placed in polyethylene baskets, equilibrated for 30 min in standard KR, and then incubated with A-23187 (30 min, 0.02 mM), before being allowed to recover in normal KR for a further 3 h. Data are means and SD; n = 6. *P < 0.04 vs. Con.

	DISCUSSION

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Three bouts of high-frequency electrical stimulation specifically increased the mRNA expression of the -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 - and -subunits. These findings are in contrast to the previously reported increase in the mRNA expression of the ₂-isoform but not of the ₁-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 ₁-, ₃-, and ₂-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 ₁-, ₂-, ₁-, ₂-, and ₃-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 -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 ₁-, ₂-, and ₃-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 -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 -isoforms, but not of the -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 -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 -subunit in mammalian skeletal muscle (18, 29). Thus only an increased expression of the -subunit may be required for the formation of additional -heterodimers. This response may also reflect the catalytic nature of the -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 ₁- and ₁-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 - and -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 ₂- and ₃-isoforms, veratridine reduced the mRNA expression of the ₂- and ₃-isoforms, and monensin also reduced the mRNA expression of the ₃-isoform. It therefore is probable that mRNA expression of the ₂-, ₂-, and ₃-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 -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 ₂-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 ₂-isoform was reduced by 35%. In the present study, Na⁺ loading with veratridine decreased mRNA expression of the ₂-isoform by 69%, whereas Na⁺ loading with ouabain induced a nonsignificant 47% reduction in mRNA expression of the ₂-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 ₂- and ₃-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 ₁-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 ₁-isoform and a tendency (P = 0.055) toward an increased mRNA expression of the ₃-isoform but had no significant effect on the mRNA expression of any of the ₂-, ₃-, ₁-, or ₂-isoforms. In rat liver cells, low [K⁺]_o (0.65 mM) was shown to increase the mRNA expression of the -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 - and -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 ₃-isoform but reduced mRNA expression of the ₁-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 ₃-isoform, reducing the mRNA expression of the ₁-isoform, and having no significant effect on the mRNA expression of any of ₁-, ₂-, ₂-_, or ₃-isoforms. These findings are in contrast to the 300% elevations in the mRNA expression of both the ₁- and ₁-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 ₃-isoform and a reduction in the mRNA expression of the ₁-isoform in response to increased intracellular Ca²⁺ content and/or Ca²⁺ influx is unclear. Nonetheless, because the relative mRNA expression of the ₃-isoform in skeletal muscle is likely to be low (29), the quantitative importance of an increase in the mRNA expression of the ₃-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 ₃-isoform and the reduction in mRNA expression of the ₁-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 ₁-isoform with an increase in extracellular Ca²⁺ concentration could almost completely be accounted for by an acceleration in the transcription rate of the ₁-isoform (37). On the other hand, the effect of an increase in extracellular Ca²⁺ concentration on the mRNA expression of the ₁-isoform was thought to be mediated by an altered degradation rate of the ₁-isoform. However, in that study, an increase in extracellular Ca²⁺ concentration was thought to attenuate the degradation rate of the ₁-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 ₂-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 ₁- and ₃-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 -isoforms but not of the -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

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
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.

	ACKNOWLEDGMENTS

 
We thank Ann-Charlotte Andersen, Marianne Stürup Johansen, Tove Lindahl Andersen, and Vibeke Uhre for skilled technical assistance and Associate Professor Ole Bækgaard Nielsen at the University of Aarhus for the planning of the Na⁺ washout experiments. We also thank Dr. Rodney Snow and Dr. Cameron-Smith at Deakin University, Melbourne, for the use of their laboratory to conduct the mRNA analyses.

	FOOTNOTES

 

Address for reprint requests and other correspondence: K. T. Murphy, Institute of Physiology and Biophysics, Univ. of Aarhus, Ole Worms Allé 160, DK-8000 Århus C, Denmark (e-mail: km{at}fi.au.dk)

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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TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

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