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¹ Muscle, Ions and Exercise Group, School of Human Movement, Recreation and Performance, Centre for Rehabilitation, Exercise and Sports Science, Victoria University of Technology, Melbourne; Australia² Exercise, Muscle and Metabolism Unit, School of Health Sciences, Deakin University, Melbourne; Australia

	Abstract

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Characterization of expression of, and consequently also the acute exercise effects on, Na⁺,K⁺-ATPase isoforms in human skeletal muscle remains incomplete and was therefore investigated. Fifteen healthy subjects (eight males, seven females) performed fatiguing, knee extensor exercise at 40% of their maximal work output per contraction. A vastus lateralis muscle biopsy was taken at rest, fatigue and 3 and 24 h postexercise, and analysed for Na⁺,K⁺-ATPase ₁, ₂, ₃, ß₁, ß₂ and ß₃ mRNA and crude homogenate protein expression, using Real-Time RT-PCR and immunoblotting, respectively. Each individual expressed gene transcripts and protein bands for each Na⁺,K⁺-ATPase isoform. Each isoform was also expressed in a primary human skeletal muscle cell culture. Intense exercise (352 ± 69 s; mean ±S.E.M.) immediately increased ₃ and ß₂ mRNA by 2.4- and 1.7-fold, respectively (P < 0.05), whilst ₁ and ₂ mRNA were increased by 2.5- and 3.5-fold at 24 h and 3 h postexercise, respectively (P < 0.05). No significant change occurred for ß₁ and ß₃ mRNA, reflecting variable time-dependent responses. When the average postexercise value was contrasted to rest, mRNA increased for ₁, ₂, ₃, ß₁, ß₂ and ß₃ isoforms, by 1.4-, 2.2-, 1.4-, 1.1-, 1.0- and 1.0-fold, respectively (P < 0.05). However, exercise did not alter the protein abundance of the ₁–₃ and ß₁–ß₃ isoforms. Thus, human skeletal muscle expresses each of the Na⁺,K⁺-ATPase ₁, ₂, ₃, ß₁, ß₂ and ß₃ isoforms, evidenced at both transcription and protein levels. Whilst brief exercise increased Na⁺,K⁺-ATPase isoform mRNA expression, there was no effect on isoform protein expression, suggesting that the exercise challenge was insufficient for muscle Na⁺,K⁺-ATPase up-regulation.

(Received 11 September 2003; accepted after revision 25 January 2004; first published online 30 January 2004)
Corresponding author M. J. McKenna: School of Human Movement, Recreation and Performance (FO22), Victoria University of Technology, PO Box 14428, MCMC, Melbourne, Victoria, Australia.  Email: michael.mckenna{at}vu.edu.au

	Introduction

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The Na⁺,K⁺-ATPase enzyme is ubiquitously expressed in mammalian tissues. In skeletal muscle, the Na⁺,K⁺-ATPase has major regulatory functions, including maintenance of intra- to extracellular [Na⁺] and [K⁺] gradients, membrane excitability and contractility, as well as secondary transport processes (Blanco & Mercer, 1998; Nielsen & Clausen, 2000; Clausen, 2003). The Na⁺,K⁺-ATPase comprises a catalytic subunit (100–112 kDa) and a glycosylated ß subunit (40–60 kDa), and belongs to a multigene family, with different genes encoding four (₁, ₂, ₃, ₄) and three ß isoforms (ß₁, ß₂, ß₃) (Blanco & Mercer, 1998). Expression of these isoforms is tissue-, developmental- and species-specific, suggesting a diversity of isoform-specific functions (Orlowski & Lingrel, 1988; Blanco & Mercer, 1998; Wang et al. 2001). Rat skeletal muscle has been reported to express the Na⁺,K⁺-ATPase ₁, ₂, ₃, ß₁ and ß₂ gene transcripts (Orlowski & Lingrel, 1988; Tsakiridis et al. 1996; Blanco & Mercer, 1998; Wang et al. 2001), and the ₁, ₂, ß₁, ß₂ and ß₃ proteins (Hundal et al. 1993; Tsakiridis et al. 1996; Arystarkova & Sweadner, 1997). Furthermore, expression of the ß₁ and ß₂ isoforms is specific to red–oxidative and to white–glycolytic muscle, respectively (Hundal et al. 1993; Tsakiridis et al. 1996). Whilst expression of the ₄ isoform was previously thought to be exclusive to the testis (Shamraj & Lingrel, 1994), the ₄ gene transcript was recently reported in human skeletal muscle of unspecified origin (Keryanov & Gardner, 2002).

In contrast to rat skeletal muscle, characterization of the Na⁺,K⁺-ATPase isoforms expressed in human skeletal muscle remains incomplete, at both gene transcription and protein levels. The ₁, ₂, ß₁ and ß₃ isoforms are the only gene transcripts to be previously investigated and detected in human skeletal muscle (Malik et al. 1998; Nordsborg et al. 2003). The first study to investigate Na⁺,K⁺-ATPase isoform protein expression in human muscle obtained soleus muscle from amputated lower limb from patients with non-specified disease (Hundal et al. 1994). They detected the ₁, ₂, ₃ and ß₁ proteins, but not the ß₂ protein, and stated that the ß₂ protein was also undetected in anterior tibialis muscle (Hundal et al. 1994). Similarly, the ß₂ protein was undetected in vastus lateralis muscle obtained from healthy humans (Juel et al. 2000). The apparent absence of ß₂ is surprising and contrasts with ß₂ protein expression in rat muscle (Hundal et al. 1993; Tsakiridis et al. 1996). Conflicting data exist on ₃ protein expression in human muscle. Whereas the ₃ protein was detected in amputated soleus muscle (Hundal et al. 1994), it was not found in human skeletal muscle of unstated origin and pathology (Wang et al. 2001). Finally, despite very low levels of ß₃ mRNA being reported in human skeletal muscle (Malik et al. 1998), ß₃ protein expression has not yet been investigated. Thus, research is required to clarify expression of the ₃, ß₂ and ß₃ isoforms. Previous studies investigating isoform protein expression have isolated sarcolemmal membranes via membrane fractionation on a sucrose gradient (Hundal et al. 1994; Tsakiridis et al. 1996) or via the formation of giant sarcolemmal vesicles (Juel et al. 2000; Juel et al. 2001), thereby restricting detection of Na⁺,K⁺-ATPase isoforms to those of sarcolemmal origin. This study therefore firstly aimed to characterize Na⁺,K⁺-ATPase isoform expression in human skeletal muscle, by measurement of both the gene transcript and protein expression, utilizing a crude muscle homogenate to enhance detection of Na⁺,K⁺-ATPase isoforms in the whole muscle.

Little is known about the effects of acute exercise on Na⁺,K⁺-ATPase isoform expression. This in part reflects the inconsistent findings regarding expression of Na⁺,K⁺-ATPase isoforms in skeletal muscle. In humans, different exercise protocols elevated the mRNA expression in muscle of a variety of genes involved in metabolism (Pilegaard et al. 2000). This raises the possibility that exercise might up-regulate many of the Na⁺,K⁺-ATPase isoforms. In rats, 1 h of continuous treadmill running elevated ₁ and ß₂ mRNA, and also the sarcolemmal membrane ₁ and ₂ isoform protein abundance in both red–oxidative and white–glycolytic fibres (Tsakiridis et al. 1996). In contrast, 1 h of intermittent treadmill running increased the sarcolemmal protein abundance of the ₁, ₂, ß₁ and ß₂ isoforms (Juel et al. 2001). Interestingly, these elevations were transient with expression of all isoforms returning to control levels at 3 h postexercise (Juel et al. 2001). The transient increase in Na⁺,K⁺-ATPase isoforms at the sarcolemma during exercise was interpreted by the authors (Juel et al. 2001) to reflect a translocation to and from the membrane, rather than an overall increased cellular Na⁺,K⁺-ATPase expression. In humans, high-intensity intermittent one-legged knee extensor exercise elevated Na⁺,K⁺-ATPase ₁ mRNA expression by 3-fold at 0 h, 1 h and 3 h postexercise, with expression returning to resting levels at 5 h postexercise (Nordsborg et al. 2003). No effect of exercise on ₂ and ß₁ mRNA expression was found, but their study was limited by low statistical power (Nordsborg et al. 2003). Furthermore, only the Na⁺,K⁺-ATPase ₁, ₂ and ß₁ gene transcripts were probed (Nordsborg et al. 2003). To date, only a single study has investigated exercise effects on Na⁺,K⁺-ATPase isoform protein expression in human skeletal muscle, and this only probed for the ₁, ₂ and ß₁ isoforms (Juel et al. 2000). They reported that intense one-legged knee extensor exercise elevated the sarcolemmal ₂ and ß₁ isoform protein abundance by 70% and 26%, respectively (Juel et al. 2000). The time course of any up-regulation in isoform expression in human skeletal muscle has not been investigated. The second aim of this study was therefore to investigate the effects of intense exercise on the Na⁺,K⁺-ATPase isoform mRNA and protein expression, probing for all isoforms expressed in human skeletal muscle.

We therefore tested two hypotheses. First, we hypothesized that each of the Na⁺,K⁺-ATPase ₁, ₂, ₃, ß₁, ß₂ and ß₃ isoforms would be expressed in the vastus lateralis muscle obtained from healthy individuals. Secondly, we hypothesized that acute intense exercise would transiently increase the mRNA and crude muscle homogenate protein abundance of the Na⁺,K⁺-ATPase ₁, ₂, ₃, ß₁, ß₂ and ß₃ isoforms within a 24 h postexercise period.

	Methods

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Subjects

Fifteen healthy subjects, comprising eight males and seven females (age, 24.7 ± 6.7 years; height, 174.5 ± 6.8 cm; body mass, 73.2 ± 11.4 kg; means ±S.D.) gave written informed consent and participated in this study. All subjects participated in regular physical activity, but none had any prior experience of isokinetic knee extension exercise. Each subject refrained from vigorous exercise for 48 h, and from caffeine and alcohol consumption for 24 h, prior to each of the exercise tests. All protocols and procedures were approved by the Human Research Ethics Committee at Victoria University of Technology and conform with the Declaration of Helsinki. Cell cultures were grown from a vastus lateralis muscle biopsy sample obtained at rest from one additional healthy female (age, 23; height, 164 cm; body mass, 55 kg). Tissue procurement for cell culture procedures was conducted separately at Deakin University and was approved by the Deakin University Human Research Ethics Committee.

Incremental exercise test

Each subject completed an initial exercise test to determine peak oxygen uptake as a marker of aerobic fitness, performing incremental exercise (25 W min^–1, 80 r.p.m.) on an electrically braked cycle ergometer (Lode Excalibur, Groningen, the Netherlands), until volitional exhaustion, with all equipment, procedures and calibration as previously detailed (Fraser et al. 2002; Li et al. 2002).

Maximal knee extensor muscle strength and muscle fatigue test

A second test was undertaken to determine maximal muscle strength during isokinetic knee extensor contractions, followed by a third test designed to induce local fatigue of the knee extensor muscles, and designated the muscle fatigue test. Two familiarization trials were conducted for the muscle strength and fatigue tests. A final third trial for the muscle fatigue test included the muscle biopsy and blood sampling procedures. Intervals of 1 week between familiarization trial days and 2 weeks before the final invasive muscle fatigue trial were used, to minimize any local training effects. Subjects consumed standardized meals and fluid intake on the day prior to, during, and on the day following the invasive muscle fatigue trial.

The muscle strength and fatigue tests were performed on an isokinetic dynamometer (Cybex Norm 770, Henley HealthCare, MA, USA), and involved isokinetic knee extensions from knee joint angles of 0 deg through to 90deg, at a speed of 180 deg s^–1. All torque data were corrected for gravity, and the dynamometer was calibrated for angle, torque and velocity immediately prior to each test. Subjects were strapped to the dynamometer adjustable chair using belts across the hips and chest to restrict movement of the upper body, and across the right thigh to stabilize the active leg. Identical positions were used for each of the muscle strength and fatigue tests for a given individual.

The muscle strength test comprised three maximal isokinetic contractions, with muscle-generated work measured for each contraction. The muscle fatigue test involved contractions repeated every 1.5 s, at a target rate corresponding to 40% of the maximal work output per contraction, as previously determined during the muscle strength test. The test was continued until fatigue, defined as the failure to maintain 90% of the target work output for three successive contractions. A visual real-time display of the torque and work for each contraction was provided to the subjects during the maximal strength and muscle fatigue tests. Verbal support was provided to encourage subjects to exert maximal torque during the muscle strength test, as well as to maintain the appropriate work and kicking frequency during the muscle fatigue test.

Muscle biopsy sampling

A muscle biopsy was taken at rest, immediately following the invasive muscle fatigue test, and at 3 h and 24 h postexercise. A local anaesthetic (1% xylocaine) was injected into the skin and subcutaneous tissue above the right vastus lateralis muscle, a small incision was made through the skin and fascia, and a muscle sample of approximately 120 mg was then excised using a Stille needle. Samples were immediately frozen in liquid N₂ until assayed later for Na⁺,K⁺-ATPase isoform mRNA and protein expression.

Real-time RT-PCR measurement of mRNA

Total RNA was extracted from 5 to 10 mg muscle using the FastRNA reagents (BIO 101, Vista, CA, USA) using methods previously employed in our laboratory (Murphy et al. 2001, 2003; Cameron-Smith et al. 2003). The resulting RNA pellet was dissolved in EDTA-treated water and stored at –80°C. Total RNA concentration was determined spectrophotometrically at 260 nm. For each sample, 1 µg of RNA was transcribed into cDNA using the Promega AMV Reverse Transcription Kit (kit A3500; Promega, Madison, WI, USA) as previously described (Murphy et al. 2001, 2003), and the resulting cDNA was stored at –20°C for subsequent analysis. Real Time-PCR (GeneAmp 5700 Sequence Detection System, Applied Biosystems, Foster City, CA, USA) 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). Fluorescence resulted from incorporation of SYBR Green (SYBR Green Master Mix, Applied Biosystems, Foster City, MA, USA) to double stranded DNA and this fluorescence was measured after each repetitive cycle. Triplicate wells were run for each sample. Measurements included a no-template control, as well as a human muscle sample endogenous control, cyclophilin (CYC). Primer sequences were designed from published sequences (Table 1), where possible spanning exon boundaries to minimize contaminant DNA amplification. Gene expression was quantified from fluorescence emission using a cycle threshold (C_T) method. The relative expression of the genes compared with resting samples was made using the expression, 2^–C_T, in which the expression of each gene was normalized for input cDNA using the housekeeping gene CYC Exercise had no significant effect on the mRNA expression of CYC, when expressed in the linear (2^–C_T) form (data not shown). The intra-assay coefficient of variation for each target gene was < 15.0% for 2^–C_T (Table 2), which is within values previously reported (Murphy et al. 2003). Muscle mRNA are presented for 14 subjects (7 males, 7 females), due to insufficient sample for one subject.

View this table: [in this window] [in a new window]   Table 1.  Na+,K+-ATPase gene 1–3 and ß1–ß3 and CYC primer sequences used for mRNA analyses

Immunoblotting methods were similar to those previously described (Murphy et al. 2001; Lee et al. 2002). Muscle samples (20–30 mg) were homogenized for 15 s at a speed rating of 4 (Polytron PT1200; Kinematica, Luzern, Switzerland) on ice in a 1: 40 dilution with extraction buffer (25 mM Tris-HCl, pH 6.8, 1% sodium dodecyl sulphate (SDS), 5 mM EGTA, 50 mM NaF, 1 mM sodium vanadate, 10% glycerol, 17.4 µg ml^–1 phenylmethylsulphonyl fluoride (PMSF), 10 µg ml^–1 leupeptin and 1 µg ml^–1 aprotinin). Studies utilizing repeated centrifugation of muscle and membrane separation result in very low recovery of Na⁺,K⁺-ATPase enzymes, thereby yielding a final sample that may be unrepresentative of the whole muscle Na⁺,K⁺-ATPase population (Hansen & Clausen, 1988). Therefore, muscle sample analyses did not include any membrane isolation steps, to maximize recovery of Na⁺,K⁺-ATPase enzymes. A portion of each sample was heated for 10 min at 90°C, and analysed for total protein content (BCA Assay Kit, Pierce, Rockford, IL, USA), with bovine serum albumin (BSA) as the standard. The remaining samples were frozen at –80°C for immunoblotting.

SDS-PAGE (10% separating gel, 5% stacking gel) was performed and gels were loaded with 20 (ß₁) or 70 (₁, ₂, ₃, ß₂, ß₃) µg protein. Following electrophoresis (20 min, 100 V and 90 min, 150 V), the protein was transferred (90 min, 100 V) to 0.45 µm nitrocellulose membrane, and blocked for 2 h with blocking buffer (5% non-fat milk in Tris-buffered saline 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 (HRP)-conjugated secondary antibodies (goat antimouse immunoglobulins or goat antirabbit 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, USA). The signal was captured and imaged (Kodak Digital Science Image Station 400_CF, Eastman Kodak Company, CT, USA). Positive control samples included rat brain and kidney homogenates and these were run on each gel to assess the reactivity and specificity of the antibody (see below). The linearity of the blot signal versus protein loaded for our experimental conditions was established for each antibody.

Preparation of cell cultures

To verify that Na⁺,K⁺-ATPase isoform expression in the crude homogenate preparation was unlikely to be a contaminant of vascular or nervous tissues, adipocytes or fibrocytes, we also investigated isoform expression in a primary human skeletal muscle cell culture. The sample (85 mg) was washed 3 times in serum-free medium to remove blood and then minced finely using a scalpel. A 15 ml solution of 0.05% trypsin and 0.53 mM EDTA was added to the muscle then transferred to a sterile flask, and following shaking at low speed for 20 min at room temperature, the supernatant was collected and placed on ice. This procedure was repeated twice more, the supernatants pooled and fetal bovine serum (FBS) was added at a final concentration of at least 10%. The supernatant was filtered through a 100 µm cell filter to remove any connective tissue, and centrifuged at room temperature. The supernatant was removed and the cell pellet resuspended in 5 ml growth medium containing 10% fetal calf serum. Cells were then seeded onto an uncoated 25 cm² flask to remove fibroblasts, and incubated for 20 min at 37°C. The medium containing myoblasts was transferred to an extracellular matrix (ECM)-coated 25 cm² flask, with the cells cultured at 37°C and 5% CO₂. The following day, the medium was changed once, and thereafter twice per week until 60–70% confluent. Cells were seeded into 6-well plates at a density of 100 000–150 000 cells per well and incubated in growth medium until they reached 80% confluency. Cells were then incubated in differentiation medium containing 2% horse serum for 4–5 days and prepared for immunoblotting as described above.

Antibodies

Blots were probed with antibodies specific to each isoform. These were for ₁: monoclonal 6F (developed by D. Fambrough and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA, USA); ₂: polyclonal anti-HERED (kindly donated by T. Pressley, Texas Tech University); ₃: monoclonal MA3-915 (Affinity Bioreagents, Golden, CO, USA); ß₁: monoclonal MA3-930 (Affinity Bioreagents); ß₂: polyclonal 610915 (Transduction Laboratories, Lexington, KY, USA); and ß₃: polyclonal 610993 (Transduction Laboratories). For comparative purposes, additional polyclonal antibodies used by other researchers who did not detect ₃ or ß₂ in human muscle (Hundal et al. 1994; Juel et al. 2000) were used for the ₃ and ß₂ isoforms; these antibodies were for ₃: 06-172 (Upstate Biotechnology, Lake Placid, NY, USA); ß₂: 06-171 (Upstate Biotechnology); and ß₃: 06-817 (Upstate Biotechnology).

BLAST analysis demonstrated that the antigen sequence of each of the antibodies employed did not cross-react with any other non-Na⁺,K⁺-ATPase proteins. Isoform specificity of the monoclonal antibodies specific to ₁ (6F) and ₃ (MA3-915) used in the present study has previously been established in control samples including rat kidney and rat brain homogenates (Arystarkhova & Sweadner, 1996). Consequently we ran rat kidney and rat brain homogenates as control samples on each gel. Rat kidney and rat brain have previously been reported to express the ₁ and the ₁–₃ isoforms, respectively (Hundal et al. 1994). Since the ₂ and ₃ isoforms are both expressed in rat brain, but not rat kidney, to differentiate between ₂ and ₃ we required further evidence of antibody specificity. BLAST analysis of the antigen sequence for each of the ₂ (anti-HERED) and ₃ (MA3-915) antibodies did not cross-react with the amino acid sequence of the ₃ and ₂ isoforms, respectively. Therefore it is unlikely that the antibodies used for ₁–₃ isoforms would demonstrate cross-reactivity. BLAST analysis of the antigen sequence for the antibody specific to the ß₁ (MA3-930) isoform indicated no cross-reactivity with the ß₂ or ß₃ isoforms. However, there was a 41% shared sequence identity between the antigen sequence of the antibodies specific to ß₂ (Transduction Laboratories 610915) and ß₃ (Transduction Laboratories 610993) and the ß₃ and ß₂ isoforms, respectively. Rat kidney and rat brain samples cannot be used as controls as both samples wouldn't necessarily differentiate between the ß₂ and ß₃ isoforms (Martin-Vasallo et al. 1989; Hundal et al. 1994; Malik et al. 1996). Thus, whilst unlikely, it is possible that the antibodies specific to ß₂ and ß₃ may cross-react with the other isoform.

Statistical analysis

All data are presented as means ±S.E.M., except population statistics where means ±S.D. are reported. Muscle data were analysed using a one-way repeated measures ANOVA, with Newman-Kuels post hoc analyses. To account for individual variability in time responsiveness of mRNA to exercise, the average postexercise mRNA was calculated as the mean of the fatigue, 3 h and 24 h recovery samples for each individual. This average was contrasted against the resting value and analysed using Student's t test for paired-samples. Correlations were determined by least squares linear regression. Significance was accepted at P < 0.05.

	Results

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Exercise results

Incremental exercise was 50.5 ± 2.8 ml kg^–1 min^–1. Maximal work performed per contraction during the maximal knee extensor muscle strength test was 164 ± 21 J. During the muscle fatigue test, time to fatigue was 352 ± 69 s, work performed per contraction was 66 ± 5 J, and 233 ± 46 contractions were performed.

Muscle Na⁺,K⁺-ATPase mRNA and protein expression in crude muscle homogenates

Real-Time RT-PCR analysis demonstrated amplification of each of the primer sets specific to each of the Na⁺,K⁺-ATPase ₁, ₂, ₃, ß₁, ß₂ and ß₃ isoforms (data not shown), no amplification of the no template control samples, and the heat dissociation curve confirmed amplification of only a single gene transcript for each primer set (data not shown). These results indicate the presence of gene transcripts for each of the Na⁺,K⁺-ATPase ₁, ₂, ₃, ß₁, ß₂ and ß₃ isoforms in human vastus lateralis muscle.

Crude muscle homogenates demonstrated protein bands of apparent molecular mass (₁–₃, 100–105 kDa; ß₁–ß₃, 45–52 kDa) specific to each of the ₁, ₂, ₃, ß₁, ß₂ and ß₃ subunit isoforms. Representative immunoblots are shown in Fig. 1. These results indicate protein expression of each of the ₁–₃ and ß₁–ß₃ isoforms in human muscle.

View larger version (59K): [in this window] [in a new window]   Figure 1.  Representative immunoblots of Na+,K+-ATPase 1, 2, 3, ß1, ß2 and ß3 isoforms in crude muscle homogenates of the human vastus lateralis muscle Values at left indicate molecular weight of bands.

Na⁺,K⁺-ATPase protein expression in human cell cultures

Immunoblots performed on crude homogenates from primary human skeletal muscle cell culture also expressed protein bands of the apparent specific subunit motility (₁–₃, 100–105 kDa; ß₁–ß₃, 45–52 kDa) for each of the Na⁺,K⁺-ATPase ₁, ₂, ₃, ß₁, ß₂ and ß₃ isoforms (Fig. 2).

View larger version (69K): [in this window] [in a new window]   Figure 2.  Representative immunoblots of Na+,K+-ATPase 1, 2, 3, ß1, ß2 and ß3 isoforms in crude homogenates of primary human skeletal muscle cell cultures derived from vastus lateralis muscle Values at left indicate molecular weight of bands.

subunit isoforms.  Exercise elevated ₁ mRNA expression by 2.5-fold at 24 h postexercise (P < 0.05), but had no effect on crude muscle homogenate ₁ protein expression (Fig. 3). The ₂ isoform mRNA expression increased by 3.5-fold at 3 h postexercise (P < 0.05), then returned to resting levels by 24 h postexercise (P < 0.05, Fig. 4). In contrast, exercise had no effect on ₂ protein expression (Fig. 4). Immediately after the cessation of exercise, ₃ mRNA expression increased by 2.4-fold (P < 0.05), then declined significantly by 24 h postexercise (P < 0.05, Fig. 5). A trend to elevated ₃ protein expression at 3 h postexercise was seen (P < 0.06, Fig. 5).

View larger version (26K): [in this window] [in a new window]   Figure 3.  Na+,K+-ATPase 1 isoform mRNA and protein expression before and after acute one-legged knee extensor exercise Na+,K+-ATPase 1 isoform mRNA and crude muscle homogenate protein expression at rest and immediately following fatigue (Fat), and 3 h (+3 h) and 24 h (+24 h) after acute intense exercise. All results were normalized against resting values. Inset shows representative immunoblot of 1 protein expression at corresponding times. Data are means ±S.E.M.; n= 14 for mRNA, n= 15 protein. *P < 0.05 greater than rest.

View larger version (27K): [in this window] [in a new window]   Figure 4.  Na+,K+-ATPase 2 isoform mRNA and protein expression before and after acute one-legged knee extensor exercise Na+,K+-ATPase 2 isoform mRNA and crude muscle homogenate protein expression at rest and immediately following fatigue (Fat), and 3 h (+3 h) and 24 h (+24 h) after acute intense exercise. All results were normalized against resting values. Insert shows representative immunoblot of 2 protein expression at corresponding times. Data are means ±S.E.M.; n= 14 for mRNA, n= 15 protein. *P < 0.05 greater than rest; P < 0.05 greater than +24 h.

View larger version (26K): [in this window] [in a new window]   Figure 5.  Na+,K+-ATPase 3 isoform mRNA and protein expression before and after acute one-legged knee extensor exercise Na+,K+-ATPase 3 isoform mRNA and crude muscle homogenate protein expression at rest and immediately following fatigue (Fat), and 3 h (+3 h) and 24 h (+24 h) after acute intense exercise. All results were normalized against resting values. Insert shows representative immunoblot of 3 protein expression at corresponding times. Data are means ±S.E.M.; n= 14 for mRNA, n= 15 protein. *P < 0.05 greater than rest; P < 0.05 greater than +24 h.

View larger version (25K): [in this window] [in a new window]   Figure 6.  Na+,K+-ATPase ß1 isoform mRNA and protein expression before and after acute one-legged knee extensor exercise Na+,K+-ATPase ß1 isoform mRNA and crude muscle homogenate protein expression at rest and immediately fatigue (Fat), and 3 h (+3 h) and 24 h (+24 h) after acute intense exercise. All results were normalized against resting values. Insert shows representative immunoblot of ß1 protein expression at corresponding times. Data are means ±S.E.M.; n= 14 for mRNA, n= 15 protein.

View larger version (26K): [in this window] [in a new window]   Figure 7.  Na+,K+-ATPase ß2 isoform mRNA and protein expression before and after acute one-legged knee extensor exercise Na+,K+-ATPase ß2 isoform mRNA and crude muscle homogenate protein expression at rest and immediately following fatigue (Fat), and 3 h (+3 h) and 24 h (+24 h) after acute intense exercise. All results were normalized against resting values. Insert shows representative immunoblot of ß2 protein expression at corresponding times. Data are means ±S.E.M., n= 14 for mRNA, n= 15 protein. *P < 0.05 greater than rest; P < 0.05 greater than +24 h.

View larger version (30K): [in this window] [in a new window]   Figure 8.  Na+,K+-ATPase ß3 isoform mRNA and protein expression before and after acute one-legged knee extensor exercise Na+,K+-ATPase ß3 isoform mRNA and crude muscle homogenate protein expression at rest and immediately following (fatigue (Fat), and 3 h (+3 h) and 24 h (+24 h) after acute intense exercise. All results were normalized against resting values. Insert shows representative immunoblot of ß3 protein expression at corresponding times. Data are means ±S.E.M., n= 14 for mRNA, n= 15 protein.

When investigating mRNA expression for each individual, it became apparent that exercise induced individual variability in isoform mRNA time responsiveness. To further investigate this observation, the average postexercise expression of the Na⁺,K⁺-ATPase ₁–₃ and ß₁–ß₃ mRNA was determined. As demonstrated in Fig. 9, for each of the six isoforms, exercise induced elevated mRNA expression in 10 or 12 of the 14 subjects. Indeed, when the group mean results were analysed, the average postexercise response in mRNA expression of the ₁, ₂, ₃, ß₁, ß₂ and ß₃ isoforms was 1.4-, 2.2-, 1.4-, 1.1-, 1.0- and 1.0-fold above resting values, respectively (all P < 0.05, Fig. 9). The relative change in mRNA expression from rest to average postexercise expression for each of the Na⁺,K⁺-ATPase ₁, ₂, ₃, ß₁, ß₂ and ß₃ isoforms were not significantly correlated with age, gender, or exercise time to fatigue (data not shown).

View larger version (31K): [in this window] [in a new window]   Figure 9.  Average postexercise response in Na+,K+-ATPase mRNA expression Individual expression of 1, 2, 3, ß1, ß2 and ß3 mRNA at rest and the average expression following acute intense exercise (average of fatigue, 3 h and 24 h-values). All symbols joined by lines indicate individual responses, while symbol on right indicates group average postexercise expressed as means ±S.E.M.n= 14. *Mean P < 0.05 greater than rest.

	Discussion

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Presence of Na⁺,K⁺-ATPase ₁-₃ and ß₁-ß₃ isoform gene transcripts and proteins

We provide novel characterization of the six Na⁺,K⁺-ATPase gene transcripts present in human vastus lateralis muscle. Our findings demonstrate expression of the previously uninvestigated ₃ and ß₂ gene transcripts, and verify expression of the ₁, ₂, ß₁ and ß₃ gene transcripts (Malik et al. 1998; Nordsborg et al. 2003).

This is also the first study to demonstrate expression of each of these six Na⁺,K⁺-ATPase isoform (₁–₃, ß₁–ß₃) proteins in human skeletal muscle. Our finding of three and three ß isoforms in human skeletal muscle greatly extends previous work. Whilst the presence of the ₁, ₂ and ß₁ proteins is consistent with previous studies (Hundal et al. 1994; Juel et al. 2000), our finding of both the ₃ gene transcript and protein resolves the apparent conflict regarding the presence of ₃ in human muscle (Hundal et al. 1994; Wang et al. 2001). We also confirmed detection of the ₃ isoform using the polyclonal antibody utilized by Hundal et al. (1994), but found that the immunoreactive band was of poor quality. The ß₂ protein had previously been undetected in human soleus and vastus lateralis muscle (Hundal et al. 1994; Juel et al. 2000). However, we detected the ß₂ protein using two separate antibodies, including those employed by previous studies that did not detect the ß₂ isoform (Hundal et al. 1994; Juel et al. 2000). Thus, a difference in antibody reactivity cannot explain our differing results. Rather, this discrepancy might be explained on the basis of ß₂ isoform localization. Importantly, the present study utilized crude muscle homogenates, which include all intracellular and sarcolemmal membranes, whilst the previous studies utilized separated membranes or membrane fractions (Hundal et al. 1994; Juel et al. 2000). However, Juel et al. (2000) also utilized a purified homogenate and did not detect the ß₂ protein in human muscle. Whilst the ß₃ protein had not been previously investigated, we also confirmed expression of the ß₃ protein with an additional polyclonal antiß₃ antibody, although the reactivity was weaker than that with the initial antibody. Therefore, when isoform composition of all membranes in human skeletal muscle were investigated via a crude homogenate, it is clear that human and rat muscle similarly express the Na⁺,K⁺-ATPase ₁–₃ and ß₁–ß₂ gene transcripts (Orlowski & Lingrel, 1988; Tsakiridis et al. 1996), and the ₁–₂ and ß₁–ß₃ proteins (Hundal et al. 1993; Tsakiridis et al. 1996; Arystarkova & Sweadner, 1997).

The specificity of our antibodies was assessed on three accounts. First, the apparent molecular masses of the protein bands detected with antibodies specific to each of the ₁, ₂, ₃, ß₁, ß₂ and ß₃ isoforms corresponded with those previously reported in mammalian skeletal muscle (Hundal et al. 1994; Arystarkova & Sweadner, 1997; Juel et al. 2000). Second, control samples including rat kidney and rat brain homogenates were run on each gel and supported previous observations of isoform-specific expression (Martin-Vasallo et al. 1989; Hundal et al. 1994; Malik et al. 1996). Third, BLAST analysis of the antigen sequence of each antibody was performed to evaluate isoform cross-reactivity. Whilst results from these assessments suggest that isoform cross-reactivity with each antibody would be unlikely, complete verification of isoform specificity will only be achieved with sequencing of the protein bands detected with each isoform-specific antibody.

The use of muscle tissue samples collected by biopsy raises the possibility that some of the six Na⁺,K⁺-ATPase isoforms detected may have originated from contamination by other tissues, such as vascular and nervous tissue, adipocytes or fibrocytes. For instance, human erythrocytes and reticulocytes express the ₁, ₃, ß₁, ß₂ and ß₃ proteins (Stengelin & Hoffman, 1997; Hoffman et al. 2002), while human leucocytes express the ₁, ₃, ß₁ and ß₃ gene transcripts (Stengelin & Hoffman, 1997). Rat adipocytes express both ₁ and ₂ isoforms (Lytton, 1985; Voldstedlund et al. 1993; Bofill et al. 1994; Sargeant et al. 1995), while ₁ and ß₁, but not ₂ and ß₂, isoforms were detected in mouse 3T3-L1 fibroblasts (Sargeant et al. 1995). Rat astrocytes and neurones express the ₁–₂ and ₃ isoforms, respectively (Blanco & Mercer, 1998). Importantly, each muscle sample from all individuals expressed all six isoforms at mRNA and protein levels. Whilst the likelihood of contamination by nervous tissue in each biopsy sample seems very low, blood contamination will certainly be present in all samples. We therefore also probed for Na⁺,K⁺-ATPase isoform expression in cell cultures derived from human vastus lateralis muscle, being careful to minimize any contamination. In the first comprehensive assessment of Na⁺,K⁺-ATPase isoform expression in human skeletal muscle cell cultures, we found expression of each of the ₁–₃ and ß₁–ß₃ proteins. Our results thus extend findings from a previous study which reported only ₁ and ₂ isoform expression in cultured human muscle cells (Al-Khalili et al. 2003). Although our cell culture results do not provide unequivocal verification, they do indicate that the novel expression of the ₃, ß₂ and ß₃ proteins in human muscle obtained via biopsy sampling, and indeed also the other isoforms, is unlikely to originate from contamination by other cell types.

Exercise effects on Na⁺,K⁺-ATPase isoform mRNA expression

An important finding was that all six of the Na⁺,K⁺-ATPase gene transcripts expressed in human skeletal muscle were elevated in response to only 6 min of exercise. Clear increases were found at specific time points for each of ₁, ₂, ₃ and ß₂ mRNA, whilst the average postexercise value was increased for both ß₁ and ß₃ mRNA. This differs from a recent study which reported that, 15 min of intense intermittent knee extensor exercise induced an elevation in only ₁ mRNA expression (Nordsborg et al. 2003). One reason for this discrepancy is that their study was limited by a low statistical power for ₂ and ß₁ mRNA, whilst ₃, ß₂ and ß₃ mRNA were not probed (Nordsborg et al. 2003). Large inter- and intraindividual variability was observed for Na⁺,K⁺-ATPase isoform mRNA expression, similar to numerous other genes (Boivin et al. 2000; Hameed et al. 2003; Psilander et al. 2003). The cause of such variability has not been established, and in the present study, was not significantly correlated to physical characteristics such as age or sex, or physiological factors underpinning exercise , and time to fatigue.

The observed increases in isoform mRNA expression following exercise may reflect increased transcription, reduced mRNA degradation or enhanced mRNA stability. Although the present study could not evaluate these mechanisms, the rapid increase in Na⁺,K⁺-ATPase ₁ and ß mRNA expression induced by veratridine in cultured chick skeletal muscle cells was due to elevated rates of gene transcription (Taormino & Fambrough, 1990). The mechanisms responsible for Na⁺,K⁺-ATPase gene activation with exercise also remain unknown, but may involve elevated intracellular concentrations of Na⁺ (Wolitzky & Fambrough, 1986) and/or Ca²⁺ (Rayson, 1991).

An important limitation of our methods utilized to assess mRNA expression is that they do not discriminate between cell types. Whilst highly unlikely, contamination of our mRNA samples by cell types other than skeletal muscle is possible.

Exercise effects on Na⁺,K⁺-ATPase isoform protein expression

In contrast to the up-regulation in mRNA expression, a single bout of one-legged knee extensor exercise had no significant effect on protein expression of any of the six Na⁺,K⁺-ATPase isoforms expressed in human skeletal muscle homogenate. These results clearly indicate that increases in mRNA were not matched by increases in Na⁺,K⁺-ATPase protein abundance, highlighting the separate and possibly independent regulation of protein translation and protein stability (Orphanides & Reinberg, 2002). It is known that repeated bouts of exercise induce a training effect of an increased [³H]ouabain binding, representing an increased total Na⁺,K⁺-ATPase content (Green et al. 1993; McKenna et al. 1993). The transient accumulation of Na⁺,K⁺-ATPase mRNA induced by exercise appears to be the initial step of this adaptive response. Interestingly, this initial response does not appear to be isoform-specific, as mRNA for all six isoforms were increased postexercise. The lack of increase in Na⁺,K⁺-ATPase isoform protein observed here suggests that the exercise challenge that we employed was insufficient to induce Na⁺,K⁺-ATPase up-regulation, which may require many hours of continuous exercise (Overgaard et al. 2002), or repeated exercise bouts (Green et al. 1993).

Expression of three (₁–₃) and three ß isoforms (ß₁–ß₃) in human muscle raises the possibility of nine different ß heterodimers in human skeletal muscle, with implications for diverse functions. Expression of three isoforms in human skeletal muscle also has implications for interpretation of [³H]ouabain binding, which quantifies total Na⁺,K⁺-ATPase content (Nørgaard et al. 1984; Clausen, 1996). In human kidney, heart and brain, all three isoforms have similar affinities for ouabain (Wang et al. 2001). If similar affinities are apparent in skeletal muscle, our results imply that the [³H]ouabain binding assay in human muscle measures the combined content of functional Na⁺,K⁺-ATPase ₁ß, ₂ß and ₃ß heterodimers. Finally, expression of six Na⁺,K⁺-ATPase isoforms in human skeletal muscle may have important implications for the proposed isoform-specific translocation with exercise (Tsakiridis et al. 1996; Juel et al. 2000; Juel et al. 2001) and insulin (Hundal et al. 1992; Marette et al. 1993). These previous studies have failed to probe for the full complement of Na⁺,K⁺-ATPase isoforms, which may challenge the reported specificity of any isoform translocation. Moreover, we have recently challenged the concept of isoform translocation as being a quantitatively important means of increasing Na⁺,K⁺-ATPase transport in rat skeletal muscle, failing to detect an increase in [³H]ouabain binding in response to either muscle contractions or insulin (McKenna et al. 2003).

In conclusion, each of the Na⁺,K⁺-ATPase ₁–₃ and ß₁–ß₃ isoforms is expressed in the vastus lateralis muscle of healthy humans, as evidenced at both the transcription and protein levels. Further, only 6 min of intense exercise was sufficient to increase the mRNA expression of each of these six isoforms, evidenced by increases either at specific time points or in the average postexercise value, suggesting a non-specific up-regulatory transcription response of Na⁺,K⁺-ATPase isoforms. In contrast, this exercise bout had no effect on isoform protein abundance, suggesting this was an insufficient stimulus for Na⁺,K⁺-ATPase translational up-regulation in muscle.

	References

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