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Am J Physiol Regul Integr Comp Physiol 281: R917-R925, 2001;
0363-6119/01 $5.00
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Vol. 281, Issue 3, R917-R925, September 2001

The alpha 1- and alpha 2-isoforms of Na-K-ATPase play different roles in skeletal muscle contractility

Suiwen He1,*, Daniel A. Shelly2,*, Amy E. Moseley1, Paul F. James1, J. Howard James3, Richard J. Paul2, and Jerry B. Lingrel1

1 Department of Molecular Genetics, Biochemistry, and Microbiology and 2 Department of Molecular and Cellular Physiology and 3 Department of Surgery, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The Na-K-ATPase, which maintains the Na+ and K+ gradients across the plasma membrane, can play a major role in modulation of skeletal muscle contractility. Although both alpha 1- and alpha 2-isoforms of the Na-K-ATPase are expressed in skeletal muscle, the physiological significance of these isoforms in contractility is not known. Evaluation of the contractile parameters of mouse extensor digitorum longus (EDL) was carried out using gene-targeted mice lacking one copy of either the alpha 1- or alpha 2-isoform gene of the Na-K-ATPase. The EDL muscles from heterozygous mice contain approximately one-half of the alpha 1- or alpha 2-isoform, respectively, which permits differentiation of the functional roles of these isoforms. EDL from the alpha 1+/- mouse shows lower force compared with wild type, whereas that from the alpha 2+/- mouse shows greater force. The different functional roles of these two isoforms are further demonstrated because inhibition of the alpha 2-isoform with ouabain increases contractility of alpha 1+/- EDL. These results demonstrate that the Na-K-ATPase alpha 1- and alpha 2-isoforms may play different roles in skeletal muscle contraction.

sodium-potassium-adenosinetriphosphatase; extensor digitorum longus muscle; ouabain; muscle fatigue


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE NA-K-ATPASE is an integral membrane protein that uses the energy from ATP hydrolysis for the countertransport of three Na+ and two K+ across the plasma membrane. This enzyme is critical for maintaining the resting membrane potential and osmotic balance of cells central to the excitability of muscle and nerve tissues. The Na+ gradient is also the driving force for numerous Na+-dependent secondary processes, including transport of amino acids, Ca2+, and other nutrients.

The Na-K-ATPase is a heteromeric enzyme composed of two polypeptide subunits: a 112-kDa alpha -subunit and a 40- to 60-kDa glycosylated beta -subunit (reviewed in Ref. 28). The alpha -subunit contains the cation- and ATP-binding sites to perform the catalytic and transport activity of the enzyme. The beta -subunit is involved in the modulation of the Na+ and K+ affinity of the enzyme (10, 11). There are distinct genes encoding multiple isoforms of both alpha - and beta -subunits in various species. Four alpha -subunits (alpha 1, alpha 2, alpha 3, and alpha 4) and three beta -subunits (beta 1, beta 2, and beta 3) have been found (16, 29, 37-39, 41). The alpha 1-isoform is expressed in nearly every tissue, suggesting a possible housekeeping role for this isoform. In contrast, expression of alpha 2-, alpha 3-, and alpha 4-isoforms is restricted to several tissues: alpha 2 in skeletal muscle, smooth muscle, heart, and brain (glial cells); alpha 3 in brain (neurons) and ovary; and alpha 4 in sperm. The unique pattern of expression suggests specific functional roles for these individual isoforms.

Among the four alpha -isoforms of the Na-K-ATPase, differences in cation affinity, ATP affinity, and other kinetic parameters of the enzymatic activity have been noted. Discrepancies in these parameters have been reported, depending on the various tissues and species (Refs. 43 and 45; reviewed in Ref. 4). The major biochemical difference among the four isoforms is their affinities to cardiac glycosides such as ouabain. In rodents, the affinity of the alpha 2- and alpha 3-isoforms for ouabain is 1,000-fold greater than the alpha 1-isoform (12, 42). Because multiple alpha -isoforms of the Na-K-ATPase display a tissue-specific expression pattern that is generally consistent across various species, the functional relevance and physiological significance of such expression patterns have been investigated in certain tissues such as heart and sperm (23, 47). However, little work has been done in skeletal muscle.

In skeletal muscle, excitability and contractility depend on the membrane potential and the Na+ and K+ gradients across the plasma membrane. During action potentials, Na+ enters the cell in the depolarization phase and K+ leaves the cell in the repolarization phase. The alteration of Na+ and K+ gradients across the plasma membrane is restored by the Na-K-ATPase. If the rate of passive leakage of Na+ and K+ through ion channels exceeds the rate of active ion transport by the Na-K-ATPase, the excitability of the skeletal muscle fibers will decrease, contributing to contractile fatigue. Alteration of skeletal muscle Na-K-ATPase activity has been observed in various physiological and pathophysiological states, such as thyroid hormone excess (reviewed in Ref. 6) or deficiency (1), training and inactivity (33, 36, reviewed in Ref. 6), K+ deficiency (31), and diabetes (26). In many studies, inhibition of the Na-K-ATPase results in perturbation of Na+ and K+ concentration both intracellularly and extracellularly. Accumulation of K+ in the extracellular space, especially around the T-tubule system, depolarizes the plasma membrane and makes the muscle cells unexcitable (7). The relationship of individual alpha -isoforms to muscle contractility has yet to be understood.

Using gene targeting to alter the expression levels of alpha 1- and alpha 2-isoforms of Na-K-ATPase in mice, we have previously demonstrated that these two isoforms have distinct physiological functions in heart (23). Hearts from mice lacking one copy of the alpha 2-isoform (alpha 2+/-) are hypercontractile. In contrast, genetic reduction of one copy of the alpha 1-isoform (alpha 1+/-) depresses cardiac contractility. To further define specific functional roles for the Na-K-ATPase alpha 1- and alpha 2-isoforms, we have analyzed skeletal muscle contractility in mice heterozygous for the alpha 1- and alpha 2-isoforms. We demonstrated here that under supramaximal stimulation conditions, the extensor digitorum longus (EDL) muscle from alpha 2+/- mice is capable of producing more force than wild type, whereas muscles from alpha 1+/- mice produce less force than wild type. These results, along with those from our previous work (23), clearly demonstrate unique roles for the Na-K-ATPase alpha 1- and alpha 2-isoforms in muscle contractility.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and genotypes. Male mice, 12-15 wk old, were used for all studies described. Heterozygous mice containing one copy of either the alpha 1- or alpha 2-isoform gene of the Na-K-ATPase were generated using gene-targeting methods described previously (23). Genomic DNA was extracted from mouse tails using Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN). To identify genotypes, isoform-specific probes for the respective alpha 1- and alpha 2-isoform gene were used in Southern blot analysis of genomic DNA (23). Mice were euthanized by CO2 inhalation followed by cervical dislocation, and EDL muscles from both legs were dissected. For preparation of crude microsomes, EDL muscles were frozen in liquid nitrogen for later use.

Intracellular Na+ and K+ contents. Measurement of intracellular Na+ and K+ was performed as described (22). Briefly, ions from the extracellular space were washed out by incubation in ice-cold Na+- and K+-free buffer. Muscles were homogenized in trichloroacetic acid followed by centrifugation to remove precipitated proteins. Na+ and K+ concentrations in the supernatant were measured with a Shimadzu AA-6200 atomic absorption spectrophotometer (Shimadzu Scientific Instruments, Columbia, MD). Ion concentration was expressed as micromoles per gram wet weight.

Preparation of crude microsomes. For each microsomal preparation, 20-30 EDL muscles from each genotype (wild type, alpha 1+/-, and alpha 2+/-) were homogenized on ice with two 30-s bursts of a Polytron homogenizer in 5 ml homogenization buffer [250 mM sucrose, 30 mM imidazole (pH 7.5), and 1 mM EDTA]. The homogenates were centrifuged at 3,000 g for 15 min at 4°C to remove cellular debris. The supernatants were centrifuged at 115,000 g for 60 min at 4°C. The pellets were resuspended in 30 mM imidazole buffer (pH 7.5) and 1 mM EDTA and centrifuged again at 115,000 g for 60 min. The pellets were finally resuspended into 250 µl of imidazole and EDTA buffer. Protein concentrations were measured using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Aliquots of microsomal preparations were stored at -80°C for Western blot analysis.

Quantitation of Na-K-ATPase isoform levels. Western blot analysis was used to quantitate protein levels of the Na-K-ATPase alpha -isoforms in EDL. Microsomal proteins were separated using SDS-PAGE as described (27). To facilitate detection of the glycosylated beta -subunits, sugar residues were removed with recombinant N-glycanase kit following manufacturer's direction (Glyko, Novato, CA). Samples were incubated for 30 min at 37°C in sample buffer containing 50 mM Tris (pH 6.9), 5% SDS, 1% beta -mercaptoethanol, and 10% glycerol and electrophoresed through 10% polyacrylamide gels. The separated proteins were transferred to polyvinylidene difluoride membranes (Amersham Life Science, Piscataway, NJ) overnight at 4°C. Membranes were blocked with 2% BSA in TBST solution [5 mM Tris (pH 7.4), 150 mM NaCl, and 0.05% Tween 20] for 1 h at room temperature and then incubated in TBST containing individual primary antibodies overnight at 4°C as follows: alpha 1-isoform-specific monoclonal alpha 6f, 1:1,000 (University of Iowa Developmental Hybridoma Bank, Iowa City, IA) (44); alpha 2-isoform-specific monoclonal McB2, 1:500 (generously provided by Dr. Kathleen Sweadner) (46); alpha 3-monoclonal antibody, 1:1,000 (Affinity Bioreagents, Golden, CO); polyclonal alpha -KETYY, 1:10,000 (generously provided by Dr. Jack Kyte); beta 1-polyclonal SpETB1, 1:1,500 (17); beta 2-polyclonal SpETB2, 1:1,500 (17); and beta 3-polyclonal RNTB3, 1:2,000 (generously provided by Dr. Kathleen Sweadner). The membranes were incubated overnight at 4°C in TBST containing 1:20,000 peroxidase-conjugated secondary antibodies (Calbiochem, La Jolla, CA). The membranes were processed using enhanced chemiluminescence system (Amersham Life Science) and visualized with Kodak BioMax MR X-ray film. Signal intensities were quantified by densitometry using ImageQuant software. Two microsomal preparations were analyzed for EDL muscles of each genotype. Samples containing various amounts of microsomal protein were analyzed in a single blot, and multiple exposures of the blot were obtained to ensure linearity of signal intensity.

Measurement of EDL contractility. After the resting length of each muscle was measured in situ, dissected muscles were mounted vertically in a sealed cylindrical chamber fitted with a magnetic stirrer. By means of a small plastic ring tied to each tendon of EDL with surgical silk, muscles were mounted at one end to a fixed stainless steel post. The other end was fixed to the lever of an isometric force transducer (Kistler Morse, Redmond, WA), and the muscle length was adjusted to produce a passive tension of 5 mN. Muscles were incubated in sterile Krebs solution (in mM: 118 NaCl, 4.7 KCl, 25 NaHCO3, 2.5 CaCl2, 1.2 MgSO4, 1.2 NaH2PO4, 0.026 EDTA, 11 glucose equilibrated with 95% CO2-5% O2) at room temperature (~22°C). Muscles were electrically stimulated via two platinum electrodes positioned along either side of the muscle.

Supramaximal voltage was determined empirically using a series of short (1.5 s) tetani and subsequently increasing the voltage for each tetani. Similarly, supramaximal frequency of stimulation was determined. Supramaximal electrical stimuli employed capacitor discharges of equal but alternating polarity (66 Hz at 15 V, 15-ms duration, circuit rate constant, 5 ms). Experiments consisted of a set (~5) of twitches and a set (2-4) of tetani with duration of 3-7 s. Digital recordings of force were obtained with the BioPac data-acquisition system (BioPac System, Goleta, CA). Measurements of force were normalized to muscle cross-sectional area estimated as muscle weight divided by muscle length for comparison among genotypes (32). Twitch and tetanus parameters were analyzed by ANOVA or Student's t-test using the SigmaStat statistical software package (Jandel Software, San Rafael, CA).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Quantitative analysis of alpha 1- and alpha 2-protein levels in EDL. EDL, a fast-twitch skeletal muscle, was selected because of ease of dissection and its small size such that it is not diffusion limited for oxygen. The lack of either the alpha 1- or alpha 2-isoform gene in alpha 1+/- or alpha 2+/- animals was expected to decrease the amount of the respective isoform by 50%. SDS-PAGE and quantitative Western blot analysis were used to quantitate levels of alpha 1- and alpha 2-proteins in EDL (Fig. 1A). The protein level of the alpha 1-isoform decreased by 48% in alpha 1+/- EDL (Fig. 1B) and that of the alpha 2-isoform decreased by 46% in alpha 2+/- EDL (Fig. 1C), close to the expected 50%. Interestingly, the alpha 1-protein level increased by 39% in alpha 2+/- EDL (Fig. 1B), while the alpha 2-protein level was not altered in alpha 1+/- EDL (Fig. 1C).


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  		Fig. 1.   Western blot analysis of the alpha -isoforms of Na-K-ATPase in extensor digitorum longus (EDL). A: microsomal proteins were separated using SDS-PAGE, and the gels were transferred to polyvinylidene difluoride (PVDF) membranes for immunoblotting. Primary antibodies of alpha 6F (1:1,000), Mcb2 (1:500), and alpha -KETYY (1:10,000) were used to detect the alpha 1-, alpha 2-, and both alpha -isoforms, respectively. Different amounts of EDL samples were loaded (150, 75, and 37.5 µg, from left to right, for each genotype). For NIH/3T3 cells, 2.5, 5, and 10 µg of proteins were loaded (left to right). WT, wild type. B: signal intensities from A were quantitated by densitometry. The amount of alpha 1- and alpha 2-protein in alpha 1+/- EDL was normalized to that of the WT and expressed as a percentage. C: amount of alpha 1- and alpha 2-protein in alpha 2+/- EDL was quantitated as in B. D: signal intensities from immunoblots using alpha 6F and alpha -KETYY in A were used to calculate the percentages of alpha 1- and alpha 2-isoforms in WT, alpha 2+/-, and alpha 1+/- EDL. Values are means ± SE (n = 8).

Besides quantitative analysis of individual alpha -isoforms in EDL from alpha 1+/- and alpha 2+/- mice, Western blot analysis was also used to quantitate the relative amounts of the alpha 1- and alpha 2-isoforms in EDL by the method developed by our laboratory (24). Because monoclonal antibodies used above exhibit different binding affinities to the alpha 1- and alpha 2-isoforms, the relative amounts of these two isoform proteins in EDL could not be directly assessed. To resolve this, we used a polyclonal antibody (alpha -KETYY) that recognizes an epitope common to all mouse alpha -isoforms, in conjunction with a standard that contains only the alpha 1-isoform and the isoform-specific monoclonal antibodies to characterize the relative amounts of each alpha -isoform protein in any sample. In this study, NIH/3T3 cells, a murine cell line that contains only the alpha 1-isoform, were used as a reference to calculate the relative amounts of alpha 1- and alpha 2-isoforms (Fig. 1A). In wild-type EDL, the alpha 2-isoform was predominant, accounting for 87% of total alpha -isoform of Na-K-ATPase, whereas the alpha 1-isoform accounted for only 13% (Fig. 1D). In alpha 2+/- EDL, the total amount of Na-K-ATPase alpha -isoforms was 66% of wild type, comprising 48% alpha 2- and 18% alpha 1-isoform (Fig. 1D). In alpha 1+/- EDL, the total amount of Na-K-ATPase alpha -isoforms was 93% of wild type, comprising 86% alpha 2-isoform and 7% alpha 1-isoform (Fig. 1D).

Western blot analysis was used to examine other Na-K-ATPase subunits in EDL. The protein levels of beta 1, beta 2, and beta 3 are similar among wild type, alpha 2+/-, and alpha 1+/- EDL (Fig. 2). The alpha 3-isoform, which is normally expressed in brain and ovary, is not expressed in either alpha 2+/- or alpha 1+/- EDL (Fig. 2). The results show that the total pool of other Na-K-ATPase subunits in the microsomal protein preparation was not altered because of decreased protein levels of alpha 1- or alpha 2-Na-K-ATPase.


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  		Fig. 2.   Western blot analysis of the beta - and alpha 3-subunits of Na-K-ATPase in EDL. Microsomal proteins were separated using SDS-PAGE, and the gels were transferred to PVDF membranes for immunoblotting. Primary antibodies of SpETB1 (1:1,500), SpETB2 (1:1,500), RNTB3 (1:2,000), and alpha 3 (1:1,000) were used to detect the beta 1-, beta 2-, beta 3-, and alpha 3-subunits, respectively. Different amounts of EDL samples were loaded (10, 20, and 40 µg, from left to right). For brain tissue as a positive control, 5 µg of protein was loaded.

EDL from alpha 1+/- and alpha 2+/- mice has distinct alterations in muscle contractility. The gross anatomy of EDL muscles from the wild-type, alpha 2+/-, and alpha 1+/- animals was examined for any alteration that might influence muscle contractility (Table 1). The weight and length of EDL from all three genotypes were similar. Cross-sectional area of muscle fiber, an important parameter used to normalize the contractile force, did not differ. Hypertrophy was not observed as assessed by the ratio of EDL weight to animal body weight. Because the Na-K-ATPase is important for controlling ionic levels in tissues, the concentrations of Na+ and K+ in EDL muscles were also measured, but no significant alteration was found.

                              
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  		Table 1.   Parameters of EDL muscle and intracellular Na+ and K+ content

Isometric force during stimulation was measured to assess the consequences of a reduction of individual alpha -isoform levels on EDL contractility. The relationship of force and voltage, determined empirically, is shown in Fig. 3. The alpha 2+/- produces more force for almost any given voltage and has higher force at supramaximal voltage than wild type. Figure 4A shows representative force traces for wild-type, alpha 2+/-, and alpha 1+/- EDL muscles. With supramaximal stimulation, the force quickly reached a peak value and declined slightly during the process of stimulation. At the end of stimulation, tension returned to the resting level. The peak value of tetanic force was 384.4 ± 13.2 mN/mm2 for alpha 2+/- EDL, i.e., 29% higher than EDL from wild-type mice shown in Fig. 4B (297.0 ± 13.6 mN/mm2, P < 0.01). On the other hand, the peak value for EDL from alpha 1+/- mice was only 238.9 ± 17.1 mN/mm2, representing a 20% reduction from the wild type (Fig. 4B; P < 0.05). Twitch force, another independent contractile parameter for EDL, was also measured. EDL from alpha 2+/- mice was able to produce 34% more twitch force (41.3 ± 1.8 mN/mm2, P < 0.01) than wild-type EDL (30.8 ± 2.6 mN/mm2). However, the twitch force of alpha 1+/- (29.2 ± 2.2 mN/mm2) was not significantly different from wild type (Fig. 4C). The greater force found in alpha 2+/- EDL and less force in alpha 1+/- EDL compared with the wild type suggest that the Na-K-ATPase alpha 1- and alpha 2-isoforms may play distinct roles in skeletal muscle contractility.


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  		Fig. 3.   Force-voltage relationship. A series of 1.5-s tetani was elicited from WT and alpha 2+/- EDL over an increasing voltage range to empirically determine the supramaximal stimulation voltage. Values are means ± SE (n = 5). Statistical analysis utilized unpaired Student's t-test. * Significant difference between WT and alpha 2+/- (P < 0.05).



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  		Fig. 4.   Tetanic and twitch contractions in EDL. A: EDL muscles from WT, alpha 2+/-, and alpha 1+/- mice were used for contractility measurement. Nonnormalized representative force recordings are shown. B: peak values of tetanic contractions are shown for WT, alpha 2+/-, and alpha 1+/- EDL. Force was normalized to the cross-sectional area of EDL muscle, which was estimated by the ratio of EDL weight (mg) and length (mm). C: twitch forces were plotted and normalized to the cross-sectional area of the muscle. Values are means ± SE (n = 8). Statistical analysis utilized ANOVA, and intergroup comparison used Scheffé's test. * Significant difference between WT and alpha 2+/- (P < 0.01). ** Significant difference between WT and alpha 1+/- (P < 0.05).

To confirm that the contractile phase, but not the relaxation phase, of skeletal muscle contraction is altered, parameters of contraction and relaxation in EDL were further analyzed (Fig. 5). The rate of force development in the tetanic contraction was increased in alpha 2+/- mice (750.8 ± 29.9 mN · mm-2 · s-1) compared with wild type (571.9 ± 28.7 mN · mm-2 · s-1, P < 0.01), whereas the rate of tetanic contraction in alpha 1+/- muscle was lower (521.9 ± 28.1 mN · mm-2 · s-1, P > 0.05), but not statistically significant (Fig. 5A). For the relaxation phase of tetanic contractions, half-relaxation time was not altered in either alpha 2+/- (0.189 s) or alpha 1+/- (0.194 s) compared with wild type (0.192 s) (Fig. 5B). Our studies further indicate that the relaxation phase, primarily controlled by sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), was not altered in either heterozygous animal.


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  		Fig. 5.   Rate of tetanic contraction (A) and half-relaxation time (B). A: rate of isometric force development (dF/dt) was obtained by dividing the normalized peak value of tetanus (mN/mm2) by the time (s) needed to reach the peak value. The force trace is nearly linear from initiation to the peak of the contraction. B: half-relaxation time (T1/2) represents time (s) required for force to decrease to one-half after termination of the stimulus. Values are means ± SE (n = 8). Statistical analysis utilized ANOVA, and intergroup comparison used Scheffé's test. * Significant difference between WT and alpha 2+/- (P < 0.01).

Cardiac glycoside inhibition of the Na-K-ATPase alpha 2-isoform increases contractility of alpha 1+/- EDL. The loss of one-half of the alpha 1-isoform in alpha 1+/- EDL results in less force. Because the protein level of the alpha 1-isoform increased by 39% in alpha 2+/- EDL (Fig. 1C), it is possible that the greater force observed in alpha 2+/- EDL resulted from secondary upregulation of the alpha 1-isoform. To establish that the greater force resulted specifically from a reduction in the alpha 2-isoform and not a compensatory upregulation of the alpha 1-isoform, ouabain (10-6 M) was applied to the alpha 1+/- EDL to specifically inhibit the Na-K-ATPase while EDL contractility was measured. This concentration of ouabain inhibits the alpha 2-isoform but is not sufficient to inhibit the alpha 1-isoform because this isoform has a much lower affinity for cardiac glycosides (12, 45). In these experiments, EDL muscles from one leg were treated with ouabain, and EDL muscles from the other leg were used as control. The EDL was stimulated to produce several tetanic contractions and then incubated in ouabain for 30 min (Fig. 6A). Stimulation was then reapplied, and force was measured. A 17% increase of peak value of tetanus (P < 0.05) was induced by ouabain treatment in alpha 1+/- EDL (Fig. 6B). These data indicate that inhibition of the alpha 2-isoform in the alpha 1+/- EDL by low concentrations of ouabain elicits increased contractile force and further support our hypothesis that greater force observed in alpha 2+/- EDL results from reduction of the alpha 2-isoform but not from a compensatory upregulation of the alpha 1-isoform in alpha 2+/- EDL.


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  		Fig. 6.   Effect of ouabain on tetanic contraction in alpha 1+/- EDL. A: nonnormalized representative force recordings before and after 10-6 M ouabain treatment from alpha 1+/- EDL. B: peak value of tetanus contraction in alpha 1+/- EDL. Peak value of isometric force before ouabain treatment was normalized to 100%. Values are means ± SE (n = 7). Statistical analysis utilized paired Student's t-test. * Significant difference (P < 0.05).

Fatigue study. In many pathological states, downregulation of the expression of Na-K-ATPase alpha -isoforms is correlated with skeletal muscle fatigue (1, 26). Strenuous muscle activity can also cause a significant flux of Na+ and K+ across the plasma membrane that exceeds the capacity of the Na-K-ATPase activity to restore the ionic gradients (8). We thus tested whether muscle fatigue in the alpha 1+/- and alpha 2+/- EDL was altered. EDL was first stimulated to produce a tetanus and then allowed to rest for 30 min. Stimuli were repetitively applied for 15 min (1 stimulus/min) to induce fatigue (Fig. 7A). At the beginning of the train of stimulation, peak values of isometric tetani were greater for alpha 2+/- EDL and lower for alpha 1+/- EDL than the wild-type EDL (Fig. 7B). However, at the end of the stimulation train, peak values for all three genotypes were similar: wild type, 54.8 ± 7.7 mN/mm2; alpha 2+/-, 76.9 ± 26.1 mN/mm2; and alpha 1+/-, 59.9 ± 7.2 mN/mm2 (Fig. 7B). EDL from all three genotypes exhibited similar time constants for fatigue measured as the time to half the maximal tetanus peak value (wild type, 5.5 ± 0.5 min; alpha 2+/-, 5.3 ± 0.6 min; alpha 1+/-, 6.6 ± 0.6 min).


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  		Fig. 7.   Muscle fatigue in EDL. A: electrical stimulation for 5 s was applied every minute for 15 min to examine fatigue in EDL muscles from WT, alpha 2+/-, and alpha 1+/- mice. A representative trace for a WT EDL is presented. B: peak value of tetanus contraction in WT, alpha 2+/-, and alpha 1+/- mice. Peak values of isometric force for each stimulation in A were plotted for the WT, alpha 2+/-, and alpha 1+/- EDL muscle. Values are means ± SE (n = 8 for the WT, and n = 7 for both the alpha 1+/- and alpha 2+/-). Statistical analysis utilized ANOVA, and intergroup comparison used Scheffé's test. * Significant difference between WT and alpha 2+/- (P < 0.01). ** Significant difference between WT and alpha 1+/- (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isoform-specific roles of Na-K-ATPase. Two observations from this study indicate that the Na-K-ATPase alpha 1- and alpha 2-isoforms may play different roles in skeletal muscle contractility. First, distinct alterations in tetanic force between alpha 1+/- and alpha 2+/- EDL were observed. Reduction of the alpha 2-isoform results in an increased isometric force. This is consistent with previous findings that the hearts from alpha 2+/- mice are hypercontractile (23). It is possible that the increased tetanic force in alpha 2+/- EDL results from an increase of sarcoplasmic reticulum (SR)/endoplasmic reticulum (ER) Ca2+ content via inhibition of the Na+/Ca2+ exchanger activity. Interestingly, reduction of the alpha 1-isoform results in a decreased isometric force in alpha 1+/- EDL that is also similar to the phenotypic change described in alpha 1+/- hearts. The mechanism for reduced force in alpha 1+/- mice is presently not clearly understood.

Second, the relative amounts of the alpha 1- and alpha 2-isoforms in skeletal muscle are strikingly different from those in cardiac muscle, further suggesting the alpha 1- and alpha 2-isoforms play different roles in muscle contractility. In EDL, the alpha 2-isoform makes up 87% of the total alpha -isoform, whereas the alpha 1-isoform only accounts for 13%. Interestingly, these percentages are almost reversed in cardiac muscle, where 70% of the total is alpha 1-isoform (unpublished observation). Skeletal muscle has a more developed T-tubule system than cardiac muscle (15). The percentage of T-tubule membrane area is up to 85% of the total sarcolemmal membrane area in mammalian skeletal muscle (9). The unique percentages of the two alpha -isoforms in these two types of striated muscle may become more understandable when the isoform-specific roles of the two alpha -isoforms are considered. Because skeletal muscle has more sarcolemmal membrane area in the T-tubule system, more Na-K-ATPase alpha 2-isoform may be required to modulate the Na+/Ca2+ exchanger activity to exert the regulation of SR/ER Ca2+ content.

Two recent findings strongly suggest that the alpha 1- and alpha 2-isoforms play different functional roles at the cellular level. First, different locations for the alpha 1- and alpha 2-isoforms in the plasma membrane of rat astrocytes and arterial myocytes have been observed (25). Although the alpha 1-isoform is uniformly distributed within the plasma membrane, the alpha 2-isoform is concentrated in restricted regions of the plasma membrane (microdomains) where the Na+/Ca2+ exchanger is also localized. These microdomains are in close proximity to SR/ER and serve as a functional compartment in which Na+ and Ca2+ concentrations can be regulated in a locally restricted cytosolic area without altering global Na+ and Ca2+ concentrations in the cytosol.

Second, this laboratory has demonstrated that alpha 1- and alpha 2-isoforms of the Na-K-ATPase play different roles in cardiac muscle contractility in mice (23). By reducing the expression levels of either alpha -isoform by gene targeting, it was found that with alpha 2+/- animals, the heart is hypercontractile, whereas hearts from alpha 1+/- are hypocontractile even though reduction of the alpha 1-isoform Na-K-ATPase in alpha 1+/- hearts does not alter Ca2+ transients (23). In hearts from alpha 2+/- animals, reduction of the alpha 2-Na-K-ATPase may result in an increase of intracellular Na+ concentration that lessens the Na+ gradient across the plasma membrane. Because the Na+ gradient is the driving force of the Na+/Ca2+ exchanger to extrude intracellular Ca2+, inhibition of Na-K-ATPase ultimately causes an increase of intracellular Ca2+ that is transported into SR/ER by SERCA. In subsequent contractions, more Ca2+ is released from SR/ER to produce higher Ca2+ transients that result in the hypercontractile state in the alpha 2+/- hearts.

Na-K-ATPase and skeletal muscle contractility. During the determination of the supramaximal stimulation parameters, the alpha 2+/- muscles generated more force at any given stimulation voltage. Thus alpha 2+/- EDL shows both an increased sensitivity to submaximal voltages and greater forces at supramaximal voltages. This is consistent with the traditional view that whole skeletal muscle regulates total force via increased recruitment of fibers. It is possible that the increased sensitivity of the alpha 2+/- muscle is due to an excitation/contraction coupling phenomenon. The loss of the alpha 2-isoform may cause a slight depolarization. Under such conditions, a muscle may be more easily stimulated, resulting in greater recruitment of muscle fibers, leading to greater force at any given voltage. However, this phenomenon cannot explain the greater force in the alpha 2+/- muscle at supramaximal voltage where all fibers would be activated.

In the present study, we also found that 10-6 M ouabain increased tetanic force in alpha 1+/- EDL. Thus inhibition of the alpha 2-isoform resulted in more contractile force, possibly through increasing Ca2+ content in SR/ER. This may be due to the putative functional association of the alpha 2-Na-K-ATPase and the Na+/Ca2+ exchanger, whose functional role has recently been elucidated in skeletal muscle (2). A study by Clausen and Everts (7) reported that 10-6 M ouabain produces a slight loss of contractility in the presence of 12.5 mM K+. In our study, a physiological concentration of 4.7 mM KCl was used that may contribute to the different observations of ouabain effect on muscle contractility between the two studies. Additionally, this discrepancy may be due to the different protocol used in our study. To facilitate intracellular Ca2+ loading, EDL muscles in our experiments were not stimulated during the preincubation with 10-6 M ouabain. The tetanic contractions were elicited after the incubation period and compared with untreated EDL, whereas the study by Clausen and Everts (7) applied several conditional stimulations of short duration (0.5-2 s) during the incubation period with ouabain. It is highly possible that any increase of SR/ER Ca2+ content may be dampened by these conditional stimulations. Therefore, increased force was never observed.

One hypothesis to explain our observations is that for any given stimulus, the alpha 2+/- EDL releases more Ca2+, resulting in greater force. This applies equally to the increased force during both the submaximal and supramaximal stages of the force-voltage curve. However, Zhao et al. (48) showed that in a single twitch, free Ca2+ concentration ([Ca2+]free) increases to ~16 µM in frog fast-twitch muscle (48). Hollingworth et al. (20) measured [Ca2+]free in fiber bundles isolated from mouse fast-twitch muscles and showed that Ca2+ release is slightly temperature dependent, increasing [Ca2+]free to 17 and 22 µM at 16 and 28°C, respectively (20). It has been shown that in frog fast-twitch skeletal muscle, the spatially averaged [Ca2+]free can increase as high as ~18 µM, which is sufficient to saturate troponin C (3, 19). Thus the actomyosin contractile apparatus would not be expected to be Ca2+ limited during a contractile event in frog muscle. Importantly, however, these authors also determined that the half-time of the [Ca2+]free transient in mice is remarkably rapid (~4 ms), approximately one-half that of the frog (20). Given the relationship of the alpha 2-isoform with the Na+/Ca2+ exchanger, it is possible that the [Ca2+]free transient is either directly prolonged in EDL from alpha 2+/- animals or that the release of Ca2+ is much larger than normal such that it requires more time to resequester effectively. In either case, this could lead to higher tetanic steady-state [Ca2+]free, resulting in potentiation of force.

Results of the fatigue study also support our hypothesis of higher [Ca2+]free in alpha 2+/- EDL. While the alpha 2+/- EDL loses more force over the entire time course of the fatigue protocol, these muscles reach the same basal level of force as the wild-type and alpha 1+/- EDL, and the time to half-maximal tetanus peak value is similar for all three genotypes. This suggests that there are no intrinsic differences in muscle ultrastructure or biochemistry but, rather, that Ca2+ handling has been normalized by the frequent stimulations.

It is possible, given the phenotypic plasticity of muscle tissue, that an increase in fast-twitch myosin isoforms in the alpha 2+/- animal could contribute to the increased force. Myosin isoform distribution can be altered by electrical stimulation (34), thyroid hormone status (21, 30), exercise (5, 18, 40), and disease (13, 14, 35). Furthermore, it has been shown that in rodent models of type I diabetes, there is often a change in Na-K-ATPase activity (36) that is concurrent with changes in myosin isoforms.

Perspectives

Cardiac glycoside treatment of congestive heart failure also inhibits the Na-K-ATPase activity in skeletal muscle and may result in loss of muscle contractility. We present for the first time that this inhibition can be therapeutically beneficial. Our study indicates that a loss of one-half of the alpha 2-isoform results in more force in skeletal muscle. Increased force may be helpful for venous return from peripheral veins to improve circulation or for preventing respiratory muscle failure. Furthermore, it needs to be noted that this study is based on mouse models whose alpha 2-isoform is at least 1,000-fold more sensitive to cardiac glycosides than the alpha 1-isoform. However, in most mammals, including human, the difference in affinities for cardiac glycosides between the alpha 1- and alpha 2-isoforms is small. Cardiac glycosides will influence the activity of both alpha 2- and alpha 1-isoforms in human skeletal muscle. The data here demonstrate that inhibition of the alpha 1-isoform results in decreased muscle contractility, contributing to possible toxic side effects of cardiac glycosides, whereas inhibition of the alpha 2-isoform results in stronger contraction. Combining the finding from skeletal muscle with the results from the heart (23), it is reasonable to speculate that identification or design of compounds that specifically inhibit the alpha 2-isoform, but not the alpha 1-isoform, may reduce the toxicity without altering the therapeutic potency of Na-K-ATPase inhibition. Specific inhibition of the alpha 2-isoform by potential compounds may be beneficial for both cardiac and skeletal muscle.


    ACKNOWLEDGEMENTS

We thank Dr. M. H. Cougnon for carefully reading and discussing the manuscript.


    FOOTNOTES

* S. He and D. A. Shelly contributed equally to this paper.

Present address of P. F. James: Dept. of Zoology, Miami University, Oxford, OH 45056.

Address for reprint requests and other correspondence: J. B. Lingrel, Dept. of Molecular Genetics, Biochemistry, and Microbiology, Univ. of Cincinnati, College of Medicine, Cincinnati, OH 45267 (E-mail: jerry.lingrel{at}uc.edu).

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.

Received 11 November 2000; accepted in final form 16 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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