Isoform-specific role of Na/K-ATPase α1 in skeletal muscle

Abstract

The distribution of Na/K-ATPase α-isoforms in skeletal muscle is unique, with α1 as the minor (15%) isoform and α2 comprising the bulk of the Na/K-ATPase pool. The acute and isoform-specific role of α2 in muscle performance and resistance to fatigue is well known, but the isoform-specific role of α1 has not been as thoroughly investigated. In vitro, we reported that α1 has a role in promoting cell growth that is not supported by α2. To assess whether α1 serves this isoform-specific trophic role in the skeletal muscle, we used Na/K-ATPase α1-haploinsufficient (α1^+/−) mice. A 30% decrease of Na/K-ATPase α1 protein expression without change in α2 induced a modest yet significant decrease of 10% weight in the oxidative soleus muscle. In contrast, the mixed plantaris and glycolytic extensor digitorum longus weights were not significantly affected, likely because of their very low expression level of α1 compared with the soleus. The soleus mass reduction occurred without change in total Na/K-ATPase activity or glycogen metabolism. Serum analytes including K⁺, fat tissue mass, and exercise capacity were not altered in α1^+/− mice. The impact of α1 content on soleus muscle mass is consistent with a Na/K-ATPase α1-specific role in skeletal muscle growth that cannot be fulfilled by α2. The preserved running capacity in α1^+/− is in sharp contrast with previously reported consequences of genetic manipulation of α2. Taken together, these results lend further support to the concept of distinct isoform-specific functions of Na/K-ATPase α1 and α2 in skeletal muscle.

INTRODUCTION

The Na/K-ATPase was discovered over 60 years ago as the membrane-bound protein complex that catalyzes the active transport of K⁺ into and Na⁺ out of the cell, thereby maintaining the resting membrane potential and excitability. The minimal functional Na/K-ATPase unit is made up of two subunits, α and β. The α-subunit is the catalytic subunit and bears the binding sites for ATP, ions, and cardiotonic steroids (CTS; 9). In addition to the ubiquitously present α1, three isoforms of the catalytic subunit have been characterized. Na/K-ATPase α2 is found mainly in muscle, adipose, and glial cells, α3 is found mainly in neurons, and α4 expression is restricted to sperm (9). This highly tissue-specific expression pattern and isoform-specific response to both physiological and pathological stimuli have long suggested that they must be serving tissue-specific functions.

Over the last 15 years, we and others have reported that Na/K-ATPase α1 serves important scaffolding and signaling functions in addition to its role as an ion pump (9). Specifically, α1 can interact with and modulate Src activity, which in turn affects EGF receptors through transactivation. This subsequently adjusts the assembly and activation levels of multiple protein/lipid kinases as well as the generation of reactive oxygen species and other intracellular messengers, allowing endogenous CTS to regulate cell growth (e.g., kidney development; 15, 16, 19, 27, 44). On the other hand, sustained and dysregulated activation of this signaling mechanism causes reactive oxygen species stress and pathological remodeling in the heart and kidneys (31, 49, 51).

In the skeletal muscle, the role of the ion-pumping function of the dominant α2-isoform in maintaining the membrane potential during contraction has been studied extensively (13, 21, 22, 32, 41, 42). Additionally, Radzyukevich et al. have described an improvement in exercise performance in mice expressing a ouabain-resistant mutant α2-isoform, suggesting a role of endogenous CTS in the regulation of muscle contraction via the α2-isoform (41). Interestingly, expression of ouabain-resistant α2 or even α2 knockout did not affect skeletal muscle mass (13, 32, 41, 42). In a renal epithelial cell knockdown and rescue system, we have obtained evidence that the α1-isoform is important for cell growth and that rescue with α2 restores ion-pumping capacity but does not restore growth or Src-dependent signal transduction in response to ouabain binding at concentrations too low to impair enzymatic activity (50). Taken together, the apparent lack of impact of α2 in skeletal muscle mass in genetic mouse models and the inability to support cell growth in the absence of α1 in vitro are consistent with a model whereby α1, but not α2, plays a role in the regulation of skeletal muscle mass. To test this model, we investigated the impact of α1 reduction on muscle mass in Na/K-ATPase α1-haplodeficient mice (α1^+/−) and control littermates (α1^+/+). This mouse model has been previously used to examine α1- and α2-isoform-specific functions in the heart (24, 34, 35) and the skeletal muscle (21). Whereas the latter study specifically focused on the glycolytic extensor digitorum longus (EDL) muscle and the respective roles of α1 and α2 in the maintenance of ion homeostasis during contraction, we extended our search for a trophic role of α1 to the three muscle types (oxidative, mixed, and glycolytic).

MATERIALS AND METHODS

Reagents.

The polyclonal anti-Na/K-ATPase α1 antiserum NASE and polyclonal anti-Na/K-ATPase α2 antiserum HERED used for Western blot analysis were raised in rabbits and were generous gifts from Drs. T. Pressley and P. Artigas at Texas Tech University Health Sciences Center (40). Antibodies for phospho-serine 9 glycogen synthase kinase-3β (GSK-3β) and total GSK-3β were from Cell Signaling Technology (cat. no. 9322-S and 9315-S, respectively). Anti-α-tubulin antibody (cat. no. T-5168; Sigma) or anti-β-actin antibody (cat. no. sc-7210; Santa Cruz Biotechnology) were used as a loading control. Secondary antibodies were horseradish peroxidase-conjugated anti-rabbit and anti-mouse from Santa Cruz Biotechnology (cat. no. sc-2004 and sc-2005, respectively).

Animals.

Mice heterozygous for the Na/K-ATPase α1-isoform were developed by Dr. J. Lingrel’s group at the University of Cincinnati (24). The colony was backcrossed to C57J/BL6 mice from Jackson Laboratories and maintained through a heterozygous × wild-type breeding scheme, resulting in α1^+/− experimental animals and littermate controls. Male α1^+/− mice and control littermates were housed in 12-h light and dark cycles at constant temperature and humidity until 6 mo of age. All animal procedures were approved by the Marshall University Institutional Animal Care and Use Committee.

Treadmill testing.

Six-month-old male α1^+/− mice and littermate controls were placed in the six lanes of an Exer 3/6 treadmill from Columbus Instruments equipped with a shock detection system. Animals were acclimated to the treadmill for 3 days at 5 m/min for 5 min at a 5° angle and were subjected to the testing protocol on the fourth day. Mice began the testing protocol running at 5 m/min for 5 min and increased by 2 m/min each minute up to 25 m/min, then continued running at 25 m/min until they reached fatigue. Each shock administered and each visit to the shock grid were recorded for each animal. Fatigue was defined as 10 consecutive seconds spent on the shock grid, and the shock was discontinued to each mouse upon reaching fatigue.

Tissue collection.

Mice were anesthetized with 50 mg/kg pentobarbital administered via intraperitoneal injection. Tissues were dissected and weighed. Muscles used for Western blot analysis or enzymatic activity assays were flash frozen in liquid nitrogen and then stored at −80°C until later use. Muscles used for histological analysis were fixed in 10% neutrally buffered formalin for 24 h and then stored in 70% ethanol until they were embedded in paraffin blocks.

Western blot analysis.

Left and right muscles of the same type from the same mouse were homogenized together in ice-cold radioimmunoprecipitation buffer (0.25% sodium deoxycholate, 1% Nonidet P-40, 1 mM EDTA, 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 150 mM NaCl, 50 mM Tris·HCl, pH 7.4, and 1% protease inhibitor cocktail) with a Fisher TissueMiser homogenizer. Homogenates were centrifuged at 14,000 g for 15 min, supernatants were collected, and the protein content was measured using DC Protein Assay Kit from Bio-Rad (cat. no. 500-0114 and 500-0113). Equal amounts of protein from each sample were loaded, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes probed for α1 and α2 were blocked in 5% milk, and then primary antibodies were added overnight at 4°C. Membranes were visualized with Western Lightning Plus-ECL (Western Lightning) and radiographic film. Densitometric quantification was performed using ImageJ software from the National Institutes of Health.

Membrane fractionation.

Crude membrane fractions were prepared from frozen α1^+/− and α1^+/+ gastrocnemius muscles following a procedure modified from Walas and Juel (47). Frozen muscles were ground into a fine powder with a mortar and pestle. The resulting powder was homogenized in ice-cold fractionation buffer (250 mM mannitol, 30 mM l-histidine, 5 mM EGTA, and 0.1% deoxycholate, adjusted to pH 6.8 with Tris-base) for 30 s with a Fisher TissueMiser handheld homogenizer. The crude homogenate was centrifuged at 3,000 g for 30 min, and the supernatant was then centrifuged at 190,000 g for 90 min. The pellet was resuspended in 30 mM histidine, 250 mM sucrose, and 1 mM EDTA, pH 7.4, and protein concentration was determined using the DC Protein Assay Kit from Bio-Rad (cat. no. 500-0114 and 500-0113).

ATPase activity assay.

Ouabain-sensitive ATPase activity in crude membrane fractions was determined by measuring ATP hydrolysis as previously described (7, 8). Released inorganic phosphate (Pi) was detected using a malachite-based Biomol Green reagent. Samples containing 10 µg of protein were added to a reaction mix containing 20 mM Tris·HCl, 1 mM MgCl₂, 100 mM NaCl, 20 mM KCl, and 1 mM EGTA-Tris, pH 7.2. Ouabain was added to the samples to a final concentration of 1 mM to completely inhibit both α1- and α2-isoforms of the Na/K-ATPase. After 10 min of preincubation at room temperature, the reaction was started by adding Mg-ATP at a final concentration of 2.25 mM and incubation at 37°C with shaking for 30 min. The reaction was stopped with the addition of ice-cold 8% TCA, and the concentration of Pi was measured spectrophotometrically at outer diameter 620 nm using Biomol Green as an indicator (cat. no. BML-AK111-250; Enzo Life Sciences). Maximal Na/K-ATPase activity was calculated as the difference between ATPase activity obtained in the absence or presence of 1 mM ouabain.

Immunohistochemistry.

Muscles were collected and then washed twice with ice-cold PBS, fixed with 10% neutrally buffered formalin for 24 h, and embedded in paraffin. Transverse sections of the midbelly were immunostained for fast and slow myosin heavy chain by Wax-It (Vancouver, BC, Canada) as described by Behan et al. (6) to differentiate between type 1 and type 2 fibers. Additional sections were stained for Na/K-ATPase α1 by Wax-It. The samples were examined on a Leica confocal SP5 microscopes (Leica Microsystems, Wetzlar, Germany). The images were processed with the Leica Application Suite for Advanced Fluorescence (Leica Microsystems), Fiji platform, and the GNU Image Manipulation Program (GIMP) to obtain maximum projections, extract lateral slices, and construct figures.

Morphometric tissue analysis (cross-sectional area and fiber types).

Images of muscles stained for fast and slow myosin heavy chain were obtained by Wax-It with digital whole slide scanning. Aperio ImageScope software was used to determine the cross-sectional area of each fiber. Fibers that had been damaged were excluded from cross-sectional area analysis. Every fiber of each type in each muscle was counted to determine the average number of fibers per muscle.

Glycogen content analysis.

Glycogen was assayed in whole muscle homogenates using a colorimetric glycogen assay kit from Abcam (cat. no. ab-169558) according to the manufacturer’s instructions.

Serum analytes.

Whole blood was collected from the hepatic portal vein and then allowed to clot for 15 min in 0.8-ml SST-MINI tubes with clot activator and gel. The blood was then centrifuged at 2,000 rpm for 15 min. Clear serum was transferred to 1.5-ml transport tubes and analyzed by IDEXX Bioresearch using a Beckman AU680 chemistry system.

Data analysis.

Data are presented are means ± SE, and statistical analysis was performed using Student’s t-test. When >2 groups were compared, one-way ANOVA was performed before post hoc comparison of individual groups using Dunnett’s multiple-comparison test. Significance was accepted at P < 0.05.

RESULTS

Skeletal muscle Na/K-ATPase in α1-haplodeficient mice.

He et al. first reported a significant decrease in Na/K-ATPase α1 expression in α1^+/− muscle, but their study focused on one of the three main types of muscle, the glycolytic EDL (21). In the present study, the extent of Na/K-ATPase α1 expression decrease was assessed in all muscle types. Specifically, Western blot analyses were performed in a representative oxidative (soleus), a mixed (plantaris), and a glycolytic (EDL) muscle. As shown in Fig. 1A, α1 expression in α1^+/− mice was decreased by 30–40% in all muscle examined (P < 0.05 vs. α1^+/+). Furthermore, no compensatory increase in α2 expression was observed (Fig. 1A), and the expression of the regulatory phospholemman (FXYD1) subunit was also unchanged (data not shown). The decrease of Na/K-ATPase α1 was also clear after immunofluorescence labeling using a α1-specific antibody in histological preparations of soleus and EDL muscles (Fig. 1, B and C). To assess the impact of this reduction of α1 on total Na/K-ATPase activity, a preparation of the gastrocnemius, a mixed muscle of larger size, was used. A decrease of α1 without detectable change in α2 expression comparable to that of the three other muscles tested was observed by Western blot analysis (Fig. 2A). As shown in Fig. 2B, maximal Na/K-ATPase activity was not different between α1^+/+ and α1^+/− crude membrane fractions (ouabain-inhibited ATPase activity of 1.91 ± 0.23 μmol·µg protein⁻¹·h⁻¹ in α1^+/+ membrane fractions compared with 2.01 ± 0.40 μmol·µg protein⁻¹·h⁻¹ in α1^+/− membrane fractions).

Fig. 1.

Na/K-ATPase α-isoform abundance in skeletal muscles from Na/K-ATPase α1-haplodeficient mice (α1^+/−) and control littermates (α1^+/+). A: representative Western blots for Na/K-ATPase α1- and α2-isoforms in soleus (oxidative), plantaris (mixed), and extensor digitorum longus (EDL, glycolytic) muscle homogenates with n = 2 per genotype. The quantitative data are means ± SE from 7–9 specimens per group normalized to the average of the α1^+/+ controls on each gel. B and C: representative immunohistochemical staining for Na/K-ATPase α1-isoform in soleus (B) and EDL (C) from α1^+/+ and α1^+/− mice. *P < 0.05.

Fig. 2.

Na/K-ATPase activity. A: representative Western blots for Na/K-ATPase α1- and α2-isoforms in gastrocnemius muscle homogenates with n = 2 per genotype. The quantitative data are means ± SE from 4 specimens per group normalized to the average of the α1^+/+ controls on each gel. B: maximal ATPase activity in crude membrane fractions from α1^+/+ and α1^+/− gastrocnemius muscles (n = 6–7). ATPase activity was measured by Pi release with a colorimetric indicator. *P < 0.05.

Na/K-ATPase α1 reduction affects oxidative but not mixed or glycolytic muscle size.

On the basis of the recent finding that removing α1 causes decreased growth in an in vitro system (50), we investigated the impact of reduced α1 expression on skeletal muscle size in α1^+/− mice compared with α1^+/+. Mice were age matched at 6 mo and exhibited no differences in body weight. Consistent with previous studies (24), there were no major abnormalities detected in basal conditions, and kidney weight-to-body weight ratio and heart weight-to-body weight ratio were comparable (Table 1). Consistent with previous observations in these animals (21), the mass-to-body weight ratio of the glycolytic EDL did not change in α1^+/− mice (Fig. 3). Likewise, the mass of the mixed-type plantaris was comparable (Fig. 3). In contrast, the mass-to-body weight ratio of the oxidative soleus muscle was decreased by 9%, from 0.230 ± 0.009 mg/g for α1^+/+ mice to 0.209 ± 0.006 mg/g for α1^+/− mice (P < 0.05).

Table 1.

Selected physiological parameters in Na/K-ATPase α1^+/+ versus α1^+/− mice

	Age, days	BW, g	Tibia Length, mm	Heart/BW, mg/g	Kidney/BW, mg/g	Adipose Weight, mg	Serum K+, mM	Serum Na+, mM	Glucose, mg/dl
α1+/+	188.1 ± 0.9	34.9 ± 0.6	16.4 ± 0.2	4.37 ± 0.08	6.82 ± 0.12	1,253 ± 180	6.15 ± 0.17	153.8 ± 1.1	232 ± 37
α1+/−	187.9 ± 0.7	35.5 ± 0.4	16.3 ± 0.2	4.25 ± 0.07	6.67 ± 0.11	995 ± 250	6.48 ± 0.84	152.0 ± 0.9	238 ± 36

Values are means ± SE. For body weight (BW), age, and tibia length, n = 30–40. For heart weight-to-BW ratio (Heart/BW) and kidney weight-to-BW ratio (Kidney/BW), n = 20–33. Adipose weight represents the combined weights of the epidydimal and inguinal fat pads (n = 7–8). Serum concentrations of K⁺, Na⁺, and glucose were measured in blood collected from the hepatic portal vein of fed mice (n = 4). No significant difference was observed.

Fig. 3.

Changes in muscle mass in α1^+/− mice. Muscle mass of soleus, plantaris, and extensor digitorum longus (EDL) in α1^+/+ and α1^+/− mice. The quantitative data are means ± SE from 23 α1^+/+ or 40 α1^+/− mice. BW, body weight. *P < 0.05.

Expression of Na/K-ATPase α1 in skeletal muscle types.

Previous studies of Na/K-ATPase isoforms have described a muscle type-specific distribution of α1 and α2 in rats, with the oxidative soleus containing more α1 than any other muscle type studied and the glycolytic EDL expressing the least (43). Hence, higher expression level could explain the relatively high impact of α1 depletion in the soleus compared with other muscle types in the mouse. This was evaluated by Western blot analysis in the oxidative soleus and red gastrocnemius, the glycolytic EDL and white gastrocnemius, and mixed plantaris muscles of C57BL/6 mice. As shown in Fig. 4, expression of α1 decreases as muscles become increasingly glycolytic, with the oxidative soleus expressing significantly more α1 than either the mixed plantaris or the glycolytic EDL. The sharp contrast between α1 expression in the soleus versus EDL was confirmed by immunofluorescence (Fig. 4C) and is consistent with a relative lack of detectable impact on growth upon reduction by 30%. A difference in α2 expression between muscle types was also detected, although not as pronounced and less systematically correlated with muscle type.

Fig. 4.

Na/K-ATPase α-isoform abundance in skeletal muscles from C57BL/6 mice. A: representative Western blots for Na/K-ATPase α1- and α2-isoforms in oxidative [soleus and red gastrocnemius (R Gastroc)], mixed (plantaris), and glycolytic [extensor digitorum longus (EDL) and white gastrocnemius (W Gastroc)] muscles with n = 2 per muscle type. B: quantitative data are means ± SE from 6 specimens per group normalized to the average of the soleus on each gel. *P < 0.05 and ***P < 0.0001 vs. soleus. C: representative micrographs of immunohistological staining for Na/K-ATPase α1 in soleus and EDL, with quantification of 3 samples per group. **P < 0.001.

Decreased cross-sectional area without change in muscle fiber number in the soleus.

To determine which structural changes were associated with the decreased muscle mass in the soleus, we examined the number and size of myofibers in α1^+/− versus α1^+/+ soleus muscles after staining for fast and slow myosin heavy chain from four muscles per group. As shown in Fig. 5A, fiber composition of the α1^+/− soleus was not different from α1^+/+. However, fiber cross-sectional area was significantly decreased by 10% in α1^+/− soleus muscles, suggesting that the decrease in muscle mass was due to changes in fiber size rather than number. This change in fiber size was observed in both type 1 and type 2a fibers of the soleus (Fig. 5A). In contrast, the EDL exhibited no change in either fiber number or fiber cross-sectional area, which is consistent with the lack of impact on overall muscle size (Fig. 5B).

Fig. 5.

Structural changes in the soleus muscle of Na/K-ATPase α1-haplodeficient mice. Soleus and extensor digitorum longus (EDL) muscles were dissected and weighed, and then cross sections of the midbelly of paraffin-embedded soleus muscles were stained for fast and slow myosin heavy chain. Cross-sectional areas of the fibers were determined using Aperio ImageScope software. A: representative micrographs of α1^+/+ and α1^+/− soleus muscles, with type I and type II fibers shown with white and black arrows, respectively. Quantifications of cross-sectional areas of type I and type II fibers from 4 soleus muscles per group and total number of fibers of each type in each soleus muscle (n = 4). **P < 0.005 and ***P < 0.0001 vs. cross-sectional area in α1^+/+ littermates. B: representative micrographs of α1^+/+ and α1^+/− soleus muscles. Quantifications of cross-sectional areas of fibers in EDL muscles (n = 4–5). No significant difference was observed.

Glycogen content and GSK-3β status in the soleus.

The Na/K-ATPase α1 signaling pathway is a modulator of glycogen synthesis and GSK-3β in skeletal muscle cells (28) and may therefore lead to a change in size, growth, and differentiation in the soleus of α1^+/− mice (1, 30, 45, 46). Accordingly, we compared glycogen content and GSK-3β in α1^+/− and α1^+/+ soleus muscles. As shown in Fig. 6A, Western blot analysis did not reveal any difference in GSK-3β content or serine 9 phosphorylation between α1^+/+ and α1^+/− muscles from fed mice. Consistent with this result, glycogen content was comparable in α1^+/+ and α1^+/− soleus muscles (Fig. 6B).

Fig. 6.

A: glycogen synthase kinase-3β (GSK-3β) and glycogen content in the soleus muscle of Na/K-ATPase α1-haplodeficient mice. Representative Western blots for phospho-serine 9 (p-Ser9) GSK-3β and total GSK-3β in α1^+/+ and α1^+/− soleus muscles. Quantitative data are means ± SE from 4 specimens per group. p-Ser9 GSK-3β-to-total GSK-3β ratios were normalized to the average of the α1^+/+ controls on each gel. B: glycogen content of soleus muscles presented as means ± SE from 6–7 samples per group.

Exercise performance.

As shown in Table 1, the 10% change in muscle mass observed in some but not all muscles of the α1^+/− mouse was not accompanied by a noticeable change in physiological parameters related to growth (tibia length) or metabolic dysregulation (glycemia, adipose tissue mass), or K⁺ homeostasis in basal conditions. To test whether the observed structural changes in α1^+/− soleus size affected exercise performance in 6-mo-old mice, two treadmill exercise paradigms were used. A gradual increase in velocity allowed us to assess changes in tolerance to high speeds and high-intensity exercise, and a prolonged time at 25 m/min allowed us to determine whether endurance exercise was affected. As shown in Fig. 7, the number of shocks at each speed remained unchanged, suggesting that the reduced amount of α1 present in the α1^+/− muscle did not prevent animals from running at high speeds. Furthermore, the distance to fatigue was unchanged.

Fig. 7.

Distance to fatigue in Na/K-ATPase α1-haplodeficient mice (α1^+/−) and control littermates (α1^+/+) during forced treadmill running. A: number of shocks administered per animal per minute at increasing speeds during testing of 6-mo-old mice (n = 7–9 per group; α1^+/+, ●; α1^+/−, gray squares). B: distance to fatigue for 6-mo-old α1^+/+ and α1^+/− animals (n = 7–9).

DISCUSSION

Based on our observations in renal epithelial cell lines, Na/K-ATPase α1 possesses isoform-specific functions that are not supported by α2, which results in a sizable effect on cell growth rate (50). Since renal epithelial cells do not normally express Na/K-ATPase α2, we took the next step of investigating this issue in a tissue expressing both isoforms. We focused on the skeletal muscle, a tissue where α2 expression uniquely predominates over the minor α1-isoform and with a regenerative process that occurs throughout life. Specifically, we used the Na/K-ATPase α1^+/− mouse model to investigate whether a downregulation of the minor pool of Na/K-ATPase α1 expressed in skeletal muscle could affect muscle mass. The data indicate that a 30–40% decrease of Na/K-ATPase α1 protein expression, which does not noticeably decrease total maximum Na/K-ATPase capacity of the muscle, induces a modest yet significant decrease of 10% in the mass of the oxidative soleus (Fig. 3). In contrast, the mass of the glycolytic EDL was not affected, nor was that of the mixed-type muscle plantaris (Fig. 3). The very low level of α1 expression in the EDL and the plantaris compared with the soleus may explain this intriguing muscle type-specific effect. Indeed, as shown in Fig. 4 and consistent with previous studies in rats by Thompson and McDonough (43) and others (10, 20), the oxidative soleus expresses substantially more α1 than the glycolytic EDL in wild-type mice. Moreover, when the Western blot analysis was extended to additional representative muscles of each type, a positive correlation between α1 content and oxidative metabolism was observed, suggesting that the role of α1 could be particularly important in oxidative fibers. Mechanistically, we speculate that endogenous CTS signaling through α1 may have a role in maintaining the growth of the soleus and the lack of this ouabain signaling could be responsible for the reduced soleus mass in α1^+/− mice (Fig. 3). It should be noted that the mouse α1-isoform has a much lower affinity for ouabain than α2 and only 0.05% of the α1-isoform is bound to ouabain at the reported endogenous ouabain concentrations (5, 37). However, through amplification of signaling cascades, concentrations of ouabain comparable to circulating endogenous ouabain can activate signaling in cells and tissue expressing such low-affinity α1 (4, 15, 16, 19). In contrast, pumping inhibition is directly related to the number of pumps bound to ouabain, which means that signaling could have a long-lasting impact at concentrations too low to affect the membrane potential or ion homeostasis. Although this remains to be specifically tested, a plausible model is that the higher expression level of α1 in the soleus enables endogenous CTS-stimulated signaling, as seen in other cell lines and tissue types (4, 12, 19, 27, 44). Consistent with our observation that a 50% reduction in α1 expression prevents ouabain from stimulating signaling and growth in renal epithelial cells (44), α1 expression in the EDL (6% of soleus) or plantaris (23% of soleus) may not allow CTS-stimulated growth through stimulation of Na/K-ATPase α1 signaling. Although signaling of the CTS ouabain through Na/K-ATPase α1 modulates glycogen synthesis through GSK-3β signaling in skeletal muscle cells (28) and may therefore lead to a change in size, growth, and differentiation in the soleus of α1^+/− mice (1, 30, 45, 46), we did not detect a significant change in the 6-mo-old mouse (Fig. 6). This result certainly warrants further investigation, as a dysregulation of GSK-3β may have occurred at an earlier time point and/or may only be discernible under agonist stimulation.

Skeletal muscle-specific ablation of Na/K-ATPase α2 does not affect muscle mass (13, 32, 42), which suggests that the observed decrease in soleus mass in α1^+/− is likely unrelated to altered ion-pumping capacity of the cell. Consistently, we did not detect any significant decrease in Na/K-ATPase activity in crude membrane preparations from α1^+/− muscles. Based on an α1 contribution of ~15% of total skeletal muscle Na/K-ATPase (21) and a decrease of ~40% of α1 in the α1^+/− skeletal muscle, the expected decrease in Na/K-ATPase activity would have been minimal (~6%) and may have remained below the limit of detection of the assay (22, 23, 47). On the other hand, it is well established that most cells have a large reserve pump capacity and that a decrease in the number of Na/K-ATPase expressed at the cell membrane can be compensated by either a substrate-mediated stimulation of existing pumps or a mobilization of the reserve pump pool (2, 3). Finally, although we consider it unlikely because a decreased myofiber diameter as observed in α1^+/− muscles is not expected as a secondary adjustment for optimization of ion-pumping capacity and maintenance of the membrane potential [in fact, fibers with larger rather than smaller diameters require significantly less energy to maintain their membrane potential (25, 26)], it should be noted that a change in Na/K-ATPase ion transport activity secondary to α1 depletion has not been specifically excluded in this study.

This model of a change in muscle mass related to α1-specific signaling function is also consistent with the concept of distinct and specific roles for α1 and α2 in skeletal muscle initially suggested by He et al. Indeed, the authors first proposed that α1 is primarily responsible for establishing a baseline membrane potential and α2 maintains the membrane potential during contraction (21). This role for α2 was subsequently confirmed by a series of studies using skeletal muscle-specific ablation of α2 in mice. In those studies, muscle mass of both EDL and soleus was unchanged, but maintenance of the membrane potential during contraction was severely impaired because of an inability to clear the excitation-dependent increase in extracellular [K⁺] (13, 32, 42). Taken together, those studies and the results presented here support a model where skeletal muscle α2 has an isoform-specific role in the maintenance of membrane potential during contraction related to its enzymatic activity, whereas α1 has, in addition to its transport function, an isoform-specific role in growth that is independent of its ion-pumping activity.

Although the effect of α1-haplodeficiency on muscle growth may seem modest at first, it is important to note that it is relevant to reported models of skeletal muscle atrophy. The soleus-specific decrease in muscle mass, accompanied by a reduction of fiber size but not fiber number, is a feature of disuse-induced atrophy induced by hind limb suspension (18, 29). Although this decrease in size is relatively minor compared with some forms of atrophy, it is comparable to the 10–15% decrease in gastrocnemius mass in burn cachexia reported by Pedroso et al. (38). Similarly, the commonly used subcutaneous inoculation model of cancer cachexia consistently decreases muscle mass by 6–15% (11, 33, 36, 48). As may have been expected with a relatively modest decrease, the α1^+/− mouse model does not present with major metabolic abnormalities in basal conditions (Table 1). Perhaps more surprising is the lack of defects in exercise capacity (Fig. 7), given that He et al. found that the tetanic force of isolated α1^+/− EDL muscles is decreased in vitro (21). This apparent discrepancy is likely due to the global haplodeficiency of the α1^+/− mouse model, which affects other systems involved in exercise, including the nervous, cardiovascular, and endocrine systems.

Clearly, inherent limitations due to global and incomplete reduction of Na/K-ATPase α1 in the α1^+/− model warrant future studies in a skeletal muscle-specific model. Although such a model seems required to fully assess the scope and significance of the proposed new isoform-specific trophic role for Na/K-ATPase α1 in the skeletal muscle, several important conclusions can be drawn from the present study. First, the α1-specific trophic role observed in vitro is relevant in vivo. Second, manipulation of Na/K-ATPase α1 content leads to morphological changes in the skeletal muscle that do not impact maximal running capacity, in contrast to manipulation of α2, which did not affect muscle size but affected running capacity. These lend further support for the concept that Na/K-ATPase α1 and α2 serve distinct and isoform-specific functions in the skeletal muscle. Finally, the findings presented here suggest a novel mechanism for exercise-induced changes in muscle size and metabolism. Physiologically, exercise increases serum concentrations of endogenous Na/K-ATPase ligands such as ouabain and leads to modifications of skeletal muscle structure and function (5, 17). Alteration of muscle activity during exercise through modulation of α2 by endogenous Na/K-ATPase ligands has been demonstrated (41), and the present study now suggests that they may also modulate changes in oxidative skeletal muscle structure in response to exercise training through the modulation of α1 signaling. These new findings suggest a possible impact of endogenous or pharmacological administration of CTS on muscle growth, which could be further investigated using the established mouse line expressing ouabain-sensitive α1 (14).

GRANTS

This work was supported by National Institutes of Health Grants HL-109015 and RO1-AR-063710 and Marshall Institute for Interdisciplinary Research funds.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

J.A.H., J.B.L., S.V.P., and Z.X. conceived and designed research; L.C.K., S.T.M., X.W., A.B., and I.L. performed experiments; L.C.K., S.T.M., X.W., and I.L. analyzed data; L.C.K., X.W., I.L., J.A.H., S.V.P., and Z.X. interpreted results of experiments; L.C.K., S.T.M., and I.L. prepared figures; L.C.K., S.V.P., and Z.X. drafted manuscript; L.C.K., S.T.M., J.A.H., S.V.P., and Z.X. edited and revised manuscript; J.A.H., J.B.L., S.V.P., and Z.X. approved final version of manuscript.