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Am J Physiol Regul Integr Comp Physiol 285: R733-R740, 2003. First published June 12, 2003; doi:10.1152/ajpregu.00266.2003
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DEVELOPMENT AND TISSUE PLASTICITY

Exercise training differentially modifies age-associated alteration in expression of Na+-K+-ATPase subunit isoforms in rat skeletal muscles

Yuk-Chow Ng,1 Murali Nagarajan,1 Korinne N. Jew,2 Lisa C. Mace,2 and Russell L. Moore2

1Department of Pharmacology, Milton S. Hershey Medical Center, College of Medicine, The Pennsylvania State University, Hershey, Pennsylvania 17033-0850; and2Department of Integrative Physiology, University of Colorado, Boulder, Colorado 80309

Submitted 13 May 2003 ; accepted in final form 5 June 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The present study tests the hypothesis that endurance exercise training (ETr) reverses age-associated alterations in expression of Na+-K+-ATPase subunit isoforms in rat skeletal muscles. Expression of the isoforms was examined in 16-mo-old sedentary middle-aged, 29-mo-old sedentary senescent, and 29-mo-old treadmill exercise-trained senescent Fischer 344 x Brown Norway rats. Levels of the {alpha}1-isoform increased with age in red gastrocnemius (GR), white gastrocnemius (GW), and extensor digitorum longus (EDL) muscles, and ETr further increased its levels. Levels of the {alpha}2-isoform were unchanged in GR, had a strong trend for a decrease in GW, and decreased significantly in EDL. ETr increased expression of the {alpha}2-isoform in all three muscle groups. There was no increase in expression of the {beta}1-isoform in GR, GW, or EDL with age, whereas ETr markedly increased its levels in the muscles. There was a marked decrease with age in expression of the {beta}2-isoform in the muscle groups that was not reversed by ETr. By contrast, {beta}3-isoform levels increased with age in GR and GW, and ETr was able to reverse this increase. Na+-K+-ATPase enzyme activity was unchanged with age in GR and GW but increased in EDL. ETr increased enzyme activity in GR and GW and did not change in EDL. Myosin heavy chain isoforms in the muscle groups did not change significantly with age; ETr caused a general shift toward more oxidative fibers. Thus ETr differentially modifies age-associated alterations in expression of Na+-K+-ATPase subunit isoforms, and a mechanism(s) other than physical inactivity appears to play significant role in some of the age-associated changes.

{alpha}-subunit; {beta}-subunit; aging; gastrocnemius; extensor digitorum longus; myosin heavy chain

SKELETAL MUSCLE DYSFUNCTION during aging is a syndrome of profound clinical importance, although the underlying mechanisms remain incompletely understood (9, 27, 46). In skeletal muscle, Na+-K+-ATPase plays a central role in the maintenance of Na+-K+ homeostasis (8, 10, 32), which in turn modulates muscle contractile function (15, 38, 39). Thus a change in the expression and/or function of the Na+-K+ pump with advancing age may alter the physiological function of aging skeletal muscle. Indeed, a recent report demonstrated that, in gene-targeted mice, skeletal muscles expressing different ratios of the Na+-K+-ATPase isoforms exhibited distinct contractile properties (18).

Na+-K+-ATPase consists of a transmembrane catalytic {alpha}-subunit and a {beta}-subunit. Multiple isoforms of the {alpha}- and {beta}-subunits have been cloned and sequenced (29-31, 40, 42). Skeletal muscle of mature rats expresses the {alpha}1- and {alpha}2-subunit isoforms and the three {beta}-subunit isoforms (1-3, 21, 28). Fast and slow oxidative-rich fibers express more {alpha}1- and {beta}1-isoforms than fast glycolytic fibers, whereas the opposite is true for the {beta}2-isoform (22, 44). Relative expression of the {beta}3-isoform in the different fibers has not been determined.

We demonstrated previously that, in 6- to 30-mo-old Fischer 344 x Brown Norway rats, advancing age is associated with increased levels of {alpha}1- and {beta}1-isoforms and decreased levels of {alpha}2- and {beta}2-isoforms in red and white gastrocnemius (41). Our data demonstrated, for the first time, dynamic regulation of skeletal muscle Na+-K+-ATPase isozymes during aging. The mechanism(s) underlying the age-associated differential expression of Na+-K+-ATPase subunit isoforms is unclear. One of the important adaptations associated with aging, common in humans and in animal models, is reduced spontaneous physical activity (20, 48). Furthermore, it has been well established that exercise training alters the abundance of Na+-K+-ATPase (11, 17, 33). Therefore, in the present study, we tested the hypothesis that increased physical activity, by endurance exercise training, attenuates or reverses age-associated changes in Na+-K+-ATPase. The corollary to this hypothesis is that physical inactivity contributes to altered expression of the Na+-K+-ATPase subunit isoform associated with advancing age. Our result shows that endurance exercise training differentially modifies expression of the isoforms in aged skeletal muscle; the training paradigm reversed the expression of some, but not all, of the subunit isoforms.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Endurance exercise training. Male 13- to 26-mo-old Fischer 344 x Brown Norway rats were housed in a 12:12-h light-dark cycle and given standard rat chow and water ad libitum. The 26-mo-old rats were arbitrarily assigned to the sedentary or the exercise-trained group. Animals in the trained group underwent 13-14 wk of running on a motorized tread-mill according to a protocol similar to that described previously (23). During the first 2 wk, running duration and speed were progressively increased. From the start of week 3 until death, the final training protocol consisted of running 5 days/wk up a 10% grade at 14 m/min for 5 min and then at 17 m/min for 40 min for a total of 45 min. The protocol was similar for the animals in the sedentary group, except they did not run. At ~24 ± 4 h after the last bout of exercise training, the rats were anesthetized with pentobarbital sodium (35 mg/kg ip) >=15 min after heparin injection (250 U). Skeletal muscles were dissected after hearts were removed. Thus the three experimental groups (n = 12-15/group) were as follows: 16-mo-old middle-aged sedentary (Ms), 29-mo-old senescent sedentary (Ss), and 29-mo-old senescent exercise-trained (St) rats. All the following measurements were performed using tissues from a group of five to six animals randomly selected from each experimental group, except the citrate synthase activity assay, which was performed using tissues from the entire groups of rats. All animal use protocols were approved by the institutional animal care committee.

Preparation of tissue homogenates. Total tissue homogenates were prepared as described previously (41). Briefly, skeletal muscles (~200-300 mg) were pulverized and homogenized with a Polytron (Brinkmann Instruments, Westbury, NY) at a speed of 6.5 (11.0 full scale) for three 20-s periods at 4°C in a buffer containing 10 mM Tris · HCl (pH 7.5), 1 mM EDTA, protease inhibitors (500 µM phenylmethylsulfonyl fluoride), 1 µM leupeptin, 1 µM pepstatin, and 10 µM E-64. Protein concentrations were determined by a protein assay (Bio-Rad, Melville, NY).

Western blot. Na+-K+-ATPase subunits were resolved by SDS-PAGE and immunoblotted as previously described (41). For analysis of the {beta}-subunits, equal amounts of homogenates (80 µg) were first deglycosylated with N-glycosidase F (Glyko, Novato, CA) for 18-20 h at 37°C according to the manufacturer's instructions. Antibodies for {alpha}1- and {alpha}2-isoforms were kindly provided by K. Sweadner (Harvard University). Antibodies for {beta}1 (SpEtB1)- and {beta}2 (GP50)-isoforms were kindly provided by P. Martin-Vasallo (Tenerife, Spain) and P. Beesley (Royal Holloway and Bedform New College, Egham, Surrey, UK), respectively, and antibody for the {beta}3-isoform was purchased from Upstate Biotechnology (Lake Placid, NY). Bound monoclonal antibodies were detected with rabbit anti-mouse IgG antibody followed by 125I-labeled protein A (ICN, Costa Mesa, CA), whereas bound polyclonal antibodies were detected with 125I-labeled protein A alone. The blots were subjected to autoradiography for the purpose of displaying the images. Subsequently, band signal intensities were quantitated by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Because of the relatively low abundance of the {beta}-subunit isoforms, a chemiluminescent detection method was used (SuperSignal West Pico, Pierce, Rockford, IL). Blots were exposed to multiple films to ensure that signals were within the linear range of the film, and relative intensity of the bands was quantitated by densitometry (Molecular Dynamics laser densitometer). Because the anti-{beta}-subunit antibodies generate a significant number of nonspecific bands, we verified the bands of interest by running skeletal muscle controls to observe band shifts at appropriate molecular sizes before and after N-glycosidase F treatment, as demonstrated previously (37) (data not shown). An N-glycosidase F-digested brain microsomal preparation, in which levels of the {beta}-subunits are much higher than in skeletal muscle, was run in the reference lane of the blots to identify the appropriate bands (data not shown). The apparent molecular size of all core {beta}-subunit isoforms is ~35 kDa as revealed by molecular mass markers. The transferred gels and portions of some of the membranes were stained with Coomassie blue to verify efficient transfer of proteins and equal loading, respectively (data not shown).

Enzyme activity. Citrate synthase activity in plantaris muscles was determined as described previously (34) in the entire group of exercise-trained and control rats. Ouabain-sensitive, Na+- and K+-stimulated hydrolysis of [{gamma}-32P]ATP was determined as described by Feschenko and Sweadner (14) with slight modifications. Briefly, tissue homogenates were diluted in 10 mM Tris with 1 mM EDTA (pH 6.8, room temperature) to 150 µg/40 µl. After 10 freeze-thaw cycles in dry ice-acetone, the homogenates were added to a buffer (0.16 ml total) containing (in mM) 100 NaCl, 15 KCl, 5 MgCl2, 5 NaN3, 50 Tris (pH 7.8, room temperature), and 1 EGTA. After a 5-min preincubation, the reaction was started by the addition of 5.5 mM [{gamma}-32P]ATP. Nonspecific enzyme activity was assayed in the presence of 3 mM ouabain. The reaction was allowed to proceed for 25 min and stopped with 0.5 ml of quenching solution (1 N sulfuric acid and 0.5% ammonium molybdate). After 1 ml of isobutanol was added, the phosphomolybdate complex was extracted into the organic phase by vigorous vortexing and separated by centrifugation, and 0.5 ml of the extract was counted with a scintillation counter. For each sample, the assay was performed in triplicate. Specific activity is calculated as the difference between total and nonspecific activity.

Separation of myosin isoforms. The method for separation of myosin isoforms is basically identical to that described by Yu et al. (49), with only slight modifications. Briefly, total tissue homogenates (0.075 µg/15 µl) were separated on 6% SDS gel (0.75 mm thick) at 15°C. Electrophoresis was performed at a constant voltage of 120 V for 24 h, and the gels were stained with silver stain (Amersham Pharmacia Biotech, Piscataway, NJ) immediately after electrophoresis. Relative intensity of the bands was quantitated by densitometry (Molecular Dynamics laser densitometer).

Statistical analysis of data. Values are means ± SE. Oneway ANOVA was used to compare group means, and Duncan's test was used for post hoc analysis. Data were examined at P < 0.05 and P < 0.10 to indicate statistical significance and trends, respectively.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Effects of exercise training on muscle weight and citrate synthase enzyme activity. Red and white gastrocnemius muscle weight decreased by 25.97 and 28.02%, respectively, in Ss compared with Ms rats [red gastrocnemius: 1.286 ± 0.071 and 0.952 ± 0.044 g (n = 6) in Ms and Ss, respectively; white gastrocnemius: 0.811 ± 0.046 and 0.583 ± 0.027 g (n = 6) in Ms and Ss, respectively]. After endurance exercise training, weight of the gastrocnemius muscle in the St rats was not significantly different from that in the Ss animals: 0.937 ± 0.045 and 0.563 ± 0.040 g (n = 5) for red and white gastrocnemius, respectively. On the other hand, the training paradigm significantly increased citrate synthase enzyme activity in the treadmill-trained rats: 21.7 ± 0.8 (n = 15), 21.4 ± 1.7 (n = 12), and 26.1 ± 2.0 (n = 13) µmol · min-1 · g wet wt-1 in Ms, Ss, and St rats, respectively (P < 0.05, Ss vs. St).

Effect of exercise training on subunit isoform expression. Expression of the subunit isoforms was examined in red gastrocnemius, white gastrocnemius, and extensor digitorum longus (EDL) muscles. Total tissue homogenates were used in the present study to avoid unintentional selection of subcellular pools. In red gastrocnemius muscle, levels of the {alpha}1-isoform increased in Ss rats compared with Ms rats (Fig. 1). Expression of the {alpha}1-isoform in senescent rats further increased after exercise training: 1.00 ± 0.06, 1.25 ± 0.08, and 1.44 ± 0.06 arbitrary densitometry units in Ms, Ss, and St rats, respectively. With age, there was a statistically insignificant decrease in expression of the {alpha}2-isoform. Exercise training increased expression of the {alpha}2-isoform in St rats to levels significantly higher than that in Ms and Ss rats: 1.00 ± 0.05, 0.88 ± 0.06, and 1.53 ± 0.07 arbitrary densitometry units in Ms, Ss, and St rats, respectively. Similar changes were observed in white gastrocnemius muscle (Fig. 1), except there was a strong trend for a decrease in the {alpha}2-isoform in Ss rats, and exercise training failed to further increase the already elevated levels of the {alpha}1-isoform in Ss rats ({alpha}1: 1.00 ± 0.11, 1.77 ± 0.15, 1.83 ± 0.26 arbitrary densitometry units in Ms, Ss, and St rats, respectively; {alpha}2: 1.00 ± 0.12, 0.74 ± 0.07, and 1.40 ± 0.05 arbitrary densitometry units in Ms, Ss, and St rats, respectively). In EDL muscle, the changes are also similar to that in the gastrocnemius muscle: there was a strong trend for an increase in the {alpha}1-isoform in Ss rats, and exercise training significantly increased the levels (1.00 ± 0.26, 2.01 ± 0.39, and 2.70 ± 0.36 arbitrary densitometry units in Ms, Ss, and St rats, respectively); there was a small, but significant, decrease in the {alpha}2-isoform in Ss rats, and exercise training reversed that decrease (1.00 ± 0.06, 0.78 ± 0.05, and 0.98 ± 0.06 arbitrary densitometry units in Ms, Ss, and St rats, respectively).



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  		Fig. 1. Levels of {alpha}-subunit isoforms in red gastrocnemius (A), white gastrocnemius (B), and extensor digitorum longus (EDL, C) muscles of rats with age. Skeletal muscle homogenate (100 µg) of mature sedentary (Ms, n = 6), senescent sedentary (Ss, n = 6), and senescent exercise-trained (St, n = 5) rats was resolved by SDS-PAGE. Transferred blots were immunoblotted with {alpha}1- or {alpha}2-isoform-specific antibodies. Top: typical autoradiograms of the blots. Bottom: radioactivity of specific bands quantitated by PhosphorImager, with data normalized to those of Ms rats. Open bars, {alpha}1-isoform; solid bars, {alpha}2-isoform. Values are means ± SE. aSignificantly different from Ms (P < 0.05); bsignificantly different from Ss (P < 0.05).

 

Levels of the {beta}1-isoform in red gastrocnemius muscle were slightly increased in Ss rats compared with Ms rats, although the difference was not statistically significant (Fig. 2). Exercise training markedly increased its expression in St rats compared with Ms and Ss rats: 1.00 ± 0.22, 1.51 ± 0.19, and 4.69 ± 0.45 arbitrary densitometry units in Ms, Ss, and St rats, respectively. Expression of the {beta}2-isoform, by contrast, was markedly decreased in Ss rats compared with Ms rats, and exercise training failed to reverse the decrease: 1.00 ± 0.10, 0.23 ± 0.07, and 0.23 ± 0.05 arbitrary densitometry units in Ms, Ss, and St rats, respectively. Expression of the {beta}3-isoform is very different from expression of the {beta}1- and {beta}2-isoforms: its levels significantly increased in Ss rats, and exercise training reversed that increase (1.00 ± 0.16, 2.16 ± 0.17, and 1.44 ± 0.17 arbitrary densitometry units in Ms, Ss, and St rats, respectively), although not completely.



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  		Fig. 2. Levels of {beta}-subunit isoforms in red gastrocnemius (A), white gastrocnemius (B), and EDL (C) muscles of rats with age. Equal amounts of tissue homogenates (80 µg) from Ms (n = 6), Ss (n = 6), and St (n = 5) rats were deglycosylated with N-glycosidase F, resolved by SDS-PAGE, transferred, and immunoblotted with {beta}1-, {beta}2-, or {beta}3-isoform-specific antibodies. Bound antibodies were detected by chemiluminescence. Top: typical image of the bands. Bottom: intensity of specific bands quantitated by densitometry, with data normalized to those of Ms rats. Open bars, {beta}1-isoform; solid bars, {beta}2-isoform; hatched bars, {beta}3-isoform. Values are means ± SE. aSignificantly different from Ms (P < 0.05); bsignificantly different from Ss (P < 0.05).

 

In white gastrocnemius muscle, expression of {beta}-subunit isoforms is almost identical to that in red gastrocnemius muscle (Fig. 2): 1.00 ± 0.35, 1.04 ± 0.36, and 3.22 ± 0.37 arbitrary densitometry units for the {beta}1-isoform in Ms, Ss, and St rats, respectively; 1.00 ± 0.19, 0.39 ± 0.18, and 0.25 ± 0.08 arbitrary densitometry units for the {beta}2-isoform in Ms, Ss, and St rats, respectively; and 1.00 ± 0.30, 3.30 ± 0.29, and 1.63 ± 0.51 arbitrary densitometry units for the {beta}3-isoform in Ms, Ss, and St rats, respectively. In EDL muscle, expression of the {beta}1- and {beta}2-isoforms is also similar to that in gastrocnemius muscle: 1.00 ± 0.18, 1.34 ± 0.30, and 3.51 ± 0.55 arbitrary densitometry units for the {beta}1-isoform in Ms, Ss, and St rats, respectively, and 1.00 ± 0.16, 0.41 ± 0.13, and 0.43 ± 0.15 arbitrary densitometry units for the {beta}2-isoform in Ms, Ss, and St rats, respectively. Because of an insufficient amount of tissue samples, expression of the {beta}3-isoform in EDL muscle was not examined. Table 1 summarizes the above data regarding expression of the subunit isoforms.


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  		Table 1. Alterations in expression of isoforms during aging and after exercise training

 

Na+-K+-ATPase enzyme activity. Ouabain-sensitive, Na+- and K+-stimulated ATPase enzyme activity was determined in total tissue homogenates. In red and white gastrocnemius muscle, there were no detectable changes in enzyme activity between Ms and Ss rats; however, enzyme activity increased significantly after exercise training (Fig. 3; red gastrocnemius: 21.63 ± 0.97, 22.51 ± 1.52, and 29.13 ± 0.37 µmol · mg protein-1 · h-1 in Ms, Ss, and St rats, respectively; white gastrocnemius: 44.93 ± 4.21, 45.14 ± 3.16, and 57.50 ± 3.30 µmol · mg protein-1 · h-1 in Ms, Ss, and St rats, respectively). In EDL muscle, enzyme activity was significantly higher in Ss than in Ms rats, and exercise training failed to further increase this elevated level of enzyme activity: 46.07 ± 2.41, 55.35 ± 3.04, and 58.45 ± 1.36 µmol · mg protein-1 · h-1 in Ms, Ss, and St rats, respectively.



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  		Fig. 3. Na+-K+-ATPase activity in skeletal muscle of rats with age. Total enzyme activity in Ms (n = 6), Ss (n = 5), and St (n = 5) rats was measured as hydrolysis of [{gamma}-32P]ATP in the presence of 100 mM NaCl and 15 mM KCl in tissue homogenates (150 µg/40 µl) from red gastrocnemius (open bars), white gastrocnemius (solid bars), and EDL (hatched bars) muscles. Nonspecific activity was assayed in the presence of 3 mM ouabain, and specific activity was calculated as the difference between total and nonspecific activity. Enzyme activity from 5 samples in each age group was assayed, and the assay was performed in triplicate. Values are means ± SE. aSignificantly different from Ms (P < 0.05); bsignificantly different from Ss (P < 0.05).

 

Expression of the myosin heavy chain isoforms. Previous studies suggested a fiber type-dependent differential expression of the Na+-K+-ATPase subunit isoforms (22, 44). To examine whether the observed alterations in expression of subunit isoforms with age and exercise training can be correlated with muscle fiber type changes, relative abundance of myosin heavy chain isoforms was determined by SDS-PAGE. Overall, there was no statistically significant change in the level of the myosin heavy chain isoforms in red gastrocnemius, white gastrocnemius, and EDL muscles between Ms and Ss animals (Fig. 4). After exercise training, there was an increase in myosin isoforms I and IIX in red gastrocnemius muscle and a statistically insignificant decrease in IIB in St rats compared with Ss rats. In white gastrocnemius muscle, exercise training increased levels of myosin isoform IIX in St rats, with no significant change in IIB. In EDL muscle, myosin isoforms IIA and IIX appear to migrate close together and could not be resolved in the gel. Exercise training caused a significant decrease in the level of myosin isoform IIB, with no detectable change in the IIA/X in St rats.



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  		Fig. 4. Expression of myosin heavy chain in red gastrocnemius (A), white gastrocnemius (B), and EDL (C) muscles of rats with age. Tissue homogenates (0.075 µg) from Ms (n = 6), Ss (n = 5), and St (n = 5) rats were subjected to SDS-PAGE in 6% gel containing 30% glycerol. Gels were stained with silver for visualization of myosin heavy chain isoforms. Top: typical silver-stained bands. Bottom: intensity of bands as quantitated by densitometry. Open bars, IIX; solid bars, IIB; hatched bars, I; cross-hatched bars, IIA/IIX. Values are means ± SE. aSignificantly different from Ms (P < 0.05); bsignificantly different from Ss (P < 0.05).

 


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
The present study was undertaken to test the hypothesis that increasing physical activity, through endurance exercise training, reverses age-associated changes in expression of the Na+-K+-ATPase subunit isoforms. The corollary to this hypothesis is that physical inactivity contributes to the age-related changes. The data demonstrated that endurance exercise training differentially modifies age-associated alterations in expression of the subunit isoforms and suggest that some of the age-associated alterations in expression of the Na+-K+-ATPase subunit isoforms involve a mechanism(s) that is unrelated to physical inactivity.

Our study shows that senescent Fischer 344 x Brown Norway rats can be trained to undergo a fairly vigorous endurance running paradigm. Because of the physical condition of the senescent rats, the animals were not trained at a higher intensity to avoid unreasonable loss of animals due to injury. It is important to note that the paradigm significantly increased citrate synthase enzyme activity in plantaris muscle of the senescent rats. In recruited skeletal muscle, citrate synthase activity is known to increase with endurance training. Therefore, increases in this activity provide a peripheral marker of skeletal muscle adaptation to exercise. Furthermore, our result showed a significant shift toward slower myosin isoforms in red and white gastrocnemius muscle after exercise training. These data strongly suggest that the senescent rats have received exercise training of sufficient intensity. Along with significant changes in expression of the Na+-K+-ATPase isoforms in white gastrocnemius muscle, our results suggest recruitment of this skeletal muscle under our training paradigm. This is somewhat unexpected, because previous studies suggested that recruitment of white gastrocnemius muscle occurs only when exercise training is at very high intensity (35, 36). Whether the advanced age of the rats is responsible for this apparent difference is unclear. On the other hand, we cannot completely eliminate the possibility that some general training effect was producing the training-induced changes. To limit the number of exercise-trained animals to a more manageable size, we chose not to include exercise-trained young or matured rats in this study. We believe that the simpler experimental design is justified, because in this study we specifically are interested in whether exercise training reverses the changes that occur during senescence, not during growth or maturation of the rats. Some important questions, such as whether exercise training has similar effects in young vs. old rats, cannot be answered by the present study.

In the present study, red gastrocnemius, white gastrocnemius, and EDL muscles were examined, because they represent different muscle types. In the rat, red gastrocnemius muscle consists of predominantly fast oxidative glycolytic fibers mixed with oxidative fibers, white gastrocnemius muscle consists of predominantly fast glycolytic fibers, and EDL muscle consists of roughly equal amounts of fast oxidative glycolytic and fast glycolytic fibers. We have detected qualitatively very similar, but less dramatic, age-associated changes in expression of Na+-K+-ATPase than we reported previously (41). In addition, the previously observed age-associated increase in enzyme activity between middle-aged and senescent rats is not apparent in this study, at least in red and white gastrocnemius muscle (a significant increase was detected in EDL muscle). The reason for these apparent differences is not clear. A careful review of the history of the rat colonies reveals that our previous experiments were performed using animals before the 1999 rederivation of the Harlan colony. We speculate that the difference could be due to subtle variations between the colonies. Lifespan data for the present colony are not available (National Institute on Aging Office of Biological Resources and Resource Development, personal communication). It is possible that a greater magnitude of changes may be observed if older rats were used. In addition, because the rats were housed and trained in Boulder, CO (altitude ~1,650 m), an unexpected influence of high altitude on expression and activity of the subunit isoforms cannot be excluded. Interestingly, Green et al. (16) showed that Na+-K+-ATPase in human skeletal muscle decreased after a 21-day expedition at high altitude. It must be emphasized, however, qualitatively, that data from the present study and our previous study are in excellent agreement. Furthermore, the small differences in no way obscure the conclusions of this study.

As mentioned above, ouabain-sensitive Na+- and K+-stimulated ATPase enzyme activity in red and white gastrocnemius muscle was unchanged between Ms and Ss rats. By contrast, enzyme activity in EDL muscle increased with age, similar to our previously reported results. Exercise training increased Na+-K+-ATPase activity in red and white gastrocnemius muscle. Thus it seems unlikely that physical inactivity plays an important role in the increased enzyme activity that was detected in the present and previous studies.

Expression of the two {alpha}-subunit isoforms in skeletal muscle responded very differently to aging and exercise training. Because increasing physical activity did not reverse the increased levels of the {alpha}1-isoform, we conclude that the age-associated increase in the {alpha}1-isoform is unlikely to be due to physical inactivity. Furthermore, endurance exercise training appears to elicit tissue-specific differential effects in expression of the {alpha}1-isoform. The mechanism(s) underlying this tissue-specific regulation of the {alpha}1-isoform is not clear. It is worth noting that the age-associated changes occurred without significant switches in myosin isoforms and that, despite a switch toward slower fibers in both tissues, exercise training affects red and white gastrocnemius muscle differently. Thus the tissue-specific effect of exercise training does not appear to be primarily due to fiber type changes.

With regard to the {alpha}2-isoform, its levels in red and white gastrocnemius muscle were not significantly different between Ms and Ss rats, in accordance with our previously reported observations, even though a trend toward decreased levels is evident. Indeed, the decrease in the {alpha}2-isoform in EDL muscle of Ss rats reached statistical significance. Previously we observed decreased expression of the {alpha}2-isoform between young (6-mo-old) and middle-aged (18-mo-old) rats (41). Thus, collectively, advancing age is associated with decreased levels of the {alpha}2-isoform. Exercise training increased levels of the {alpha}2-isoform in senescent rats in all three muscle groups examined. The reversal could be due to a switch of fiber types, because the present study shows that exercise training increases the relative amount of oxidative fiber and that expression of the {alpha}2-isoform is higher in this fiber type. However, it appears unlikely that this is the primary, or sole, reason, because data from our previous study and the present study ({alpha}2-isoform in EDL muscle) showed that decreased expression of the {alpha}2-isoform with advancing age is not dependent on fiber type changes.

How these changes in the Na+-K+-ATPase {alpha}-subunit isoforms ultimately affect function of the skeletal muscle in aging and after endurance exercise training remains to be elucidated. The {alpha}-subunit isoforms appear to have different affinities for Na+ and K+ (6, 13, 24), and the {alpha}1-isozyme is a better substrate for phosphorylation by kinase (4, 14). Insulin and exercise appear to selectively translocate the {alpha}2-isozyme from the intracellular site to the plasma membrane (21, 25). Importantly, He and co-workers (18) recently demonstrated that skeletal muscle from a mouse lacking one copy of the {alpha}1-isoform gene showed lower force than skeletal muscle from a wild-type mouse. Conversely, mouse skeletal muscle lacking one copy of the {alpha}2-isoform gene showed greater force. Although it is difficult to extrapolate data from a genetically manipulated animal model, it may be speculated that an increase in {alpha}1-isoform level and a decrease in {alpha}2-isoform level in skeletal muscle with advancing age may result in altered contractile function.

Similar to the {alpha}-subunits, the three {beta}-subunit isoforms also exhibited unique adaptations in their expression during aging and in response to exercise training. Our data demonstrated that expression of the {beta}1-isoform is highly sensitive to exercise training. Whether the increase is primarily due to an increase in oxidative fiber after exercise training cannot be definitively determined in the present study, although our previous study demonstrated that increased expression of the {beta}1-isoform in aged skeletal muscle is not associated with a significant switch in expression of myosin isoforms.

Levels of the {beta}2-isoform decreased dramatically with age, and exercise training failed to reverse or modify the decrease, making it the only subunit isoform examined in the present study in which expression is not modified by exercise training. The data suggest that factors other than physical inactivity are important for its altered expression with age or that, in aged skeletal muscle, expression of the {beta}2-isoform lost its responsiveness to increased physical activity. Levels of the {beta}3-isoform increased in aged skeletal muscles, and exercise training substantially reversed that increase. Thus physical inactivity may be responsible, at least in part, for increased expression of the {beta}3-isoform with aging. Expression of the {beta}3-isoform in skeletal muscle is among the highest in the various tissues examined (1). Whether the {beta}3-isoform performs a special function in skeletal muscle is an interesting question to be explored in future studies. It remains to be determined in skeletal muscle whether the {beta}-subunit isoforms play different roles in the assembly of the {alpha}{beta}-complex or during differentiation of muscle cells. The age-associated changes in levels of {beta}2- and {beta}3-isoforms may be a compensatory response to aging of skeletal muscle or may signify a pathological maladaptation process. However, lower levels of the {beta}3-isoform in younger rats and reversal of increased levels of the {beta}3-isoform in aged skeletal muscle by exercise training seem to suggest that, in rat skeletal muscle, a low level of the {beta}3-isoform is preferred.

In skeletal muscle of exercise-trained senescent rats, as the result of a marked increase in the {beta}1-isoform, a decrease in the {beta}3-isoform, and unchanged levels of the {beta}2-isoform, a significant shift toward more {beta}1-isozyme may be expected, if the elevated levels of {beta}-subunits indeed combine with {alpha}-subunits to form functional units. Interestingly, Klip and co-workers demonstrated that intracellular membranes of red skeletal muscles contain primarily {alpha}2- and {beta}1-isoforms (28) and that insulin stimulates their translocation from the internal membrane to the plasma membrane (21). Thus one may speculate that levels of the {alpha}2{beta}1-type Na+ pump in the internal membrane may be elevated in aged skeletal muscle after exercise training. Such an elevated pool of intracellular Na+ pump may boost availability of the plasma membrane Na+ pump when there is acute demand. In addition, it appears that isozymes with the {beta}2-isoform have lower apparent affinity for K+ (12) and higher affinity for Na+ (5, 7). Thus the age-associated decrease in the {beta}2-isoform could significantly affect the kinetics of ion transport in aging skeletal muscle.

Helwig et al. (19) demonstrated exercise-induced tissue- and isoform-specific alterations in expression of the Na+-K+-ATPase subunit isoforms in rats with myocardial infarction. In their study, exercise training failed to alter expression of all the subunit isoforms examined, except the {beta}2-isoform. The reason(s) for these apparent differences between their study and ours is not clear but could be due to a difference in the exercise-training paradigm or, perhaps more interestingly, could suggest some fundamental differences in responses of the subunit isoforms to exercise training between aged rats and rats with heart failure.

Cellular mechanisms responsible for the exercise-induced altered expression of the subunit isoforms in senescent rats remain unclear. Previous study in young rats demonstrated that an acute bout of exercise increased mRNA content of {alpha}1- and {beta}2-isoforms, but not {alpha}2- and {beta}1-isoforms (45). It was speculated that an increase in intracellular Na+ content could have played a role. Indeed, we and others reported that elevation of intracellular Na+ in various cell types in culture increased expression of the subunit isoforms and/or pump units (26, 43, 47). Nevertheless, it is interesting to note that, in our study using the skeletal muscle C2C12 cell line, increased Na+ transport is associated with increased expression of the {alpha}2-isoform but not the {alpha}1-isoform. These data suggest that the response of skeletal muscle cells in culture to the change in Na+ is different from that of intact skeletal muscle or that a mechanism(s) other than changes in intracellular Na+ plays an important role in expression of the subunit isoforms. Cellular mechanisms responsible for the divergent responses of the three {beta}-subunit isoforms clearly cannot be explained by changes in intracellular Na+ alone and remain to be elucidated.

In conclusion, aging of skeletal muscle is associated with distinct patterns of alterations in expression of the Na+-K+-ATPase subunit isoforms, and exercise training differentially modifies these age-associated alterations. Because endurance exercise training was able to reverse only some of the age-associated changes, physical inactivity appears to play, at most, only a partial role in the age-associated changes. Future investigations will explore the role of other well-known aging-related factors, including hormonal changes and oxidative stress, in altering expression of the Na+-K+-ATPase subunit isoforms during aging.


   DISCLOSURES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This work was supported by National Institute on Aging Grants AG-16822-01A2 (Y. C. Ng) and AG-13981-04 (R. L. Moore).


   ACKNOWLEDGMENTS
 
We thank Drs. K. Sweadner, P. Martin-Vasallo, and P. Beesley for generously providing the antibodies. We thank L. Chung for critical reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y.-C. Ng, Dept. of Pharmacology, College of Medicine, The Pennsylvania State University, Milton S. Hershey Medical Center, Hershey, PA 17033 (E-mail: ycn1{at}psu.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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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