INTRODUCTION

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CHRONIC HEART FAILURE (CHF) produces a reduction in exercise capacity that is commonly associated with the early onset of muscular fatigue (19). Initially, it was proposed that the exercise deficits associated with CHF were primarily the result of skeletal muscle blood flow abnormalities produced in this disease state (19, 68, 69). However, more recent studies have suggested that abnormalities intrinsic to skeletal muscle may be associated with the early onset of fatigue in CHF, including muscle atrophy, reductions in oxidative enzyme capacity, along with changes in fiber type composition, myosin heavy chain expression, and excitation-contraction coupling (4, 18, 25, 39, 43, 52, 59, 61-63).

In addition, sarcoplasmic reticulum (SR) Ca²⁺ release and reuptake are perturbed in CHF such that muscle twitch and tetanic force generation are reduced (29, 52, 67). Moreover, these perturbations coincide with a downregulation in sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) gene expression, and it has been postulated that the downregulation of the SERCA gene may be related to the contractile dysfunction found in the CHF state (53). Along with these changes in SR function, it has been suggested that perturbations in Na⁺ and K⁺ balance across the sarcolemmal membrane could also be contributing factors to the decrements in muscle performance found in CHF (41). Consistent with this hypothesis, a number of studies have shown that skeletal muscle Na⁺-K⁺ pump number is reduced in CHF (49, 51, 56). These pumps are essential in restoring the sarcolemmal transmembrane potential during repeated muscular contractions, and a reduction in the number of these pumps could interfere with contractile performance via reductions in membrane excitability (50). However, contrary to this hypothesis, recent studies on humans (24) and rats (40) have shown that skeletal muscle Na⁺-K⁺ pump number is maintained in the CHF condition. Therefore, the premise that CHF results in a reduction in the number of sarcolemma Na⁺-K⁺ pumps remains controversial.

The Na⁺-K⁺ pump consists of a catalytic alpha ()-subunit and a glycosylated accessory beta ()-subunit (33, 64). Thus far, two isoforms of the -subunit (₁ and ₂) have been found to be expressed in rat skeletal muscle, with the ₂-isoform of the enzyme being more abundant than the ₁-isoform (64). Both of the -isoforms are expressed at greater levels in individual muscles that possess a high-oxidative capacity [i.e., soleus, red portion of the gastrocnemius (gastrocnemius_red) muscle] compared with their low-oxidative highly glycolytic [i.e., white portion of the gastrocnemius (gastrocnemius_white) muscle] counterparts (31). Moreover, the ₂-subunit of the enzyme has a much greater affinity for ouabain than the ₁-subunit (64), and there is evidence suggesting that functional differences may exist between the two isoforms (17).

The exact role that the -subunit has in defining sarcolemmal Na⁺-K⁺ pump activity is not known. However, pump stability, correct folding, and transport of the pump to the plasma membrane are dependent on the interaction of the - and -subunits (11, 14, 44). Presently, three -isoforms have been identified (₁, ₂, and ₃) and are known to exist in rat skeletal muscle (5, 37). The ₁-isoform is highly expressed in slow-twitch oxidative fibers (SO; i.e., soleus), whereas the ₂-isoform is found predominantly in fast-twitch glycolytic fibers (FG; i.e., gastrocnemius_white muscle)(32). In comparison, both ₁- and ₂-isoforms are expressed in muscle fibers that are fast-twitch, highly oxidative, and glycolytic in nature (FOG; i.e., gastrocnemius_red muscle) (32). Studies in both human and rats show that each of the -subunits imparts unique pharmacological and transport properties to the pump (12, 17).

Recently, our laboratory demonstrated that the number of ouabain binding sites was reduced in the soleus, plantaris, and the gastrocnemius_red muscle of rats with CHF (49). Because these reductions in the number of ouabain binding sites coincided with decrements in maximal oxygen uptake (O_2 max) and endurance capacity, it suggested that a reduction in the number of Na⁺-K⁺ pumps (Na⁺-K⁺-ATPase) produced in CHF may be a contributing factor to the reductions in exercise performance found in this disease state. The [³H]ouabain binding assay used in our previous investigations primarily reflects changes in the expression of the ₂-subunit of the enzyme (22, 49, 56, 64). Therefore, the possibility exists that a compensatory increase in the ₁-subunit could have occurred such that Na⁺-K⁺-ATPase activity was maintained in the CHF animals. Moreover, changes in -isoform expression could significantly impact the number of functional pumps found at the sarcolemma, thereby influencing total enzyme activity via - interaction (9, 10, 26).

The present investigation was undertaken to determine whether the expression of the - and -subunits of the Na⁺-K⁺ pump is modified in the skeletal muscle of rats with CHF. On the basis of results from our laboratory (49, 56), we tested the hypothesis that the expression of the ₂-subunit would be reduced in muscle that possessed a high oxidative capacity (i.e., the gastrocnemius_red). In addition, we expected that ₁-subunit expression would not be significantly changed in either the gastrocnemius_red or the gastrocnemius_white muscle. In regard to -isoform expression, we tested the hypothesis that -subunit expression would remain stable in the CHF condition. This hypothesis is based on the recent findings of Lunde and colleagues (40) where these investigators demonstrated that the expression of the - and -subunit of the Na⁺-K⁺ pump did not change in the muscles of rats with CHF compared with non-CHF controls. Finally, we tested the hypothesis that exercise training would increase the number of ouabain binding sites along with - and -subunit expression in the muscle of rats with CHF. This hypothesis is based on the fact that endurance exercise training has been shown to increase Na⁺-K⁺ pump number (16, 21, 23, 36) along with the finding that acute exercise has been shown to increase - and -subunit expression in the skeletal muscle of normal rats (66).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Female Wistar rats were obtained from Charles River Laboratories. All rats were housed in 6 × 9-in. cages, received rat chow and water ad libitum, and were maintained on a 12:12-h light-dark cycle. All experimental procedures were approved by the Institutional Animal Care and Use Committee at Kansas State University.

Surgically induced myocardial infarction. Rats were anesthetized initially with a 5% halothane-95% oxygen mixture. Rats were intubated, connected to a rodent respirator (model 680, Harvard Apparatus), and maintained on 2% halothane-98% oxygen mixture. The heart was exposed through a left-sided thoracotomy between the fifth and sixth rib. The pericardial sac was opened, and the heart was exteriorized. Rats received either a myocardial infarction (MI) or a sham operation (sham) as described previously (48). In rats receiving a MI, the left main coronary artery was ligated between the pulmonary artery and the left atrium. A 6-0 suture was placed around the left main coronary artery and tied. Sham operations were completed by using the same surgical procedure except that the coronary artery was not ligated. After these procedures, the lungs were hyperinflated and the ribs approximated with 3-0 gut. The muscles of the thorax were sewn together with 4-0 gut, and the skin incision was closed with 3-0 silk. Analgesic agents were applied to each animal. Anesthesia was withdrawn, and the animals were extubated. All rats were returned to individual cages. Postoperatively, each rat received ampicillin (50 mg/kg sc) for each day for 5 days.

Determination of endurance exercise capacity. Six weeks after the MI or sham operation, all rats performed a treadmill exercise test to fatigue. Our laboratory's use of this exercise test has previously demonstrated that rats with CHF suffer from reduced exercise capacity as indicated by the early onset of fatigue compared with noninfarcted sham-operated control animals (1). The exercise protocol consisted of a graded running test in which each rat initially ran up a 5% grade at a speed of 25 m/min for 15 min. Thereafter, the treadmill speed was increased 5 m/min every 15 min until each animal reached the point of fatigue. The criterion for fatigue was the rat's inability to keep pace with the treadmill, even though the animal was encouraged to run by applying bursts of high-pressure air at the hindquarters. At the end of each exercise test, the end point of fatigue was confirmed by the loss of the animal's righting reflex. Time from the beginning of the exercise to the removal of the rat from the treadmill was measured and recorded to the nearest half minute.

Determination of O_2 max . O_2 max was determined for all rats according to previously established methods that have been used extensively in our laboratory (46). This method uses a metabolic chamber (14.5 × 43 × 7 cm) designed to fit into a stall of a 10-channel rodent treadmill and utilizes the standard techniques described by Brooks and White (13) for determining oxygen uptake (O₂) and carbon dioxide production (CO₂).

Exercise training protocol. After the endurance capacity and O_2 max had been determined for each animal, the MI rats were separated randomly into exercise training and sedentary control groups. The rats in the exercise training (MI-T) group were subjected to an exercise training protocol that consisted of having each animal run on a motor-driven treadmill at a speed of 27 m/min up a 10% grade for 60 min/day (47). The MI-T rats ran 5 days/wk and trained for 6 wk. Familiarity with treadmill running was maintained in both the sedentary control groups of MI and sham rats by having each animal run on the treadmill for 5 min/day at a treadmill speed of 20 m/min up a 10% grade. At the conclusion of the 6-wk training period or sedentary control period, all rats had their endurance capacity and O_2 max redetermined using the same protocols as described above. During this time, the rats in the MI-T group continued training until all measurements of exercise capacity were complete.

Determination of left ventricular dysfunction. Left ventricular (LV) function was determined for each rat after posttraining or postsedentary control measurements of endurance capacity and O_2 max had been completed (6-8 wk after training and 12-14 wk after the initial sham or MI surgery had been performed).

[³H]ouabain binding. The number and affinity of Na⁺-K⁺ pumps in the soleus and plantaris muscles were determined by using a radiolabeled ([³H]ouabain) binding assay (51, 56). These muscles of the ankle extensor group of the rat's hindlimb were selected because they are recruited significantly during exercise (48). In addition, the soleus muscle contains nearly 100% SO fibers, whereas the plantaris muscle contains a mixed fiber type composition (SO, FG, and FOG) (3). Moreover, previous results from our laboratory have demonstrated that the number of Na⁺-K⁺ pumps that have a high affinity for ouabain found in these muscles was significantly reduced in MI rats with CHF (49).

Assay protocol. The preincubation wash in the standard solution alone was performed on ice for 20 min to remove extracellular K⁺. Slices were incubated in fresh standard solution with radiolabel and gently shaken at 37°C for 3 h in a Dubnoff metabolic incubator. Total binding was obtained over the concentration range of 5-1,000 nM ouabain. Nonspecific binding was determined by using an excess of unlabeled ouabain (10⁴ M). All wells contained 5 nM [³H]ouabain titrated to the appropriate concentration with unlabeled ouabain. Free ouabain in the incubation solution was determined at the end of the incubation by removing 250 µl of the incubation medium from the wells with the most dilute concentration of ouabain (5 nM).

4

Isolation of membrane proteins. Frozen gastrocnemius_white and gastrocnemius_red muscle samples were homogenized (Pro Scientific) in 5 vol of cold (4°C) isolation buffer (10 mM imidazole and 0.3 M sucrose, pH 7.4) containing protease inhibitor cocktail (1:500 vol/vol). The gastrocnemius_red and gastrocnemius_white muscles are from the ankle extensor group of the rat's hindlimb and were selected because they are recruited significantly during exercise (48). Moreover, the gastrocnemius_red muscle contains ~91% SO and FOG fibers (3). In contrast, the gastrocnemius_white muscle contains ~91% FG fibers (3).

Immunoblotting and densitometry. Skeletal muscle proteins (30 µg) were incubated in Laemmli sample buffer and heated at 37°C for 30 min. Proteins were run on a 4-20% acrylamide gel (Bio-Rad). On completion of the run, proteins were transferred to a nitrocellulose membrane by electrophoretic transfer in a tank system with plate electrodes. Membranes were incubated at room temperature with a primary polyclonal antibody (1:2,000 vol/vol) in 5% nonfat milk and Tris-buffered saline (TBS: 100 mM Tris and 0.9% NaCl, pH 7.5) containing 0.1% Tween 20. After incubation, membranes were washed four times with 0.1% TBS Tween. All antibodies used in this investigation [₁, ₂, ₁, ₂, ₃, and dihydropyridine receptor (DHPR)] were obtained from Upstate. The membrane was incubated at room temperature in a horseradish peroxidase-labeled secondary antibody diluted (1:25,000) in TBS with 0.1% Tween. After four washes with TBS Tween, blots were visualized with chemiluminescence (West Femto, Pierce Chemical) and recorded on radiographic film. After visualization, membranes were stripped of all antibodies (Restore Western blot stripping buffer, Pierce Chemical) via incubation at 37°C for 1 h. Membranes were probed to ensure complete removal of antibodies and then probed with a second primary antibody as described above. Densitometry was performed by using AlphaEase Image software. Densitometry was compared within groups and assigned arbitrary units. Linear range of protein expression was established by loading 0-80 µg of membrane protein on gels followed by subsequent exposure of the blots to the radiographic film at timed intervals to ensure that the signals were in the linear range. All analyses shown are of the 30 µg of membrane protein, which was shown to be within the linear range of detection. In addition, all immunoblots were run with prepared brain tissue as a positive control.

Enzyme-coupled assay of ouabain-sensitive Na+-K+-ATPase activity. NADH was measured to assess ouabain sensitive activity as described by Schwinger et al. (58). Samples (with and without ouabain) were treated with either 0.5% saponin or assay buffer and incubated on ice for 1 h before spectrophotometry. The assay was started by adding 50 µl of NADH (1.28 µM) to a cuvette containing 900 µl of assay buffer (150 mM NaCl, 10 mM KCl, 100 mM NH₄Cl, 5 mM MgCl₂, 5 mM ATP, 2.5 mM phosphoenolpyruvate, and 150 mM immidazole HCl, pH 7.25). In addition 50 µl of phosphoenolpyruvate (58 units/mg protein) were added to the assay buffer. A 50-µl protein sample was added to the cuvette, and the decrease in absorbance was measured every 10 s for 1 min at a wavelength of 340 nm on a spectrophotometer (Shimadzu UV-120). The difference between maximal activity and ouabain-sensitive activity was used to determine Na⁺-K⁺-ATPase activity.

Determination of CS activity. CS activity, an index of oxidative capacity, was determined for the plantaris muscle of each rat. Tissue samples were homogenized at 0°C in a volume of 100 mM KPO₄ buffer such that a 1:20 (wt/vol) homogenate was obtained. CS activity was measured according to the spectrophotometric method of Srere (60). All assays were linear with respect to time and dilution, and each sample was analyzed in duplicate.

Statistical analysis. Structural and hemodynamic indexes, plantaris CS activity, the maximal number of specific ouabain binding cites (B_max), the apparent dissociation constant (K_d), densitometry results from the - and - subunit protein expression and the Na⁺-K⁺-ATPase activity measured with the enzyme-coupled assay were compared between sham, MI, and MI-T rats with a one-way ANOVA. Indexes of exercise performance (O_2 max and run time to fatigue) were analyzed with a two-way ANOVA with a repeated-measures design. When a significant F value was demonstrated by the ANOVA, a Student-Newman-Kuels post hoc test was performed to detect differences between mean values.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Structural and hemodynamic indexes indicative of LV dysfunction and CHF were prevalent in both MI and MI-T rats. Accordingly, the MI and MI-T group of rats demonstrated elevations in LVEDP compared with sham rats along with reductions in mean arterial pressure and LV dP/dt (Table 1). LV weight normalized to body weight was elevated in both the MI and MI-T groups of rats. Moreover, these increases in LV weight coincided with increases in lung weight-to-body weight ratio, demonstrating that these animals had significant pulmonary congestion. Because increases in RV weight normalized to body weight were also found in these animals, they appeared to be in a chronic state of compensated congestive heart failure.

Decrements in exercise performance were found in both the MI and MI-T groups of rats before the training protocol was initiated. These decrements were manifested as reductions in O_2 max and exercise endurance capacity (run time to the point of fatigue) found in both groups of MI rats compared with their sham counterparts (Fig. 1). Moreover, these decrements in exercise performance before training were similar for both the MI and MI-T groups.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Maximal oxygen uptake (O2 max) was determined for sham sedentary control rats (sham; n = 10), myocardial infarction sedentary control rats (MI; n = 16), and myocardial infarction endurance-trained rats (MI-T; n = 16) both before (solid bars) and after 6-8 wk of endurance training or sedentary control conditions (hatched bars) (A). Similarly, run time to fatigue was determined for the same animals (B). Values are means ± SE. *P < 0.05 compared with sham.  P < 0.05 compared with pretraining value.

The training regimen used in this study produced a 34% increase in the CS activity of the plantaris muscle of the MI-T group of rats compared their sedentary counterpart (Fig. 2). Sham and MI groups did not differ in CS activity.

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Citrate synthase (CS) activities measured in the plantaris muscle of sham rats (n = 10), MI rats (n = 16), and MI-T rats (n = 16). Values are means ± SE. *P < 0.05 compared with sham.  P < 0.05 compared with MI.

The decrements in exercise performance that were found in the MI rats were retained in this group of animals after the 6-8 wk of sedentary control conditions (Fig. 1). However, the MI-T group of rats demonstrated significant increases in exercise performance after training because O_2 max increased to the same level as the sham group of rats after 6-8 wk of sedentary control conditions. In addition, run time to fatigue for the MI-T group of rats increased after 6-8 wk of training and actually exceeded those found for their sham counterparts.

The number of [³H]ouabain binding sites (B_max) was reduced in the soleus (14%) and plantaris (13%) muscles of the MI rats compared with their sham counterparts (Table 2). On the other hand, B_max was increased (+20%) in the soleus in the MI-T group of rats compared with their sedentary MI counterparts. Moreover, B_max was increased (+45%) in the plantaris of the MI-T group of rats compared with their sedentary MI counterparts and actually exceeded those found for the sedentary sham rats. In comparison, the affinity (K_d) of the single population of [³H]ouabain binding sites found for all muscles examined was similar across the different groups of animals.

Both - and -subunits of the Na⁺-K⁺-ATPase were found to be expressed in both the gastrocnemius_white and gastrocnemius_red muscles of the sham, MI, and MI-T group of rats (Fig. 3). DHPR expression was used as a positive control and to ensure that our results were not a result of differences in protein loading of the gels because DHPR expression in skeletal muscle remains unchanged in the CHF condition (40). DHPR expression was similar for all three groups of rats in both the gastrocnemius_white and gastrocnemius_red muscle.

View larger version (69K): [in this window] [in a new window]   Fig. 3.   Representative immunoblots of the - and -isoforms expressed in the white portion of the gastrocnemius (gastrocwhite) and red portion of the gastrocnemius (gastrocred) muscles of sedentary sham control rats (SH), sedentary MI control rats (MI), and MI endurance-trained rats (MIT). Arrows, location of the molecular mass marker in kDa. Both 1- and 2-isoforms along with 1-, 2-, and 3-isoforms were expressed in both muscles. Dihydropyridine receptor (DHPR) expression was used as a positive control. Densitometry results indicated no differences in DHPR expression for either the gastrocwhite or the gastrocred muscle.

In the gastrocnemius_white muscle, -isoform expression along with -isoform expression was similar for the sham, MI, and MI-T groups of rats (Fig. 4). In contrast, - and -isoform expression in the gastrocnemius_red muscle was significantly different between these groups (Fig. 5). In the gastrocnemius_red muscle, ₁-isoform expression was similar for the sham, MI, and MI-T groups of rats (Fig. 5A). However, ₂-isoform expression was reduced in the MI group of rats compared with the sham, but at the same time, ₂-isoform expression found in the MI-T group of rats was not significantly different from those found in the MI or the sham group of rats (Fig. 5A). Similar to that found in the gastrocnemius_white, ₁- and ₃-isoform expression in the gastrocnemius_red muscle was similar between the sham, MI, and MI-T groups of rats (Fig. 5B). In addition, ₂-isoform expression was similar between the sham and MI group of rats. However, ₂-isoform expression was significantly greater in the MI-T group of rats compared with the sham, and there was a trend for the ₂-isoform expression to be greater (P = 0.09) in the MI-T group of rats compared with their sedentary MI counterparts (Fig. 5B).

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Densitometry results of the immunoblot analysis of the  (A)- and -isoforms (B) measured in the gastrocwhite muscle of sham, MI, and MI-T rats. Values are means ± SE. Number of observations in each group of sham, MI, and MI-T rats are the following: 1, n = 6; 2, n = 8; 1, n = 6; 2, n = 5; 3, n = 5.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Densitometry results of the immunoblot analysis of the  (A)- and -isoforms (B) measured in the gastrocred muscle of sham, MI, and MI-T rats. Values are means ± SE. Number of observations in each group of sham, MI, and MI-T rats are the following: 1, n = 7; 2, n = 8; 1, n = 6; 2, n = 5; 3 n = 4. *P < 0.05 vs. sham group of rats. § MI-T and sham group of rats are not significantly different, P > 0.05.

Na⁺-K⁺-ATPase activity was measured by using an enzyme-coupled assay in the same gastrocnemius_red muscle samples in which the expression of - and -subunits had been performed. Unfortunately, only limited amounts of tissue remained after immunoblot analysis and the Na⁺-K⁺-ATPase activity could be determined for only three animals in each group. No statistical differences in activity were detected between the sham, MI, and MI-T groups of rats. However, the amount of Na⁺-K⁺ activity measured in the MI group of rats showed a strong trend to be reduced compared with their sham counterparts. Furthermore, the amount of activity measured in the MI-T group of rats appeared to be similar to that found in the sham group after 6-8 wk of endurance exercise training (Fig. 6).

View larger version (36K): [in this window] [in a new window]   Fig. 6.   Enzyme-coupled assay of ouabain-sensitive ATPase activity with NADH. Values are means ± SE. The ouabain-sensitive ATPase activity was decreased in the gastrocnemiusred of MI rats with severe left ventricular dysfunction and chronic congestive heart failure (n = 3) compared with sham counterparts (n = 3). This decrease in activity was ameliorated in the MI-T rats (n = 3).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The present investigation is the first to demonstrate that CHF alters the ₂-isoform expression of the Na⁺-K⁺ pump within rat skeletal muscle and that endurance exercise training can partially reverse these alterations. Specifically, in CHF rats, we found a downregulation of the ₂-subunit (without a compensatory upregulation of the ₁-subunit) within high-oxidative muscle (gastrocnemius_red) that was associated with a reduced exercise capacity (i.e., O_2 max and exercise endurance). These changes were not found in the low-oxidative highly glycolytic gastrocnemius_white muscle. Training-induced normalization of ₂-subunit expression and increased ₂-subunit expression occurred in CHF rats concomitant with an elevation of ouabain binding sites to control levels in soleus and above control levels in plantaris muscles in conjunction with an augmented exercise capacity. Within the muscles examined, results suggest that exercise training is an effective stimulus for reversing CHF-induced Na⁺-K⁺ pump activity impairment and also that maintenance of Na⁺-K⁺ pump activity is associated with exercise capacity.

Effects of CHF on the Na+-K+ pump. Previous results from our laboratory have shown that the number of ouabain binding sites is reduced in the soleus, plantaris, and gastrocnemius_red but maintained in the gastrocnemius_white muscle of rats with severe LV dysfunction and CHF (49). The present investigation confirmed these observations because we found the number of ouabain binding sites to be reduced in the soleus and plantaris muscles of the MI group of rats compared with their sham counterparts. Moreover, the amount of ₂-isoform that was expressed in the gastrocnemius_red muscle was significantly reduced in these animals (Figs. 3 and 5) and is consistent with a reduction in Na⁺-K⁺-ATPase activity that was measured in the same muscle samples (Fig. 6). In contrast, ₂-subunit expression was maintained in the gastrocnemius_white muscle of the MI rats compared with their sham counterparts. This maintenance of the ₂-subunit of the pump is consistent with previous findings from our laboratory where the number of ouabain binding sites was maintained in the gastrocnemius_white muscle of rats with a similar degree of LV dysfunction and CHF (49).

Effects of endurance exercise training on the Na+-K+ pump. Studies have shown that acute exercise and/or muscle contractions will significantly increase skeletal muscle Na⁺-K⁺ pump activity (16) along with inducing the expression of both ₁- and ₂-subunits of the enzyme (66). Similarly, chronic exercise or endurance exercise training has been shown to increase the number of Na⁺-K⁺ pumps as indicated by the number of ouabain binding sites found in skeletal muscle (23, 35, 42, 45). Based on these studies, one would expect the expression of the ₂-subunit to increase with chronic treadmill exercise training. However, the question as to whether chronic endurance exercise training would significantly increase the expression of the ₁-subunit as suggested by the effects of acute exercise was central to the design of the present investigation. As expected, endurance exercise training (chronic treadmill running) increased the number of ouabain binding sites found in the soleus and plantaris muscles (Table 2) along with the expression of the ₂-isoform in the gastrocnemius_red muscle (Fig. 5). However, contrary to our expectations endurance exercise training did not increase the expression of the ₂-isoform in the gastrocnemius_white muscle (Fig. 4), nor did this type of training significantly increase the expression of the ₁-subunit in either of these muscles (Figs. 4 and 5).

Model considerations or limitations. Relevant to the present investigation, a number of limitations in regard to data interpretation should be acknowledged. First, only limited amounts of gastrocnemius_red and gastrocnemius_white muscle were obtained from the animals used in these experiments. To ensure that the tissues harvested from the gastrocnemius muscle were truly representative of the red and white regions, we attempted to minimize the opportunity for any fiber type contamination from adjacent regions of the muscle. Because of limited amounts of tissue, Na⁺-K⁺-ATPase activity (using a coupled-enzyme assay) was measured on the same gastrocnemius_red muscle samples that were subjected to immunoblot analysis from only a very limited number of animals (n = 3) found in each experimental group. Thus the Na⁺-K⁺-ATPase activities expressed in the present investigation (Fig. 6) should be construed as preliminary in nature.