INTRODUCTION

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THE MOST COMMON SYMPTOM EXPERIENCED by individuals with chronic heart failure (CHF) is a reduced exercise capacity that is associated with the early onset of muscular fatigue (12). Initially, it was thought that early onset of fatigue found in the CHF state was primarily the result of a reduced skeletal muscle blood flow response to exercise (12, 58, 59). However, more recent investigations have shown that abnormalities intrinsic to skeletal muscle may enhance muscle fatigability (3, 17, 30, 34, 36, 52).

Accordingly, a large number of investigators have demonstrated that the oxidative capacity of skeletal muscle is reduced in the CHF state (3, 36, 51). In addition, skeletal muscle atrophy and changes in fiber type composition and myosin heavy chain (MHC) expression have been shown to occur in CHF (11, 50, 51, 53). Along with these changes in skeletal muscle biochemistry and morphology, functional changes in excitation-contraction coupling have been shown to occur in CHF. Sarcoplasmic reticulum (SR) Ca²⁺ release and reuptake are perturbed in the CHF state such that twitches and tetanic force generation are reduced (20, 46, 57). Moreover, these perturbations in SR function coincide with a downregulation in SR Ca²⁺-ATPase gene expression, and it has been postulated that this downregulation of the SR Ca²⁺-ATPase gene may be related to the contractile dysfunction found in CHF (48).

Although the decrements in contractile function found in CHF may be partially ascribed to changes in SR function, it has been suggested that perturbations in Na⁺ and K⁺ balance across the sarcolemmal membrane of exercising muscle could contribute to the early onset of muscular fatigue found in individuals with CHF (31). Consistent with this hypothesis, studies have shown that the number of Na⁺-K⁺ pumps in skeletal muscle are reduced in CHF (44, 47), and, considering that these pumps are essential in restoring the sarcolemmal transmembrane potential during repeated muscular contractions, it has been postulated that the downregulation of these Na⁺-K⁺ pumps can interfere with contractile performance via reductions in membrane excitability (43).

In a recent investigation, Green et al. (16) found that the Na⁺-K⁺-ATPase concentration in the vastus lateralis muscle of patients with moderate CHF was significantly correlated with the CHF patient's ability to perform exercise as indicated by the individual's peak oxygen uptake (O_2 peak). This correlation between Na⁺-K⁺ pump activity and O_2 peak suggests that 1) pump activity or number may be related to the severity of CHF and 2) reductions in pump activity or number may be contribute to the decrements in exercise performance commonly found with this pathological condition. On the basis of the results of Green and colleagues, the present investigation was undertaken to determine whether the reduction in the number of skeletal muscle Na⁺-K⁺ pumps that have a high affinity for ouabain (i.e., the concentration of [³H]ouabain binding sites) found during CHF is related to 1) the severity of the disease state, 2) the fiber type composition of the muscle being investigated, and 3) exercise performance as tested by the duration of an endurance run to the point of fatigue along with the measurement of the animal's maximal oxygen uptake (O_2 max). We tested the hypothesis that the concentration of [³H]ouabain binding sites (B_max) would be reduced in rats with severe left ventricular (LV) dysfunction and CHF but not in rats with moderate LV dysfunction and CHF. We also tested the hypothesis that the reduction in B_max found in the individual muscles examined in this investigation would be significantly correlated with O_2 max and/or each animal's endurance capacity as designated by the time to fatigue during a progressive treadmill exercise test. Four muscles were chosen that represented slow-twitch oxidative (SO), fast-twitch oxidative glycolytic (FOG), fast-twitch glycolytic (FG), and mixed fiber types.

	METHODS

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Female Wistar rats were obtained from Charles River Laboratories. All rats 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 oxygen mixture. They were intubated, connected to a rodent respirator (model 680, Harvard Apparatus), and maintained on 2% halothane-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 as described previously (42). In rats receiving an 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 were 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. Postoperatively, each rat received ampicillin (50 mg/kg sc) for each day for 5 days. All rats were returned to individual cages. Each cage was 6 in. wide and 9 in. long.

Determination of endurance exercise capacity. Six weeks after the MI or sham operation, all rats performed a treadmill exercise test to fatigue. Our 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 application of bursts of high-pressure air at the hindquarters. At the end of each exercise test, the end point of fatigue was confirmed by 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 9 sham and 15 MI rats according to previously established methods that have been used extensively in our laboratory (39). 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 (6) for determining oxygen uptake (O₂) and carbon dioxide production (CO₂).

Determination of LV dysfunction. The presence and severity of CHF were evaluated in each rat by using the following structural indexes: changes in LV, right ventricular (RV), and total lung weight normalized to body weight. Hemodynamic indexes included changes in mean arterial pressure and LV end-diastolic pressure (LVEDP). Approximately 8-10 wk after the initial surgery, each rat was anesthetized (pentobarbital sodium, 25 mg/kg ip), and the right carotid artery was cannulated with a 2-Fr catheter-tip pressure manometer (Millar Instruments) for the recording of arterial pressure and heart rate. While the rat was breathing spontaneously, the micromanometer was advanced into the LV in a retrograde fashion for measuring ventricular systolic and diastolic pressures. Immediately after measurement of ventricular pressures, the micromanometer was removed from the animal, and the soleus, plantaris, and red and white portions of the gastrocnemius muscles were harvested. After the skeletal muscles were removed, the rats were killed with an overdose of anesthetic (pentobarbital sodium, 100 mg/kg ip).

Ouabain binding assay. The number of Na⁺-K⁺ pumps that have a high affinity for ouabain in the soleus, plantaris, and the red and white portions of the gastrocnemius muscles was determined by using a radiolabeled ([³H]ouabain) binding assay (44, 47). These muscles or muscle parts of the ankle extensor group of the rat's hindlimb were selected because they are recruited significantly during exercise (42) and they represent muscles containing a majority of SO (soleus), FOG (red portion of the gastrocnemius), and FG (white portion of the gastrocnemius) types of fibers along with a muscle (plantaris) containing a mixed fiber type composition (2). In addition, these muscles or muscle parts contain a significant range of oxidative capacity as indicated by their citrate synthase (CS) activity (11). Each muscle was cut into 800-µm-thick transverse sections by use of a McIlwain tissue chopper (Brinkman Instruments). Each muscle slice was placed in a separate well of a tissue culture plate. Each well was filled with 2 ml of a standard solution (in mM): 10 Tris · HCl, 3 MgSO₄, 1 sodium vanadate, and 250 sucrose (pH 7.3).

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

Statistical analysis. Structural and hemodynamic indexes, and indexes of exercise performance (O_2 max and run time to fatigue) were compared between noninfarcted sham-operated control rats and rats with either moderate (LVEDP < 20 mmHg) or severe (LVEDP > 20 mmHg) LV dysfunction (11) with a one-way ANOVA. When a significant F value was demonstrated by the one-way ANOVA, a Student-Newman-Keuls post hoc test was performed to detect differences between mean values. B_max and the apparent dissociation constant for ouabain were determined for each rat and each muscle by using Scatchard analysis. These results were also analyzed with a one-way ANOVA. To determine whether a relationship existed between exercise performance parameters (O_2 max and run time to fatigue) and the degree of LV dysfunction that developed in each animal (LVEDP) and/or the B_max found in each muscle, the results were examined by use of linear and curvilinear regression analysis. P < 0.05 was considered to be statistically significant. Group data for each variable are expressed as mean ± SE.

	RESULTS

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MIs were induced in 23 rats, and sham operations were performed in 14 rats. Of the 23 rats that received an MI, 18 were categorized as having moderate LV dysfunction (LVEDP < 20 mmHg) whereas 5 were categorized as having severe LV dysfunction (LVEDP > 20 mmHg). LVEDP in sham rats was significantly lower than either group of MI rats. Body weights were not significantly different between the groups (Table 1).

Structural and hemodynamic indexes indicative of heart failure were prevalent in the MI rats. LV weight normalized to body weight was elevated in rats with moderate and severe LV dysfunction. Moreover, these increases in LV weight coincided with increases in LVEDP compared with sham rats, but the rats with severe LV dysfunction also demonstrated significantly greater increases in LVEDP compared with their counterparts with moderate LV dysfunction. Rats with severe LV dysfunction demonstrated increased lung weight normalized to body weight, suggesting that these animals had significant pulmonary congestion. Because increases in RV weight normalized to body weight were also found in these animals, they were likely in a chronic state of compensated congestive heart failure.

Decrements in exercise performance were found in rats with severe LV dysfunction as indicated by reductions in exercise endurance (run time to the point of fatigue) and O_2 max (Fig. 1). Interestingly, these decrements in exercise performance were not found in rats with moderate LV dysfunction. However, if run time to the point of fatigue and O_2 max were plotted as a function of LVEDP measured in MI rats, both indexes of exercise performance were significantly correlated with this index of LV dysfunction (Fig. 2). In addition, the endurance capacity of these MI rats was highly correlated with their O_2 max (Fig. 3).

View larger version (42K): [in this window] [in a new window]   Fig. 1.   A: exercise performance as measured by run time to fatigue during a progressive treadmill test for sham (n = 14) and myocardial infarction (MI) rats with moderate (n = 18) and severe (n = 5) left ventricular (LV) dysfunction. B: similarly, maximal oxygen uptake (O2 max) was determined for sham (n = 9) and MI rats with moderate (n = 10) and severe (n = 5) LV dysfunction. Values are means ± SE. * P < 0.05 compared with sham.  P < 0.05 compared with moderate group.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   A: relationship between run time to fatigue and LV end-diastolic pressure (LVEDP) measured in MI rats with moderate (n = 16) and severe (n = 5) LV dysfunction. B: relationship between O2 max and LVEDP in MI rats with moderate (n = 8) and severe (n = 5) LV dysfunction.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Relationship between run time to fatigue and O2 max measured in MI rats with moderate (n = 10) and severe (n = 5) LV dysfunction.

The concentration of [³H]ouabain binding sites (B_max) was significantly reduced in the soleus (27%), plantaris (22%), and red portion of the gastrocnemius muscle (20%) of the rats with severe LV dysfunction compared with sham rats and rats with moderate LV dysfunction (Table 2). In comparison, the concentration of [³H]ouabain binding sites found in the white portion of the gastrocnemius muscle and the affinity of the single population of [³H]ouabain binding sites found for all muscles examined were similar across the different groups of animals. If run time to the point of fatigue was plotted as a function of B_max found in each muscle, it was demonstrated that the concentration of [³H]ouabain binding sites was not correlated with this index of exercise performance in the MI rats. However, if O_2 max was plotted as a function of B_max found in each muscle, regression analysis demonstrated that the results were best fit and linearly correlated with this index of exercise performance (Fig. 4).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Relationship between O2 max and number of Na+-K+ pumps (Bmax) measured in the soleus (A), plantaris (B), and red (C) and white (D) portions of the gastrocnemius (Gastroc) muscles of chronic heart failure rats with moderate and severe LV dysfunction.

	DISCUSSION

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The present investigation demonstrates, for the first time, that severe LV dysfunction (defined as LVEDP > 20 mmHg) reduces the concentration of [³H]ouabain binding sites selectively in muscles comprised predominantly of oxidative (SO and FOG) fibers (i.e., soleus, plantaris, and red portion of gastrocnemius) but not the glycolytic (FG) fibers (i.e., white portion of the gastrocnemius). In contrast, moderate LV dysfunction (defined as LVEDP < 20 mmHg) did not reduce the [³H]ouabain B_max in muscle of any fiber type. In addition, severe LV dysfunction reduced O_2 max in proportion to the reduction in B_max found within the oxidative muscles examined. These results suggest that downregulation of Na⁺-K⁺ pumps that possess a high affinity for ouabain in oxidative skeletal muscle may play an important role in the exercise intolerance that attends severe LV dysfunction in CHF.

The factors that regulate the concentration of Na⁺-K⁺ pumps in skeletal muscle remain unclear at this time. Clausen (9) and others have shown that the greatest activation of the pump occurs during exercise and/or muscle contraction, and it has been demonstrated that the number of Na⁺-K⁺ pumps increases with exercise training and decreases with deconditioning (15, 25, 26, 32, 37, 55). On the basis of these results, one might expect that the reductions in Na⁺-K⁺ pump number in CHF to be attributed to a decrease in physical activity (i.e., deconditioning). However, our results do not support this conclusion. In this regard, all the rats used in this study were housed individually in cages that were 6 in. wide and 9 in. long. Although physical activity was not measured, we are confident that the size of the cage was sufficient to restrict the amount of activity of each animal such that the amount of deconditioning was similar for all rats (50). Therefore, the reduced number of Na⁺-K⁺ pumps that have a high affinity for ouabain (i.e., the concentration of [³H]ouabain binding sites) found in this study is not likely the consequence of physical deconditioning. This conclusion is consistent with previous investigations in which CHF-induced changes in skeletal muscle morphology and biochemistry could not be explained by reductions in animal activity (11, 50), and other factors associated with CHF must therefore be contributing to downregulation of the Na⁺-K⁺ pump. Related to this possibility, it has been shown that muscle sympathetic nerve activity is increased in CHF (35). In conjunction with increased levels of plasma catecholamines, skeletal muscle norepinephrine concentrations are elevated in rats with CHF, specifically in muscles that have a high oxidative capacity (41). The possibility exists that chronic stimulation of pump activity via -adrenergic receptors (10) could have resulted in the downregulation of Na⁺-K⁺ pump density.

Previous studies have suggested that the number of Na⁺-K⁺ pumps found in skeletal muscle is related to the oxidative capacity of the muscle (7, 23). Consistent with this observation, we found that the [³H]ouabain B_max measured in the present investigation correlates highly with the CS activities measured in the soleus, plantaris, and red and white portions of the gastrocnemius muscles of both sham and MI rats with moderate and severe LV dysfunction from a previous investigation from our laboratory (see Fig. 5; CS activities taken from Ref. 11). Although these results suggest that the downregulation of B_max and mitochondrial function coincide with one another in CHF, the precise mechanisms that contribute to each of these phenomena remain unknown, and further research in each of these investigative areas is clearly needed.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Relationship between citrate synthase (CS) activity and Bmax measured in the present investigation for the soleus, plantaris, and red and white portions of the gastrocnemius muscles of sham rats and MI rats with moderate and severe LV dysfunction. CS activities are taken from data from similar groups of rats published previously from our laboratory (11). Values are group means ± SE.

Reductions in exercise performance are well documented in CHF (52). In this regard, Weber and colleagues (56) have demonstrated that patients with CHF suffer from decrements in their maximal aerobic work capacity (O_2 max) compared with normal individuals. Furthermore, these reductions in O_2 max (or O_2 peak) have been shown recently to correlate with the amount of LV dysfunction that develops in the individual (i.e., decreases in ejection fraction) along with the reduction in skeletal muscle endurance capacity found in CHF patients (33, 38). We have shown previously that a similar phenomenon occurs in rats with CHF (40), and the results from this study confirm that a similar relationship exists between exercise performance and the degree of LV dysfunction in the rat CHF model (see Figs. 2 and 3).

To our knowledge, this investigation is the first to demonstrate that reductions in peak exercise performance (i.e., O_2 max) are correlated significantly with the reduction in the number of Na⁺-K⁺ pumps that possess a high affinity for ouabain found in skeletal muscles of rats with CHF. These results are consistent with previous reports that have shown that the number of Na⁺-K⁺ pumps in skeletal muscle is related to exercise/skeletal muscle performance (13, 26, 27) and also with the proposed hypothesis that electrolyte disturbances found in CHF may lead to an increased skeletal muscle fatigability (31, 43). However, some investigations have not been able to demonstrate this relationship in longitudinal exercise training studies (32, 37), and the possibility exists that the increases in pump number produced by training and their quantitative effect on exercise/skeletal muscle performance may be significantly different from those produced by the downregulation of pump number in CHF. Because exercise training increases the number of skeletal muscle Na⁺-K⁺ pumps that possess a high affinity for ouabain, it would be interesting to see whether a program of training would ameliorate the decreases in B_max found in the present investigation because of the development of CHF. It would also be interesting to determine whether the anticipated training-induced increases in Na⁺-K⁺ pump number would correlate with increases exercise performance in these individuals.

Green and colleagues (16) found recently that the number of Na⁺-K⁺ pumps measured in the vastus lateralis muscle of the leg did not change in patients with moderate CHF compared with normal controls. However, within the CHF population there was a significant correlation between O_2 peak and the number of skeletal muscle Na⁺-K⁺ pumps measured with the [³H]ouabain binding assay. The present investigation corroborates these findings and extends them to rats with severe LV dysfunction where significant reductions in the [³H]ouabain B_max were found in muscles that are characterized by a high oxidative capacity based on their fiber type composition and CS activity (see Fig. 5). Two important points can be made from these observations. First, it would appear that the downregulation of B_max in skeletal muscle does not occur in CHF until individuals develop severe LV dysfunction and congestive failure. In the present investigation reductions in B_max did not occur until LVEDP was >20 mmHg and rats demonstrated signs of chronic congestive failure as indicated by the increases in lung weight (pulmonary edema) and RV weight (RV hypertrophy). It is worth noting that a previous study (47) demonstrated decreases in B_max with LVEDPs <20 mmHg but compensated congestive failure was evident based on lung and RV weight compared with sham-operated controls. Second, muscle fiber type composition may be important in understanding the skeletal muscle adaptations that occur in CHF. Specifically, the reductions in B_max produced in CHF were clearly fiber type dependent in the present investigation. Because the vastus lateralis muscle contains a wide range of fiber type composition within and between humans (49), the possibility exists that a single biopsy from this muscle may not be representative of the whole muscle. In comparison, the skeletal muscle of rats is highly compartmentalized such that a large number of muscles contain a high proportion of a single fiber type (2, 11). This model therefore provides the unique opportunity to determine whether muscles of a certain fiber type composition are more susceptible to the adaptations produced in CHF.

Limitations of the study. Relevant to the present investigation, the Na⁺-K⁺ pump (Na⁺-K⁺-ATPase) has been shown to consist of a catalytic alpha () and a glycosylated beta () subunit (29, 54). At least three isoforms of the subunits have been identified (₁, ₂, ₃ and ₁, ₂, ₃), and, thus far, two isoforms of the  subunit (₁ and ₂) have been shown to be expressed in rat skeletal muscle (22, 45, 54). Results suggest that the ₁ subunit plays a major role in maintaining basal pump activity whereas the regulation and catalytic activity of the ₂ subunit can be influenced significantly by different hormones (4, 14, 21, 24). In addition, the ₂ subunit of the enzyme has a high affinity for the Na⁺-K⁺ pump inhibitor ouabain whereas the ₁ subunit does not (54). Because of this difference in affinity for ouabain, the [³H]ouabain binding assay used in the present investigation recognizes only the ₂ subunit of the enzyme, and therefore the reductions in the skeletal muscle Na⁺-K⁺ pump number found in the present investigation primarily reflect a reduction in the ₂ isoform. What may have occurred with the Na⁺-K⁺ pumps that contain ₁ isoform remains a matter of speculation.