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J Appl Physiol 92: 2326-2334, 2002. First published February 1, 2002; doi:10.1152/japplphysiol.00686.2001
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Vol. 92, Issue 6, 2326-2334, June 2002

Skeletal muscle ouabain binding sites are reduced in rats with chronic heart failure

Timothy I. Musch, Swen Wolfram, K. Sue Hageman, and Joel G. Pickar

Departments of Anatomy, Physiology, and Kinesiology, Kansas State University, Manhattan, Kansas 66506-5802


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Intrinsic skeletal muscle abnormalities decrease muscular endurance in chronic heart failure (CHF). In CHF patients, the number of skeletal muscle Na+-K+ pumps that have a high affinity for ouabain (i.e., the concentration of [3H]ouabain binding sites) is reduced, and this reduction is correlated with peak oxygen uptake. The present investigation determined whether the concentration of skeletal muscle [3H]ouabain binding sites found during CHF is related to 1) severity of the disease state, 2) muscle fiber type composition, and/or 3) endurance capacity. Four muscles were chosen that represented slow-twitch oxidative (SO), fast-twitch oxidative glycolytic (FOG), fast-twitch glycolytic (FG), and mixed fiber types. Measurements were obtained 8-10 wk postsurgery in 23 myocardial infarcted (MI) and 18 sham-operated control (sham) rats. Eighteen rats had moderate left ventricular (LV) dysfunction [LV end-diastolic pressure (LVEDP) < 20 mmHg], and five had severe LV dysfunction (LVEDP > 20 mmHg). Rats with severe LV dysfunction had significant pulmonary congestion and were likely in a chronic state of compensated congestive failure as indicated by an approximately twofold increase in both lung and right ventricle weight. Run time to fatigue and maximal oxygen uptake (VO2 max) were significantly reduced (down-arrow 39 and down-arrow 28%, respectively) in the rats with severe LV dysfunction and correlated with the magnitude of LV dysfunction as indicated by LVEDP (run time: r = 0.60, n = 21, P < 0.01 and VO2 max: r = 0.93, n = 13, P < 0.01). In addition, run time to fatigue was significantly correlated with VO2 max (r = 0.87, n = 15, P < 0.01). The concentration of [3H]ouabain binding sites (Bmax) was significantly reduced (21-28%) in the three muscles comprised primarily of oxidative fibers [soleus: 259 ± 14 vs. 188 ± 17; plantaris: 295 ± 17 vs. 229 ± 18; red portion of gastrocnemius: 326 ± 17 vs. 260 ± 14 pmol/g wet tissue wt]. In addition, Bmax was significantly correlated with VO2 max (soleus: r = 0.54, n = 15, P < 0.05; plantaris: r = 0.59, n = 15, P < 0.05; red portion of gastrocnemius: r = 0.65, n = 15, P < 0.01). 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.

Na+-K+ pump; exercise; performance; oxygen uptake; congestive failure


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (VO2 peak). This correlation between Na+-K+ pump activity and VO2 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 (VO2 max). We tested the hypothesis that the concentration of [3H]ouabain binding sites (Bmax) 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 Bmax found in the individual muscles examined in this investigation would be significantly correlated with VO2 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

All exercise tests were initiated between 9 and 10 AM to prevent the confounding effects from the diurnal variation in tissue glycogen (8). The exercise test was administered by an observer blinded to the animal's condition. Therefore, the observer did not know whether the animal being tested was an MI rat with CHF or a noninfarcted sham control rat.

Determination of VO2 max. VO2 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 (VO2) and carbon dioxide production (VCO2).

VO2 max was determined by having each rat perform a maximal exercise test. This test consisted of a 2-min warm-up at a treadmill grade and speed of 0% and 15 m/min, respectively. The treadmill speed and/or grade were increased every 2 min. VO2 max was defined as the point at which the VO2 did not increase with further increases in workload or when the rat was unable to or unwilling to continue running. These criteria have been shown to produce similar VO2 max values in untrained rats (5). However, confirmation that VO2 max was truly attained in each animal was demonstrated by having each rat perform a subsequent maximal exercise test after 48 h of recovery from the initial maximal test. With the second maximal test, each rat was given a 2-min warm-up at a treadmill grade and speed of 0% and 15 m/min. The treadmill grade and speed were then increased to the highest workload each animal was able to sustain during the initial maximal test. VO2 and VCO2 were recorded. The treadmill speed was then increased by 3-5 m/min, and VO2 and VCO2 were recorded. If the measured VO2 was similar between the two workloads, the animal was considered to be at VO2 max, and the exercise test was terminated. If the rat demonstrated an increase in VO2 during the second maximal exercise test, the test was terminated and the same procedure was repeated after 48 h of recovery. This procedure was repeated until comparable VO2 values were found between the initial and second (greater) workloads during each subsequent maximal exercise test, thus ensuring an accurate assessment of VO2 max in each animal (39).

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).

The binding assay was accomplished in five stages: 1) preincubation wash, 2) incubation with radiolabel, 3) washout of unbound radiolabel, 4) weighing and digestion, and 5) counting of radiolabel. Stages 1-3 were performed in the standard solution with appropriate concentration of ouabain at constant temperature (37°C). All measurements were performed in triplicate.

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 (10-4 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).

Slices were washed on ice for a total of 20 min (2 washes × 10 min). Slices from the total binding wells were washed in standard solution; slices from nonspecific binding wells were washed with an excess of unlabeled ouabain (10-4 M) during the assay. Individual slices were blotted, weighed, placed in liquid scintillation vials, and digested overnight (at least 12 h) in 250 µl of 1 M NaOH.

Three milliliters of liquid scintillation cocktail (Ecolite+: ICN Biomedicals) were added to the scintillation vials, and counting was performed in a Minaxi Tri-carb scintillation counter (4000 series, Packard). Specific binding was determined from the difference between total and nonspecific binding normalized to grams of wet muscle weight. Quench curves were established for the standard and NaOH solutions to accurately calculate specific [3H]ouabain binding sites.

Statistical analysis. Structural and hemodynamic indexes, and indexes of exercise performance (VO2 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. Bmax 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 (VO2 max and run time to fatigue) and the degree of LV dysfunction that developed in each animal (LVEDP) and/or the Bmax 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).

                              
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  		Table 1.   Structural and hemodynamic variables measured in noninfarcted sham-operated control rats and in MI rats with moderate and severe left ventricular dysfunction

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 VO2 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 VO2 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 VO2 max (Fig. 3).


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  		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 (VO2 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. dagger  P < 0.05 compared with moderate group.



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  		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 VO2 max and LVEDP in MI rats with moderate (n = 8) and severe (n = 5) LV dysfunction.



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  		Fig. 3.   Relationship between run time to fatigue and VO2 max measured in MI rats with moderate (n = 10) and severe (n = 5) LV dysfunction.

The concentration of [3H]ouabain binding sites (Bmax) 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 Bmax 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 VO2 max was plotted as a function of Bmax found in each muscle, regression analysis demonstrated that the results were best fit and linearly correlated with this index of exercise performance (Fig. 4).

                              
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  		Table 2.   3[H]ouabain binding sites and binding affinity in muscles from rats with and without moderate and severe LV dysfunction



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  		Fig. 4.   Relationship between VO2 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Bmax in muscle of any fiber type. In addition, severe LV dysfunction reduced VO2 max in proportion to the reduction in Bmax 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 beta -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 Bmax 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 Bmax 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.


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  		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 (VO2 max) compared with normal individuals. Furthermore, these reductions in VO2 max (or VO2 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., VO2 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 Bmax 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 VO2 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 Bmax 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 Bmax in skeletal muscle does not occur in CHF until individuals develop severe LV dysfunction and congestive failure. In the present investigation reductions in Bmax 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 Bmax 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 Bmax 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 (alpha ) and a glycosylated beta (beta ) subunit (29, 54). At least three isoforms of the subunits have been identified (alpha 1, alpha 2, alpha 3 and beta 1, beta 2, beta 3), and, thus far, two isoforms of the alpha  subunit (alpha 1 and alpha 2) have been shown to be expressed in rat skeletal muscle (22, 45, 54). Results suggest that the alpha 1 subunit plays a major role in maintaining basal pump activity whereas the regulation and catalytic activity of the alpha 2 subunit can be influenced significantly by different hormones (4, 14, 21, 24). In addition, the alpha 2 subunit of the enzyme has a high affinity for the Na+-K+ pump inhibitor ouabain whereas the alpha 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 alpha 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 alpha 2 isoform. What may have occurred with the Na+-K+ pumps that contain alpha 1 isoform remains a matter of speculation.

Finally, the beta  subunit is required for the functional expression of the enzyme (19) and is thought to be important in preserving the stability of the heterodimer complex along with playing a regulatory role in processing and transporting the mature enzyme complexes from the intracellular compartment to the plasma membrane (14, 22, 28). The possibility exists that modifications in the expression of the beta  subunit could alter both Na+-K+ pump activity (28) concomitant with changes in skeletal muscle metabolic function (24). Consequently, changes in the combined expression of alpha  and beta  subunits of the Na+-K+ pump might be contributing factors to the decrements in skeletal muscle contractile function found in the CHF state (18).


    ACKNOWLEDGEMENTS

The authors thank Steven Lloyd for technical contributions to this project.


    FOOTNOTES

The studies were supported by research funds from the American Heart Association-Kansas Affiliate.

Address for reprint requests and other correspondence: T. I. Musch, Dept. of Anatomy & Physiology, 228 Coles Hall, Kansas State Univ., Manhattan, KS 66506-5802 (E-mail: musch{at}vet.ksu.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.

First published February 1, 2002;10.1152/japplphysiol.00686.2001

Received 5 July 2001; accepted in final form 25 January 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aaker, A, McCormack JG, Hirai T, and Musch TI. Effects of ranolazine on the exercise capacity of rats with chronic heart failure induced by myocardial infarction. J Cardiovasc Pharmacol 28: 353-362, 1996.

2.   Armstrong, RB, and Phelps RO. Muscle fiber type composition of the rat hindlimb. Am J Anat 171: 259-272, 1984.

3.   Arnolda, L, Brosnan J, Rajagopalan B, and Radda GK. Skeletal muscle metabolism in heart failure in rats. Am J Physiol Heart Circ Physiol 261: H434-H442, 1991.

4.   Azuma, KK, Hensley CB, Tang MJ, and McDonough AA. Thyroid hormone specifically regulates skeletal muscle Na+-K+-ATPase alpha 2- and beta 2-isoforms. Am J Physiol Cell Physiol 265: C680-C687, 1993.

5.   Bedford, TG, Tipton CM, Wilson NC, Opplinger RA, and Gisolfi CV. Maximal oxygen consumption of rats and its changes with various experimental procedures. J Appl Physiol 47: 1278-1283, 1979.

6.   Brooks, GA, and White TP. Determination of metabolic and heart rate responses of rats to treadmill exercise. J Appl Physiol 45: 1009-1015, 1978.

7.   Chin, ER, and Green HJ. Na+-K+ ATPase concentration in different adult rat skeletal muscles is related to oxidative potential. Can J Physiol Pharmacol 71: 615-618, 1993.

8.   Clark, JH, and Conlee RK. Muscle and liver glycogen content: diurnal variation and endurance. J Appl Physiol 47: 425-428, 1979.

9.   Clausen, T. Long- and short-term regulation of the Na+-K+ pump in skeletal muscle. News Physiol Sci 11: 24-30, 1996.

10.   Clausen, T, and Flatman JA. The effect of catecholamines on Na,K transport and membrane potential in rat soleus muscle. J Physiol (Lond) 270: 383-414, 1977.

11.   Delp, MD, Duan C, Mattson JP, and Musch TI. Changes in skeletal muscle biochemistry and histology relative to fiber type in rats with heart failure. J Appl Physiol 83: 1291-1299, 1997.

12.   Drexler, H, and Coats AJS Explaining fatigue in congestive heart failure. Annu Rev Med 47: 241-256, 1996.

13.   Evertsen, F, Medbo JI, Jebens E, and Nicolaysen K. Hard training for 5 mo increases Na+-K+ pump concentration in skeletal muscle of cross-country skiers. Am J Physiol Regulatory Integrative Comp Physiol 272: R1417-R1424, 1997.

14.   Ewart, HS, and Klip A. Hormonal regulation of the Na+-K+-ATPase: mechanisms underlying rapid and sustained changes in pump activity. Am J Physiol Cell Physiol 269: C295-C311, 1995.

15.   Green, HJ, Chin ER, Ball-Burnett M, and Ranney D. Increases in human skeletal muscle Na+-K+-ATPase concentration with short-term training. Am J Physiol Cell Physiol 264: C1538-C1542, 1993.

16.   Green, HJ, Duscha BD, Sullivan MJ, Keteyian SJ, and Kraus WE. Normal skeletal muscle Na+-K+ pump concentration in patients with chronic heart failure. Muscle Nerve 24: 69-76, 2001.

17.   Harrington, D, and Coats AJS Skeletal muscle abnormalities and evidence for their role in symptom generation in chronic heart failure. Eur Heart J 18: 1865-1872, 1997.

18.   He, S, Shelly DA, Moseley AE, James PF, James JH, Paul RJ, and Lingrel JB. The alpha 1- and alpha 2-isoforms of Na-K-ATPase play different roles in skeletal muscle contractility. Am J Physiol Regulatory Integrative Comp Physiol 281: R917-R925, 2001.

19.   Horowitz, B, Eakle KA, Scheiner-Bobis G, Randolph GR, Chen CY, Hitzeman RA, and Farley RA. Synthesis and assembly of functional mammalian Na,K-ATPase in yeast. J Biol Chem 265: 4189-4192, 1990.

20.   Howell, S, Maarek JM, Fournier M, Sullivan K, Zhan W, and Sieck GC. Congestive heart failure: differential adaptation of the diaphragm and latissimus dorsi. J Appl Physiol 79: 389-397, 1995.

21.   Hundal, HS, Marette A, Mitsumoto Y, Ramlal T, Blostein R, and Klip A. Insulin induces translocation of the alpha 2 and beta 1 subunits of the Na+/K+-ATPase from intracellular compartments to the plasma membrane in mammalian skeletal muscle. J Biol Chem 267: 5040-5043, 1992.

22.   Hundal, HS, Marette A, Ramlal T, Liu Z, and Klip A. Expression of beta  subunit isoforms of the Na+, K+-ATPase is muscle type-specific. FEBS Lett 328: 253-258, 1993.

23.   Ianuzzo, CD, and Dabrowski B. Na+/K+-ATPase activity of different types of striated muscles. Biochem Med Metab Biol 37: 31-34, 1987.

24.   James, JH, Fang CH, Schrantz SJ, Hasselgran PO, Paul RJ, and Fischer JE. Linkage of aerobic glycolysis to sodium-potassium transport in rat skeletal muscle. Implications for increased muscle lactate production in sepsis. J Clin Invest 98: 2388-2397, 1996.

25.   Kjeldsen, K, Richter EA, Galbo H, Lortie G, and Clausen T. Training increases the concentration of [3H]ouabain-binding sites in rat skeletal muscle. Biochim Biophys Acta 860: 708-712, 1986.

26.   Kjeldsen, K, Bjerregaard P, Richter EA, Thomsen PEB, and Nogaard A. Na+,K+-ATPase concentration in rodent and human heart and skeletal muscle: apparent relation to muscle performance. Cardiovasc Res 22: 95-100, 1988.

27.   Klitgaard, H, and Clausen T. Increased total concentration of Na-K pumps in vastus lateralis muscle of old trained human subjects. J Appl Physiol 67: 2491-2494, 1989.

28.   Lavoie, L, Levenson R, Martin-Vasallo P, and Klip A. The molar ratios of alpha  and beta  subunits of the Na+-K+-ATPase differ in distinct subcellular membranes from rat skeletal muscle. Biochemistry 36: 7726-7732, 1997.

29.   Levenson, R. Isoforms of the Na,K-ATPase: family members in search of function. Rev Physiol Biochem Pharmacol 123: 1-45, 1994.

30.   Lipkin, DP, Jones DA, Round JM, and Poole-Wilson PA. Abnormalities of skeletal muscle in patients with chronic heart failure. Int J Cardiol 18: 187-195, 1988.

31.   Lunde, PK, Verburg E, Vollestad NK, and Sejersted OM. Skeletal muscle fatigue in normal subjects and heart failure patients. Is there a common mechanism? Acta Physiol Scand 162: 215-228, 1998.

32.   Madsen, K, Franch J, and Clausen T. Effects of intensified endurance training on the concentration of Na,K-ATPase in human skeletal muscle. Acta Physiol Scand 150: 251-258, 1994.

33.   Magnusson, G, Kaijser L, Rong H, Isberg B, Sylvén C, and Saltin B. Exercise capacity in heart failure patients: relative importance of heart and skeletal muscle. Clin Physiol 16: 183-195, 1996.

34.   Mancini, DM, Coyle E, Coggan A, Beltz J, Ferraro N, Montain S, and Wilson JR. Contributions of intrinsic skeletal muscle changes to 31P NMR skeletal muscle metabolic abnormalities in patients with chronic heart failure. Circulation 80: 1338-1346, 1989.

35.   Mark, AL. Sympathetic dysregulation in heart failure: mechanisms and therapy. Clin Cardiol 18: I-3-I-8, 1995.

36.   Massie, B, Conway M, Yonge R, Frostick S, Ledingham J, Sleight P, Radda G, and Rajagopalan B. Skeletal muscle metabolism in patients with congestive heart failure: relation to clinical severity and blood flow. Circulation 76: 1009-1019, 1987.

37.   McKenna, MJ, Schmidt TA, Hargreaves M, Cameron L, Skinner SL, and Kjeldsen K. Sprint training increases human skeletal Na+-K+-ATPase concentration and improves K+ regulation. J Appl Physiol 75: 173-180, 1993.

38.   Minotti, JR, Christoph I, Oka R, Weiner MW, Wells L, and Massie BM. Impaired skeletal muscle function in patients with congestive heart failure. Relationship to systemic exercise performance. J Clin Invest 88: 2077-2082, 1991.

39.   Musch, TI, Bruno A, Bradford GE, Vayonis A, and Moore RL. Measurements of metabolic rate in rats: a comparison of techniques. J Appl Physiol 65: 964-970, 1988.

40.   Musch, TI, Moore RL, Riedy M, Burke P, Zelis R, Leo ME, Bruno A, and Bradford GE. Glycogen concentrations and endurance capacity of rats with chronic heart failure. J Appl Physiol 64: 1153-1159, 1988.

41.   Musch, TI, and Zelis R. Norepinephrine response to exercise of rats with a chronic myocardial infarction. Med Sci Sports Exerc 23: 569-577, 1991.

42.   Musch, TI, and Terrell JA. Skeletal muscle blood flow abnormalities in rats with a chronic myocardial infarction: rest and exercise. Am J Physiol Heart Circ Physiol 262: H411-H419, 1992.

43.   Nielsen, OB, and Clausen T. The Na+/K+-pump protects muscle excitability and contractility during exercise. Exerc Sport Sci Rev 28: 159-164, 2000.

44.   Nogaard, A, Bjerregaard P, Baandrkup U, Kjeldsen K, Reske-Nielsen E, and Bloch Thomsoen PE. The concentration of the Na K-pump in skeletal and heart muscle in congestive heart failure. Int J Cardiol 26: 185-190, 1990.

45.   Orlowski, J, and Lingrel JB. Tissue-specific and developmental regulation of rat Na,K-ATPase catalytic alpha  isoform and beta  subunit mRNAs. J Biol Chem 263: 10436-10442, 1988.

46.   Perreault, CL, Gonzales-Serratos H, Litwin SE, Sun X, Franzini-Armstrong C, and Morgan JP. Alterations in contractility and intracellular Ca2+ transients in isolated bundles of skeletal muscle fibers from rats with chronic heart failure. Circ Res 73: 405-412, 1993.

47.   Pickar, JG, Mattson JP, Lloyd S, and Musch TI. Decreased [3H]ouabain binding sites in skeletal muscle of rats with chronic heart failure. J Appl Physiol 83: 323-329, 1997.

48.   Peters, DG, Mitchell HL, McCune SA, Park S, Williams JH, and Kandarian SC. Skeletal muscle sarcoplasmic reticulum Ca2+-ATPase gene expression in congestive heart failure. Circ Res 8l: 703-710, 1997.

49.   Saltin, B, and Gollnick PD. Skeletal muscle adaptability: significance for metabolism and performance. In: Handbook of Physiology. Skeletal Muscle. Bethesda, MD: Am. Physiol. Soc, 1983, sect. 10, chapt. 19, p. 555-631.

50.   Simonini, A, Long CS, Dudley GA, Yue P, McElhinny J, and Massie BM. Heart failure in rats causes changes in skeletal muscle morphology and gene expression that are not explained by reduced activity. Circ Res 79: 128-136, 1996.

51.   Sullivan, MJ, Green HJ, and Cobb FR. Skeletal muscle biochemistry and histology in ambulatory patients with long-term heart failure. Circulation 81: 518-527, 1990.

52.   Sullivan, MJ, and Hawthorne MH. Exercise intolerance in patients with chronic heart failure. Prog Cardiovasc Dis 38: 1-22, 1995.

53.   Sullivan, MJ, Duscha BD, Klitgaard H, Kraus WE, Cobb FR, and Saltin B. Altered expression of myosin heavy chain in human skeletal muscle in chronic heart failure. Med Sci Sports Exerc 29: 860-866, 1997.

54.   Sweadner, KJ. Isozymes of the Na+/K+-ATPase. Biochim Biophys Acta 988: 185-220, 1989.

55.   Tsakiridis, T, Wong PPC, Liu Z, Rodgers CD, Vranic M, and Klip A. Exercise increases the plasma membrane content of the Na+-K+ pump and its mRNA in rat skeletal muscles. J Appl Physiol 80: 699-705, 1996.

56.   Weber, KT, Kinasewitz GT, Janicki JS, and Fishman AP. Oxygen utilization and ventilation during exercise in patients with chronic cardiac failure. Circulation 65: 1213-1223, 1982.

57.   Williams, JH, and Ward CW. Changes in skeletal muscle sarcoplasmic reticulum function and force production following myocardial infarction in rats. Exp Physiol 83: 85-94, 1998.

58.   Wilson, JR, Martin JL, Schwartz D, and Ferraro N. Exercise intolerance in patients with chronic heart failure: role of nutritive flow to skeletal muscle. Circulation 69: 1079-1087, 1984.

59.   Zelis, R, Nellis SH, Longhurst J, Lee G, and Mason DT. Abnormalities in the regional circulations accompanying congestive heart failure. Prog Cardiovasc Dis 18: 181-199, 1975.

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