Potassium depletion improves myocardial potassium uptake in vivo

Henning Bundgaard

Medical Department B 2142, Heart Centre, Rigshospitalet, National University Hospital, University of Copenhagen, 2100 Copenhagen, Denmark

Submitted 23 December 2003 ; accepted in final form 27 February 2004


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Potassium depletion (KD) is a very common clinical entity often associated with adverse cardiac effects. KD is generally considered to reduce muscular Na-K-ATPase density and secondarily reduce K uptake capacity. In KD rats we evaluated myocardial Na-K-ATPase density, ion content, and myocardial K reuptake. KD for 2 wk reduced plasma K to 1.8 ± 0.1 vs. 3.5 ± 0.2 mM in controls (P < 0.01, n = 7), myocardial K to 80 ± 1 vs. 86 ± 1 µmol/g wet wt (P < 0.05, n = 7), increased Mg, and induced a tendency to increased Na. Myocardial Na-K-ATPase {alpha}2-subunit abundance was reduced by ~30%, whereas increases in {alpha}1- and K-dependent pNPPase activity of 24% (n = 6) and 13% (n = 6), respectively, were seen. This indicates an overall upregulation of the myocardial Na-K pump pool. KD rats tolerated a higher intravenous KCl dose. KCl infusion until animals died increased myocardial K by 34% in KD rats and 18% in controls (P < 0.05, n = 6 for both) but did not induce different net K uptake rates between groups. However, clamping plasma K at ~5.5 mM by KCl infusion caused a higher net K uptake rate in KD rats (0.22 ± 0.04 vs. 0.10 ± 0.03 µmol·g wet wt–1·min–1; P < 0.05, n = 8). In conclusion, a minor KD-induced decrease in myocardial K increased Na-K pump density and in vivo increased K tolerance and net myocardial K uptake rate during K repletion. Thus the heart is protected from major K losses and accumulates considerable amounts of K during exposure to high plasma K. This is of clinical interest, because a therapeutically induced rise in myocardial K may affect contractility and impulse generation-propagation and may attenuate increased myocardial Na, the hallmark of heart failure.

Na-K-ATPase; ion homeostasis; heart failure; iatrogenic potassium depletion


POTASSIUM (K) uptake in the myocardium depends on Na-K-ATPase transport of Na outward and K inward across the cell membrane in a 3:2 relationship. Factors affecting density or activity of the Na-K pump may alter the myocardial K homeostasis and secondarily the intracellular homeostasis of other ions. Such changes may affect secondary active transport processes (9) and membrane potential and impulse generation and propagation and under certain circumstances cause arrhythmia (13, 29) and affect myocardial systolic (27) as well as diastolic (30) properties. In some tissues primary disturbances in K homeostasis, e.g., K depletion, can alter Na-K-ATPase concentration and activity per se.

Malnutrition, gastrointestinal loss and—within the field of cardiovascular medicine—mainly non-K-sparing diuretic treatment (8, 19) are the most common causes of K depletion (for review, see Ref. 14). After the onset of a negative K balance the decline in tissue K is highly differentiated. The skeletal muscle K store composes ~75% of total body K and is considered to have an "altruistic" (21) buffer role, protecting brain and myocardium from major K losses (23). This differentiation has partially been interpreted as a result of differentiated Na-K pump isoform distribution and density among tissues (21). In skeletal muscles K depletion with K reductions of up to 40% (24) has been shown to be associated with a considerable decline in Na-K-ATPase density in animals (for review, see Ref. 6) as well as in humans (12). In rats this decline is strictly confined to the {alpha}2-isoform of the Na-K pump, whereas the {alpha}1-isoform abundance is not reduced (3, 33). In response to K depletion myocardial Na-K pump density has been reported to be reduced in rats (1, 24, 33), increased in rabbits (28, 34), and not associated with any overall change in ferrets (22). In these studies K depletion reduced myocardial K from ~0% to 13%. On the other hand, chronic K loading leading to a 6% increase in myocardial K content reduced myocardial Na-K pump density in rats (2).

It is well established that in vitro there is a positive linear correlation between Na-K pump concentration and maximum K uptake capacity (7). However, we recently reported (3) that, in contrary to findings in vitro (23), a markedly increased skeletal muscle K clearance capacity was present in vivo in K-depleted rats despite a decrease in skeletal muscle Na-K-ATPase density of up to 68%. This finding is in agreement with preliminary results reported by McDonough and Youn's group (5), showing that in K-depleted rats substantially higher K infusion rates were needed to clamp plasma K at control levels. K depletion markedly increases skeletal muscle Na that was acutely reduced by intravenous K repletion (3). If K depletion also increases myocardial K clearance capacity, then acute K repletion—often used in clinical practice—may also cause immediate reductions in intracellular Na, and thus in intracellular Ca via increased activity of the Na/Ca exchanger. This may reduce systolic but improve diastolic cardiac function. In heart failure and myocardial hypertrophy the intracellular myocyte Na concentration is increased (for recent review, see Ref. 25). In keeping with this, it is tempting to speculate that, e.g., in a patient with K depletion due to treatment of heart failure or arterial hypertension with K-wasting diuretics, K repletion may not only normalize myocardial K but also reduce myocardial Na, which may improve cardiac performance. This study was designed to evaluate the relation between myocardial Na-K-ATPase regulation in response to K depletion and myocardial K uptake in vivo.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Female Wistar rats bought at the age of 8 wk (~125 g) (Møllegaard Breeding Centre, Lille Skensved, Denmark) were used in the study. The study was conducted in agreement with legislation for experimental animals given by the Danish Ministry of Justice. The animals were kept in animal housing facilities at 22°C with a 12:12-h light-dark cycle (light 8 AM to 8 PM). Animals assigned to K depletion were housed in cages without access to urine and feces, were fed a low-K content diet containing (in mmol/100 g chow; n = measurements on 4 chow samples) 0.11 ± 0.01 K (mean ± SE), 11.52 ± 2.00 Na, and 3.30 ± 0.23 Mg, and had free access to distilled water. Control animals had reduced access to standard chow containing (in mmol/100 g chow; n = measurements on 4 chow samples) 22.87 ± 0.55 K, 9.09 ± 1.83 Na, and 7.37 ± 0.49 Mg to keep body weights comparable between the two groups (17). Animals were taken for experiments after 2 wk of treatment. After the animals were euthanized without any other instrumentation, myocardial ion and Na-K-ATPase measurements were also performed after 1, 3, and 7 days, and after 2 wk.

Instrumentation. The instrumentation of the animals was previously described in detail (4). In brief, animals were anesthetized by intraperitoneal injection of 0.05 mg/g body wt pentobarbital sodium (Mebumal, 50 mg/ml; Nycomed DAK) before catheterization of the jugular vein for infusion and the carotid artery for blood sampling. Fifteen minutes after completion of surgical procedures, the first blood sample (t = 0 min) was taken and continuous intravenous infusion of 1.5 ml·100 g body wt–1·h–1, or as indicated, of a solution of 0.5 mol/l KCl (i.e., 0.75 mmol KCl·100 g body wt–1·h–1) was initiated. During the infusion arterial blood samples of 0.2 ml were drawn at t = 5, 15, and 30 min and thereafter at 30-min intervals, or as indicated. After each sampling, blood that was drawn before sampling to avoid dilution was reinjected in addition to 0.2 ml of heparinized isotonic NaCl for intravascular volume compensation. During infusions a reduction in hemoglobin level was observed. Thus, after 60 min of infusion, reductions in hemoglobin of 0.3 ± 0.3 mmol/l in control rats and 0.4 ± 0.3 mmol/l in K-depleted rats were observed (n = 5–6, P > 0.8). During infusions animals were well oxygenated. Thus, after, e.g., 60 min, arterial oxygen saturations of 99 ± 2% (n = 6) in controls and 100 ± 3% (n = 6) in K-depleted rats were measured with an OSM3 (Radiometer, Copenhagen, Denmark). KCl infusions were continued until the animals died or as otherwise indicated. KCl infusions were then immediately stopped, the animals were decapitated, and the heart and in some instances, to verify the level of K depletion, the hind leg gastrocnemius muscles were excised and immediately taken for measurements. In some animals ouabain (12.5 nmol/g body wt) was administered intraperitoneally to block the ouabain-sensitive Na-K-ATPase subunits.

K, Na, and Mg. Arterial blood samples were immediately analyzed for plasma K and Na concentrations by ion-sensitive electrodes with an ABL 605 (Radiometer). Heart ventricular myocardium, gastrocnemius muscle, and chow K and Na contents were measured by flame photometry with an FLM3 (Radiometer) with lithium as an internal standard. A sample of ~25 mg wet wt was dissolved in 1 ml of 30% H2O2, and the suspension was placed at 90°C for 12 h to allow complete evaporation. After addition of 2 ml of trichloroacetic acid (TCA; 5% wt/vol), 0.5 ml of the solution was used for flame photometry after final addition of a further 0.5 ml of 5% TCA and 1.5 ml of 5 mmol/l LiCl. Myocardial, skeletal muscle, and chow Mg contents were measured by atomic absorption (Perkin Elmer AAnalyst 100; Norwalk, CT) at a wavelength of 285.2 nm with a solution as used for flame photometry except that LiCl was replaced by 1.5 ml of redistilled water. Measurements were performed in duplicate.

3H-labeled ouabain binding. Measurements of [3H]ouabain binding were performed as previously described in detail for intact tissue samples (16). In brief, all procedures were performed by using freshly made vanadate (Merck, Darmstadt, Germany) buffer containing (in mmol/l) 10 Tris·HCl, 250 sucrose, 3 MgSO4, and 1 vanadate. pH was adjusted to 7.3 with Tris·HCl. Samples of ~4 mg wet wt were cut from the ventricular myocardium or gastrocnemius muscles and prewashed in unlabeled buffer at 0°C for 20 min (2 x 10 min). Incubations took place at 37°C in buffer containing [3H]ouabain (2.1 µCi/ml; Amersham International, Little Chalfont, UK) and ouabain (Sigma, St. Louis, MO) added to a final concentration of 1 x 10–6 mol/l for 2 h (2 x 1 h). Thereafter, a washout at 0°C in unlabeled buffer for 2 h (4 x 30 min) was performed to reduce the amount of [3H]ouabain in the extracellular space, thereby enhancing the precision of the method. After washout, samples were blotted, weighed, and soaked overnight in vials containing 0.5 ml of 5% (wt/vol) TCA. Thereafter, 2.5 ml of Opti-fluor scintillation mixture (Packard Instruments, Downers Grove, IL) was added and [3H]activity in samples and incubation medium was assayed by liquid scintillation counting (Tri-Carb, 1600TR; Packard Instruments). On the basis of sample wet weight, 3H activity in the incubation medium, and 3H activity retained in the samples, the concentration of [3H]ouabain binding sites in the sample was calculated and expressed as picomoles per gram of wet weight. Na-K pumps internalized to endosomal pools or the ouabain-resistant {alpha}1-isoform of the rat myocardium Na-K pump are not detected by [3H]ouabain binding. Myocardial water and protein contents were determined as previously described in detail (4). Heart weight was determined immediately after the heart was dissected out.

Immunoblotting. Immunoblotting was performed with crude homogenates (10 mg tissue/ml in His buffer; Ref. 20) as previously described in detail (2). Isoform-specific antibodies McK1 and McB2 against the {alpha}1 and {alpha}2 Na-K-ATPase subtypes, respectively, were kindly provided by K. Sweadner (Harvard University). After protein concentrations were measured, equal amounts of protein were dissolved in Laemmli buffer (Bio-Rad), and loaded on a 7.5% Tris·HCl gel together with molecular weight standards (Precision Protein Standards, Bio-Rad), and run on Mini-Protean 3 cell electrophoresis system (Bio-Rad). Gels were blotted onto Immobilon 0.45-µm polyvinylidene difluoride (PVDF) membranes (Bio-Rad) with a Trans-Blot Semi-Dry transfer system (Bio-Rad) according to the manufacturer's instructions. Membranes were blocked in PBS, 0.2% Tween 20, and 5% bovine albumin fraction V (AppliChem, Darmstadt, Germany) overnight at 4°C. Membranes were incubated with isotype-specific antibodies diluted 1:2,500 in blocking buffer overnight at 4°C, washed in PBS and 0.2% Tween 20, and incubated with anti-mouse IgG horseradish peroxidase-linked whole antibody (Amersham Life Science) diluted 1:2,500 in PBS and 0.2% Tween. Membranes were then washed in PBS and 0.2% Tween, followed by a wash in PBS only. The signal was detected with an enhanced chemiluminescence (ECL) kit and Hybond ECL film (Amersham Life Science). Multiple exposures were made to ensure that signals were within the linear range of the film. Immunoblots were quantified by scanning densitometry with Kodak Digital Science 1D Image Analysis Software (Rochester, NY). Changes in isoform abundances in K-depleted animals were expressed relative to values obtained in controls.

Calculations and statistics. Myocardial net K uptake rate during KCl infusions was calculated as the difference between mean myocardial K content without KCl infusion and K content in each heart after KCl infusion. Results are expressed relative to duration of KCl infusion (mol K·g wet wt–1·min–1). Results are given as means ± SE. Statistical significance among groups was ascertained by Student's two-tailed t-test for unpaired observations. Linear regression analysis was performed by the method of least squares. Bonferroni's correction was applied to correct for multiple comparisons. P < 0.05 is regarded as significant in all comparisons.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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K depletion caused a 7% weight loss (P < 0.05, n = 6), but otherwise the dietary regimens were well tolerated and animals were clinically unaffected. Between K-depleted and control rats, i.e., rats with limited access to standard chow, no significant differences in body weight [118 ± 2 vs. 125 ± 4 g; P = not significant (NS), n = 6] or heart weight (0.39 ± 0.02 vs. 0.38 ± 0.02 g; P = NS, n = 6) were observed.

Plasma K. Plasma K was reduced to 1.8 ± 0.1 mmol/l in the K-depleted group compared with 3.5 ± 0.2 mmol/l in the control group, i.e., by 51% (P < 0.01, n = 7). No significant difference in plasma K was observed between normal control rats (3.6 ± 0.1 mmol/l) and weight-matched control rats. No difference in plasma Na was observed between the two groups. Plasma K changes in response to KCl infusions are described below.

Myocardial K, Na, and Mg content. K depletion induced a gradual decrease in myocardial K content, reaching 80 ± 1 µmol/g wet wt after 2 wk compared with 86 ± 1 µmol/g wet wt in control rats (P < 0.05, n = 7). This decrease (~6%) was considerably less than the decrease observed in gastrocnemius muscle K, to 84 ± 2 µmol/g wet wt compared with 111 ± 2 µmol/g wet wt in controls, i.e., by 23% (P < 0.01, n = 6). This reduction is in agreement with previous findings (3). Only a tendency to an increase in myocardial Na was seen in K-depleted animals compared with controls (40 ± 2 vs. 38 ± 2 µmol/g wet wt; P > 0.4, n = 7), whereas a significant increase in gastrocnemius muscle Na to 36 ± 1 compared with 22 ± 1 µmol/g wet wt, i.e., by 60% (P < 0.01, n = 6), was seen. K depletion was associated with an increase in myocardial Mg content to 11.0 ± 0.4 vs. 9.6 ± 0.2 µmol/g wet wt in controls, i.e., by 14% (P < 0.05, n = 6), and an increase in gastrocnemius muscle Mg to 12.9 ± 0.1 vs. 12.0 ± 0.3 µmol/g wet wt in controls, i.e., by 8% (P < 0.05, n = 6).

Myocardial Na-K-ATPase. In the K-depleted rats myocardial K-dependent pNPPase activity showed a tendency to increase after K depletion for 3 days, reaching the level of significance after 2 wk at 2.7 ± 0.1 vs. 2.4 ± 0.1 µmol·min–1·g wet wt–1 in controls, i.e., an increase of 13% (P < 0.05, n = 6; Fig. 1). This indicates that the total myocardial Na-K pump pool was upregulated. Linear regression analysis of data from K-depleted and control rats obtained from the start of the dietary regimens until day 14 showed a negative linear relationship between mean myocardial K-dependent pNPPase activities and mean myocardial K (y = –0.05x + 6.44; r2 = 0.8, P < 0.01, n = 7; Fig. 2). Thus K-dependent pNPPase activity was higher in the samples with lower K content.



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Fig. 1. Relative changes in myocardial Na-K-ATPase densities measured by immunoblotting, vanadate-facilitated 3H-labeled ouabain binding, and K-dependent pNPPase activity in rats K depleted for 2 wk compared with weight-matched controls. Mean ± SE values are indicated; n = 6. *P < 0.05.

 


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Fig. 2. Myocardial K vs. myocardial K-dependent pNPPase activity measured in K-depleted rats and in their weight-matched controls. Each point represents mean + SE myocardial K vs. mean + SE K-dependent pNPPase activity, with measurements at different times from commencement of the dietary regimens and after 3, 8, and 14 days. Values from K-depleted as well as control animals are included. Each point represents measurements in 5–7 animals. The equation for the linear regression line and the r2 value are shown. The linear correlation was significant (P < 0.01).

 
To quantify putative K depletion-induced changes in the abundance of the ouabain-sensitive {alpha}2-isoform of myocardial Na-K-ATPase, [3H]ouabain binding measurements were performed. [3H]ouabain binding site concentration was reduced to 106 ± 11 pmol/g wet wt compared with 149 ± 9 pmol/g wet wt in control rats, i.e., by 29% (P < 0.05, n = 6). In gastrocnemius muscle a significant [3H]ouabain binding site concentration decrease of 67% was seen. Further assessment was obtained by immunoblotting. Thus, in accordance with the decline in myocardial [3H]ouabain binding site concentration of 29%, a decrease in {alpha}2-isoform abundance of 28% (P < 0.05, n = 6) was observed. The by far most abundant {alpha}1-isoform of the rat myocardial Na-K-ATPase was increased by 24% (P < 0.05, n = 6; Fig. 1). It should be noted that no significant differences were observed between hearts from K-depleted rats and control rats concerning water content (76.1 ± 0.1% vs. 76.8 ± 0.3%; P = NS, n = 5 or 6) or protein content (233 ± 2 vs. 228 ± 4 mg/g wet wt; P = 0.2, n = 6).

K clearance capacity: Plasma K. During intravenous KCl infusion, plasma K increased rapidly and almost linearly in the control group whereas a lower increase rate was observed in the K-depleted group. Thus after the first 15 min of infusion plasma K had increased by 3.3 ± 0.3 mmol/l in K-depleted rats compared with 4.3 ± 0.2 mmol/l in control rats (P < 0.05, n = 6). After ~30-min infusion the plasma K increase rate leveled off in the K-depleted group. K-depleted rats died after 118 ± 16 min of KCl infusion, whereas control rats died after 53 ± 4 min (P < 0.01, n = 6). The last plasma K value measured before the animals died was 11.8 ± 0.8 mmol/l in K-depleted rats and 11.9 ± 0.9 mmol/l in control rats (P > 0.9, n = 6), indicating that a similar level of hyperkalemia was tolerated in the two groups.

Myocardial K content after maximum KCl infusion. From KCl infusion until the animals died (see above) myocardial K increased to 108 ± 2 µmol/g wet wt, i.e., by 34% (P < 0.05, n = 6), in the K-depleted group and to 101 ± 2 µmol/g wet wt, i.e., by 18% (P < 0.05, n = 6), in control rats (Fig. 3). The rates of net myocardial K uptake during KCl infusions were 0.26 ± 0.03 µmol·g wet wt–1·min–1 in K-depleted animals vs. 0.30 ± 0.05 µmol·g wet wt–1·min–1 in controls (P > 0.4, n = 6). It should be noted that before (see above) as well as after infusions no significant differences in myocardial water content were observed between K-depleted and control rats (73.5 ± 0.6% vs. 73.9 ± 0.7%; P = 0.7, n = 6).



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Fig. 3. Myocardial K measured in rats K depleted for 2 wk and in their weight-matched controls before and after intravenous infusion of different doses of KCl (0.75 mmol KCl·100 g body wt–1·h–1). KCl infusions were continued (from left to right) until animals died (after intravenous KCl), until KCl doses were administered for equal periods of time (~1 h) in the 2 groups, after KCl infusion until plasma K was kept at ~5.5 mmol/l for ~15 min, and after intraperitoneal injection of ouabain (12.5 nmol/g body wt). Mean ± SE values are indicated; n = 6. *P < 0.05 between the 2 groups.

 
Myocardial K after equal KCl dosages. The higher increase in myocardial K in K-depleted rats after maximum KCl infusion may have been due to the more than twice as long KCl infusion period in the K-depleted rats compared with control rats. Therefore, further assessment of myocardial K uptake was performed by infusing KCl for equal periods of time in the two groups. After infusion for 65 ± 5 min in the K-depleted group and for 65 ± 6 min in the control group (P = 1, n = 6), myocardial K had reached 92 ± 2 and 103 ± 3 µmol/g wet wt, respectively, in the two groups (Fig. 3), corresponding to net K uptake rates of 0.18 ± 0.02 and 0.27 ± 0.04 µmol·g wet wt–1·min–1 (P < 0.05, n = 6), respectively.

Myocardial K after clamping of plasma K by KCl infusion. KCl infusion for equal periods of time resulted in a higher plasma K level in control rats compared with K-depleted rats. This might have caused differences in extracellular K stimulation of Na-K pump activity between the two groups. Therefore, finally, KCl infusions in both groups were adjusted to obtain similar plasma K levels in the upper normal range, i.e., ~5.5 mmol/l. Plasma K was maintained at this level for ~15 min before infusions were stopped and the heart was immediately excised for ion measurements. After infusion for 39 ± 3 and 43 ± 6 min (P > 0.4, n = 8) in K-depleted and control rats, respectively, final plasma K values of 5.5 ± 0.2 mmol/l in K-depleted rats and 5.5 ± 0.1 mmol/l in control rats (P > 0.9, n = 8) were obtained. Infused KCl dosages were 0.37 ± 0.03 and 0.13 ± 0.02 mmol (P < 0.01, n = 8), respectively. After infusions myocardial K contents reached 89 ± 2 µmol/g wet wt in K-depleted rats vs. 90 ± 1 µmol/g wet wt in control rats (P > 0.3, n = 8; Fig. 3). This corresponds to net myocardial K uptake rates of 0.22 ± 0.04 vs. 0.10 ± 0.03 µmol·g wet wt–1·min–1 (P < 0.05, n = 8; see Calculations and statistics). Thus, with plasma K clamped within the upper normal range at equal levels in the two groups, a higher net K uptake was observed in the K-depleted rats compared with control rats.

KCl infusions were not associated with changes in myocardial Mg, whereas a general tendency to a reduction in myocardial Na was seen. Thus, comparing values before and after KCl infusions for equal periods of time in the two groups, there was a tendency to a reduction in myocardial Na in the K-depleted rats (to 36 ± 3 µmol/g wet wt; P > 0.15, n = 6) as well as in the control rats (to 34 ± 2 µmol/g wet wt; P > 0.2, n = 6).

Influence of ouabain on myocardial K content. It is reasonable to think that the K depletion-induced reductions in the myocardial and skeletal muscle {alpha}2-isoform of the Na-K pump can partially be mimicked by administration of ouabain, i.e., by functional inhibition of the ouabain-sensitive {alpha}2-isoform. On this basis a semiqualitative assessment of which isoforms are active in K homeostasis in vivo was performed. After intraperitoneal injection of 12.5 nmol ouabain/g body wt, plasma K increased from 1.8 ± 0.1 mmol/l to a maximum of 3.4 ± 0.3 mmol/l after 105 min in K-depleted rats (n = 6 at t = 0 min, P < 0.01) and from 3.3 ± 0.1 mmol/l to a maximum of 7.6 ± 0.8 mmol/l after 90 min in control rats (n = 7 at t = 0 min, P < 0.01) (Fig. 4). The plasma K rise is most likely due to inhibition of the {alpha}2-isoform of the Na-K pump in skeletal muscles. The plasma K increases were associated with increases in myocardial K content to 87 ± 2 µmol/g wet wt in K-depleted rats (P < 0.05, n = 6) and to 92 ± 2 µmol/g wet wt in control rats (P < 0.05, n = 6) (Fig. 3). In gastrocnemius muscle no significant changes were seen in K-depleted rats (86 ± 2 µmol/g wet wt; P > 0.5, n = 6) or in control rats (110 ± 2 µmol/g wet wt; P > 0.9, n = 6).



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Fig. 4. Plasma K measured in rats K depleted for 2 wk and in their weight-matched controls before (t = 0 min; n = 6) and after intraperitoneal injection of 12.5 nmol ouabain/g body wt. In the control group, n = 4 at t = 105 min and n = 2 at t = 120 min. In the K-depleted group, n = 4 at t = 105 and 120 min. Mean ± SE values are indicated.

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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The present study demonstrates an increase in rat myocardial Na-K-ATPase concentration in response to a minor decrease in myocardial K after 2 wk of K depletion. This K depletion-induced upregulation was shown for the first time to be associated with an increase in net myocardial K uptake during intravenous K repletion in vivo.

Na-K pump regulation in K depletion. Na-K pump regulations in response to K depletion were measured longitudinally, i.e., during the course of gradually developing K depletion. The overall upregulation of myocardial Na-K pump density of 13%, as measured by K-dependent pNPPase activity, after 2 wk of K depletion was the differentiated outcome of a 30% reduction in {alpha}2-isoform abundance—composing ~10% of the myocardial Na-K pump pool (31)—and a 24% upregulation of the vast majority of rat myocardial Na-K pumps, i.e., {alpha}1. The downregulation of {alpha}2 was of same order of magnitude as previously seen (1). In that study K depletion of rats for 2 wk reduced plasma K to 3.1 ± 0.3 mmol/l (n = 4–7) and reduced body weight by 12%. The presently observed increase in {alpha}1 is in acceptable agreement with results obtained in the former study (1) of no significant changes in {alpha}1, considering the less pronounced hypokalemia achieved and that the weight loss may have reduced Na-K-ATPase density (17). A K depletion-induced reduction in rat myocardial K-dependent pNPPase activity of 14% (n = 4) was previously reported (24). However, again, factors like weight loss, the use of older rats, and K depletion for twice as long a period inducing a myocardial K loss of 13% compared with 6% in the present study may all have influenced the results. Extreme K depletion may cause generalized catabolism, including reductions in Na-K pump density. Furthermore, in the two former studies (1, 24) no longitudinal measurements were reported. The presently seen {alpha}1 upregulation is in agreement with observations in K-depleted rabbits of a ~15% upregulation of {alpha}1-isoform as assessed by [3H]ouabain binding (34) and a 13% increase as assessed by patch clamping with patch pipette Na concentrations at a near-saturating level (28).

The effects of K depletion on myocardial Na-K-ATPase show similarities with changes in skeletal muscle Na-K-ATPase. Thus {alpha}2 in skeletal muscles is reduced, whereas {alpha}1 is unaltered (1, 3, 6). Because {alpha}2 composes the majority (75–85%) of the Na-K pumps in rat skeletal muscles (15), K depletion may cause a marked reduction in skeletal muscle Na-K-ATPase concentration—up to 70% in rats (23). In humans a K depletion-induced reduction of up to 19% has been reported (12). Taken together, whereas K depletion causes Na-K-ATPase downregulation in skeletal muscles, myocardial Na-K-ATPase seems to be upregulated. Furthermore, the present results illustrate how a quantitatively differentiated response to a stimulus between tissues can be achieved by differentiated Na-K-ATPase isoform distribution. Between species the myocardial response to K depletion may depend on which isoforms are expressed. For instance, rabbit myocardium only expresses the {alpha}1-isoform, which seems to protect from any K depletion-induced downregulation (28, 34). Interestingly, this phenotype also seems to protect from any myocardial K loss during K depletion (34). In humans, {alpha}1-, {alpha}2-, and {alpha}3-isoforms of the Na-K pump are expressed in myocardium (26). If regulations in humans in response to K depletion are similar to those seen in rats and rabbits, the balance between an upregulation of {alpha}1 and downregulation of {alpha}2 may be decisive for the clinically adverse cardiovascular effects of K depletion. However, a prediction of clinical effects is hampered by the lack of knowledge about regulation of {alpha}3 in K depletion.

Myocardial ion homeostasis in K depletion-repletion. The present results confirm that in response to hypokalemia there is a marked difference in K loss between myocardium (6%) and skeletal muscles (23%) (23). On the other hand, the present results show that in response to increased extracellular K—during intravenous KCl infusion—there was a considerable myocardial net K uptake of ~34% after ~2 h of KCl infusion in K-depleted rats and 18% after ~1 h in control rats. It is important to assess how much of these increases can be accounted for by the increase in extracellular K. Thus during these ~2 h of KCl infusion in K-depleted rats an increase in myocardial K of ~28 µmol/g wet wt and a rise in plasma K from 1.8 to 11.8 mmol/l were seen. Assuming identical K concentrations in plasma and in the extracellular space and an extracellular volume (ECV) of 20% of tissue weight, it can be calculated that ~7.1% [(11.8 – 1.8 mmol/l) x 0.20 ml ECV/g wet wt ÷ 28 µmol/g wet wt x 100] of the increase in myocardial K content is accounted for by increased ECV K concentration. In controls the corresponding value is 11.2%. Thus the majority of the KCl infusion-induced increase in myocardial K content was due to increased intracellular K. We previously reported (3) an increase in skeletal muscle K of 41–56% in K-depleted rats and 10% in control rats after KCl infusions for ~3.5 and 1 h, respectively. Thus, during maximum KCl infusion, skeletal muscle and myocardial K rises are of the same order of magnitude in K-depleted rats, whereas in controls the K increase in the myocardium is about twice as high as in skeletal muscle (3). This indicates that in contrast to the differentiated tissue K losses developing during K depletion, the heart participates—on a weight basis—in buffering of increases in plasma K to an extent comparable to skeletal muscles, i.e., the heart is not only "passively" protected by K buffering in skeletal muscles. This high net K uptake in the heart is in agreement with a previously reported 10% increase in net myocardial K content only 2 days after commencement of oral K loading (2).

The maximum net myocardial K uptake rate of ~0.3 µmol·g wet wt–1·min–1 was similar to the previously observed gastrocnemius muscle K uptake rate of ~0.3 µmol·g wet wt–1·min–1 (3). These net K uptake rates in myocardium and skeletal muscles do not reflect that the Na-K-ATPase density in myocardium is approximately six times higher than in skeletal muscles (24). This indicates that differences in net K uptake rates between tissues are not determined by Na-K-ATPase densities, whereas differences in Na-K-ATPase densities within tissues affect maximum net K uptake rates. Differences in K uptake between tissues are likely to be due to differences in tissue function and differences in the extent of secondary active transport of other ions and nutrients and thus differences in magnitude of cellular loss of K and gain of Na. The myocardial K uptake rate was significantly higher in K-depleted rats with increased Na-K-ATPase density compared with control rats when plasma K levels were kept equal by KCl infusion in the two groups. This confirms the concept of a positive correlation between Na-K-ATPase density and K uptake capacity (7), and the finding is also in agreement with K depletion-induced increases in Na-K pump affinity for extracellular K and for intracellular Na (28). Finally, the intracellular reduction in K and increase in Na reduce the K inhibition at the cytoplasmic Na sites of the Na-K ATPase (32). This mechanism favors preservation of cellular K during K depletion and increases K uptake during K repletion.

The ouabain-induced increases in plasma K and myocardial K without changes in skeletal muscle K content were most likely caused by near saturation of the skeletal muscle {alpha}2-isoform of the Na-K pump by ouabain. These findings give an indication of the magnitude of the ongoing skeletal muscle K leakage—K uptake at rest—even in the K-depleted animals. The ouabain-induced increase in myocardial K indicates that myocardial {alpha}1—composing 90% of the rat myocardial Na-K pump pool (31)—has a higher K affinity than skeletal muscle {alpha}1—composing 15–25% of the skeletal muscle Na-K pump pool–or reflects the difference in Na-K pump density between myocardium and muscle. Thus it is of major interest that differences between tissues in Na-K-ATPase isoform concentration, distribution, and K affinity may have marked effects on K homeostasis in vivo. Indeed, the changes in tissue K induced by ouabain injection illustrate how a functional inhibition of {alpha}2, mimicking K depletion-induced {alpha}2 downregulation, seems to favor protection of myocardial K at the expense of skeletal muscle K. The present results are not easily translatable into effects of digoxin in humans, because all three Na-K pump isoforms in human skeletal muscle and myocardium are ouabain sensitive (11, 26).

We previously reported (3) that Mg content in skeletal muscles increases in response to K depletion, and in this study we observed an increase in myocardial Mg as well. This increase was not caused by decreased myocardial water content or increased Mg content in the chow. Clinically, muscular K and Mg are generally considered positively correlated (10), and in patients with cardiac arrhythmias suspected to be associated with K/Mg depletion, K as well as Mg is generally acutely administered. However, whereas this positive correlation between K and Mg applies to K depletion induced by K-wasting diuretics (12), the present results suggest that this is not the case when a selective negative K balance induces K depletion.

Myocardial Na was not significantly affected by K depletion but only showed a tendency to be increased (by ~5%). A similar tendency has been observed by others (34). It cannot be ruled out that this tendency might be related to a higher Na content in the chow administered to the K-depleted animals, but it is more likely that an increase represents a compensatory mechanism for the decrease in cellular K. Such a compensation may be caused by a plasma K-induced reduction in Na-K pump activity, which, in turn, may not be fully compensated for by the increase in myocardial Na-K pump density. As previously reported for skeletal muscles (3), a tendency to a reduction in myocardial Na was seen after KCl infusion. Myocardial hypertrophy and heart failure are associated with increased intracellular myocyte Na in animals and in humans (for review, see Ref. 25). Because of the interplay between intracellular Na and Ca, changes in Na may have profound effects on myocardial contractility and arrhythmogenesis. Pogwizd et al. (25) suggested that increased myocardial Na in heart failure might be offset by a fall in myocardial K. It is of major interest that K depletion seems to induce a similar pattern of myocardial Na/K changes as seen in heart failure. However, it is not clear whether an increase in myocardial Na, e.g., caused by K depletion, may cause heart failure—or whether a myocardial Na increase in heart failure only develops as a homeostatic compensation to increase contractility. On the basis of the present results it is tempting to suggest that the intracellular Na and K changes seen in K depletion could in some instances play a primary role in development of cardiomyopathy and that iatrogenic K depletion might worsen heart failure. In keeping with this it is of considerable clinical interest that drugs that have been shown to improve morbidity and mortality in patients with heart failure, i.e., angiotensin-converting enzyme inhibitors, aldosterone antagonists, and {beta}-adrenoceptor antagonists (for recent review, see Ref. 18), all tend to increase plasma K, whereas K-wasting diuretics, which may cause significant skeletal muscle and myocardial K depletion, may be deleterious (8). Further attention to K homeostasis may prove to be of utmost clinical value in cardiovascular medicine.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The study was supported in part by the Danish Heart Foundation and Director Emil C. Hertz and wife Inger Hertz's foundation.


    ACKNOWLEDGMENTS
 
The author thanks Keld Kjelden for being a scientific mentor, for utmost inspiration and valuable discussions, and for providing laboratory facilities. The author thanks Ulla Steen Pedersen for skilled technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: H. Bundgaard, Medical Dept. B 2142, Heart Centre, Rigshospitalet, National University Hospital, Univ. of Copenhagen, Blegdamsvej 9, 2100 Copenhagen, Denmark (E-mail: henningbundgaard{at}dadlnet.dk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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