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Cardiovascular Research 1998 38(2):451-462; doi:10.1016/S0008-6363(98)00007-8
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Modifications of myocardial Na+,K+-ATPase isoforms and Na+/Ca2+ exchanger in aldosterone/salt-induced hypertension in guinea pigs

Juan Fernando Ramñrez-Gila,c, Pascal Trouvéc, Nathalie Mougenota, Alain Carayonb, Philippe Lechata and Danièle Charlemagnec,*

aLaboratoire de Pharmacologie Cardiovasculaire, Service de Pharmocologie, IFR Génétique et Physiopathologie Cardiovasculaire, Hôpital Pitié-Salpêtrière, 47 Bd de l'Hôpital, 75651 Paris Cedex 13, France
bService de Biochimie, Faculté de Médecine Pitié-Salpêtrière, IFR Génétique et Physiopathologie Cardiovasculaire, Hôpital Pitié-Salpêtrière, Faculté de Médecine Pitié-Salpêtrière, 91 Bd de l'Hôpital, 75013 Paris Cedex 13, France
cINSERM-U127, Biologie et Physiopathologie du coeur et des vaisseaux, IFR Circulation Lariboisière, Université D. Diderot, 41 Bd de la Chapelle, 75475 Paris Cedex 10, France

* Corresponding author. Tel.: +33 (1) 4285 8065; Fax: +33 (1) 4874 2315; E-mail: daniele.charlemagne@inserm.lrb.ap-hop-paris.fr

Received 13 June 1997; accepted 17 December 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The aim of this study was to determine whether changes in cardiac Na+,K+-ATPase subunits and Na+/Ca2+ exchanger expression are regulated in aldosterone-salt hypertensive guinea pigs. Methods: Guinea pigs (GP) were unilaterally nephrectomized and randomized into three groups (aldosterone-salt; control-salt; control). After 90 days of treatment, echocardiographic M-mode assessment and right carotid arterial catheterization were performed in vivo, and plasma hormones and electrolytes were measured. mRNA and protein levels were studied by Northern and Western blot analysis. Results: Aldosterone-salt treatment induced, (1) arterial hypertension (+40%) and LV hypertrophy (+60%) without altering LV-fractional shortening, (2) an increase in plasma norepinephrine levels (+262%) and suppression of renin activity. Northern blot analysis showed the presence of the mRNA encoding the three {alpha} isoforms and the β1 subunit of Na+,K+-ATPase in GP myocardium. In the aldosterone-salt group, levels of {alpha}1 and β1 mRNAs were unchanged. {alpha}2 mRNA was increased in both ventricles, whereas {alpha}3 mRNA was increased in hypertrophied LV only. Furthermore, levels of the Na+/Ca2+ exchanger mRNA were decreased in both ventricles. At protein level, the two major transcripts ({alpha}1 and {alpha}2) were detected but {alpha}3 isoform was not. Parallel changes in protein and mRNA accumulation of {alpha}1 and {alpha}2 isoforms were observed in hypertrophied LV. Conclusion: These results show that {alpha}1 and {alpha}2 isoforms are expressed in GP heart and that they are independently regulated in aldosterone-salt hypertension. Like the {alpha}1 isoform in renal tissue, {alpha}2 isoform is the main target of aldosterone-salt. Reciprocal expression of the Na+/Ca2+ exchanger and Na+,K+-ATPase suggests an adaptational mechanism which maintains an appropriate sodium gradient and calcium concentration in hypertensive myocardium.

KEYWORDS Aldosterone; Na+,K+-ATPase; Na+/Ca2+ exchanger; Hypertension; Heart; Guinea pigs


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The sodium pump, or Na+,K+-ATPase, generates and maintains the concentration gradients of Na+ and K+ across the plasma membrane. In the heart, Na+,K+-ATPase is particularly important in membrane repolarization and the regulation of cardiac contractility. Specific inhibition of the Na+ pump by cardiac glycosides such as digitalis leads to a positive inotropic effect by increasing intracellular Na+ concentration and decreasing the driving force for Ca2+ extrusion via the Na+/Ca2+ exchanger, thus leading to increased intracellular stores of Ca2+ [1]. Na+,K+-ATPase is composed of an {alpha}-catalytic subunit and a smaller β-glycoprotein subunit [2]. Expression of the three isoforms of the {alpha}-catalytic subunit is species- and tissue-specific and varies during ontogenic development [3–5]. The three isoforms differ in their affinity for cardiac glycosides and, according to their relative cardiac expression, contribute to differences in the inotropic response to digitalis among mammalian species. The {alpha}1 isoform is expressed ubiquitously in adult mammalian heart, as the ‘house-keeping’ form of the enzyme. It has a low affinity for ouabain in rat [4, 6], ferret [7]and dog [8]and a high affinity in sheep [9]and humans [10, 11]. Expression of the {alpha}2 and {alpha}3 isoforms varies between species: the {alpha}2 isoform has been clearly identified in adult rat [3, 5, 12], and human myocardium [10, 11, 13], and the {alpha}3 isoform is expressed in adult human [10], dog [14–16], and ferret [17]heart and in neonatal rat heart [3, 5]. These isoforms always have a high affinity for ouabain. Cardiac expression of the {alpha} isoforms is subject to hormonal regulation [18–21]and has also been reported to vary during hypertension and heart failure [12, 14, 16, 22–25]. However, it is unclear whether an increase in plasma aldosterone, which induces hypertension and cardiac hypertrophy [24, 26]and stimulates Na+,K+-ATPase {alpha}1 isoform expression in renal tubules [27]and isolated myocytes [19], might alter the myocardial expression of the {alpha} isoforms of Na+,K+-ATPase.

Our aim was to determine whether cardiac Na+,K+-ATPase isoform expression is regulated by aldosterone. The guinea pig was chosen for this study instead of the rat because it has a much greater affinity for cardiac glycosides [28, 29]and its contractility and calcium handling are more dependent upon external calcium [30, 31], characteristics shared by the human heart [10]. Hypertension was induced by aldosterone-salt administration and the model was characterized by echocardiographic and hemodynamic studies, and hormone and electrolyte measurements. Besides the determination of mRNA and protein levels of Na+,K+-ATPase isoforms, mRNA levels of the Na+/Ca2+ exchanger were also studied, because changes in Na+,K+-ATPase are related to changes in Na+/Ca2+ exchanger expression [23]and lead to altered Na+ homeostasis and contractility. Furthermore, mRNA levels were studied in both ventricles to discriminate between hormonal and hypertension-related effects.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Animals and experimental protocol
Adult male guinea pigs (350±50 g) were used throughout. The investigation was conducted under guidelines established by the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH publication No. 85-23, revised 1985).

2.1.1 Surgical procedure
All animals were fasted overnight before surgery. Anesthesia consisted of intramuscular (i.m.) ketamine hydrochloride (43 mg/kg)+chlorpromazine (0.65 mg/kg). Left nephrectomy was performed after laparotomy one centimeter below the rib cage. Cefalotine (70 mg/kg) was administered i.m. Guinea pigs were randomly assigned to one of three groups:

1. Aldosterone-salt group (n=7); an osmotic minipump was implanted subcutaneously (s.c.) to deliver 0.75 µg/h d-aldosterone and animals were given salt water (1% NaCl+0.3% KCl) immediately after nephrectomy. Minipumps were changed every 15 days.
2. Control-salt group (n=5); an osmotic minipump containing normal saline solution was implanted s.c. and animals were given salt water.
3. Control group (n=5); an osmotic minipump containing normal saline solution was implanted s.c. and the guinea pigs received normal drinking water.

Preliminary studies have shown that longer treatment (120 days) leads to the death of 50% of operated animals. Therefore all animals were studied after 90 days of treatment and there were no deaths.

2.2 Echocardiographic assessment of left ventricular size and function
Echocardiographic studies were performed on all animals. Guinea pigs were lightly anesthetized with intramuscular ketamine hydrochloride (40 mg/kg)+chlorpromazine (0.5 mg/kg), then placed in a proneposition. Imaging was performed via the anterior chest wall using a commercial echocardiographic device equipped with a 7.5-MHz transducer (Kontron Instruments, France) with M-mode acquisition. The best acoustic window was from the left parasternal position, which gave long- and short-axis views of the left ventricle (LV), equivalent to the left parasternal window in humans. M-mode tracings were recorded through the anterior and posterior LV walls at a paper speed of 50 mm/s. Anterior and posterior wall thickness (end-diastolic) and LV internal dimensions were measured using the American Society for Echocardiography method [32], adapted to rats by Litwin et al. [33], and averaged by a single blinded observer. LV fractional shortening was calculated as: (LVDD-LVSD)/LVDDx100, where LVDD is LV internal end-diastolic dimension and LVSD is LV internal end-systolic dimension. Following the study, animals were rested for 48 h before hemodynamic exploration and sacrifice.

2.3 Hemodynamic studies
The animals were weighed prior to the study. Hemodynamic studies were performed under anesthesia (ketamine+chlorpromazine, i.m.). The right carotid artery was cannulated with a polyethylene catheter connected to a pressure transducer (Gould Electronic, Cleveland, Ohio, USA). Systolic blood pressure and heart rate were allowed to stabilize for approximately 1 min and pressure tracings were then recorded on a strip chart recorder at a paper speed of 100 mm/s (Gould 2000 series, Gould Electronic, Cleveland, Ohio, USA). Guinea pigs were allowed to breathe spontaneously during recordings. At end of the study the transducer was removed and blood samples were collected. The animals were then killed for RNA extraction and quantification.

2.4 Hormone and electrolyte assays
Blood samples were collected in tubes containing 100 mmol/l EDTA for aldosterone, plasma renin activity (PRA), angiotensin I (AI), and angiotensin II (AII) assays, and in tubes containing 43 lithium heparin u.s.p. units for Na+,K+,HCO3–, epinephrine, norepinephrine and dopamine assays. After centrifugation the plasma samples were removed and stored at –20°C. Hormones of the renin–angiotensin–aldosterone system were measured as described by Schaison et al. [34]. Briefly, PRA and plasma immunoreactive AI were determined by radioimmunoassay (RIA) with [125I]AI (CEA, France). The detection limit of AI RIA was 6 pg/tube, corresponding to 0.12 ng/ml/h for PRA determination. Plasma immunoreactive AII was measured by RIA with [125I]AI (CEA, France) after blood ethanol extraction. The detection limit of AII RIA was 4 pg/tube. The cross-reactivity (%) of various peptides was Val5-AII, 100; Ile5-AII, 100; Ile5-AI, 13.6; 2-10-AI, 1.6; 2-8-AII (AIII), 45; 1-7-AII,105; 3-8-AII, 177;4-8-AII, 96; angiotensinogen, 0.001; Leu-enkephalin, 0.001; and substance P, 0.0013%. AII was expressed in picograms per milliliter. Plasma concentrations of aldosterone were measured by RIA with [125I]aldosterone (CEA, France) and expressed in picograms per milliliter. The detection limit of the method was 1 pg/tube. Plasma concentrations of norepinephrine, epinephrine and dopamine were measured by radioenzymatic methods with [3H] S-adenosyl–L-methionine (Amersham, Les Ulis, France) and expressed in picograms per milliliter. The detection limit of the method was 5 pg/ml for norepinephrine and epinephrine, and 15–20 pg/ml for dopamine. Na+,K+ and HCO3– concentrations were assayed by flame photometry and expressed in mEq/l.

2.5 Tissues
The brain and heart were removed and rinsed in ice-cold saline solution. The LV with the septum, and the right ventricle (RV) were separated, weighed, frozen in liquid nitrogen and stored at –80°C until use.

2.6 Total RNA extraction
Total RNAs from guinea pig brain, LV and RV were prepared according to the method of Chirgwin et al. [35]. RNA concentration was calculated by measuring absorbance at 260 nm, assuming 40 µg/ml for 1 absorbance unit. The RNAs were resuspended in Tris. HCl-EDTA (TE, pH 7.4) and aliquots were stored at –80°C until use.

2.7 Northern and slot blots
For Northern blots, 20 µg of total RNA was denatured in 50% formamide, 2.2 mol/l formaldehyde, and 1xMOPS buffer (pH 7.4) and electrophoresed in 1% agarose gel. Total RNA was then transferred to a nylon Hybond-N membrane (Amersham, Les Ulis, France). For slot blot analysis, 2.5, 5, 10 and 15 µg RNA of each sample (n=7 in aldosterone-salt group; n=5 in control-salt and control groups) was denatured and applied directly to nylon Hybond-N membranes. All blots were submitted to ultraviolet irradiation to covalently link the RNA samples.

Blots were subsequently hybridized with the following probes: rat {alpha} isoform Na+,K+-ATPase-specific cDNAs ({alpha}1, NarI-StuI, nucleotides 89-491; {alpha}2 ScaI-NheI, nucleotide 121-502) [22]; a guinea pig {alpha}3 cDNA probe (PstI-SmaI, nucleotides 57-331) prepared by nested PCR [36], sequenced and tested by RNase protection assay (RPA) with brain and LV GP mRNA (data not shown); a rat β1-subunit cDNA probe (HindIII-PstI, nucleotides 914-1184); a rat β2-subunit cDNA probe (EcoRI, nucleotides 1-1331, a gift from Dr. Martin-Vassalo); a rat Na+/Ca2+ exchanger cDNA probe (SacI-KpnI, 580 base pairs, a gift from Dr. K. Boheler), and a rat glyceraldehyde-3 phosphate dehydrogenase (GAPDH) cDNA probe (PstI, 1300 base pairs, a gift from Dr. F. Moreau-Gachelin). Hybridization with Na+,K+-ATPase, Na+/Ca2+ exchanger and GAPDH cDNAs was carried out in 50% formamide, 5xDenhardt's solution, 5xstandard saline phosphate ethylene-diamine-tetra-acetic acid (SSPE), 0.1% sodium dodecyl sulfate (SDS), 200 µg/ml herring sperm DNA, and 20 µg/ml poly (A+) at 42°C. Blots were prehybridized in this solution for 12 h and hybridized for 24 h with cDNA probes radiolabeled by random primer extension with the Amersham Megaprime DNA labeling system. {alpha} (32P) dCTP (3000 Ci/mmol, Du Pont-New England Nuclear, Boston, MA, USA) was incorporated to obtain a specific activity of 2–8x108 counts per µg. Blots were washed in 2xSSC (0.03 M NaCl, 0.03 M Na citrate, pH 7.0) and 0.1% SDS at room temperature for 30 min, and then twice for 45 min in 0.5xSSC with 0.1% SDS at 45°C. Northern blots were exposed to X-ray films (Hyperfilm, Amersham, Les Ulis, France) at –80°C with Quanta III intensifying screens. Slot blots were used to quantify mRNA levels and were exposed to intensifying screens (Fuji imaging plate type BAS IIIS, Fuji, Tokyo, Japan) and then analyzed in a Bio-Imaging Analyser System (BAS 1000 Mac BAS, Fuji, Tokyo, Japan); the densitometric scores of each specific mRNA were normalized to GAPDH mRNA to correct for sample loading.

2.8 Crude particulate preparation
Crude particulate preparations (CPP) were obtained according to the method of Rannou et al. [37]. Briefly, tissue samples (300 mg of guinea pig brain and LV) were thawed and homogenized in 10 ml of buffer (200 mmol/l sucrose, 20 mmol/l HEPES, pH 7.4) containing protease inhibitors (1.1 mmol/l leupeptin, 0.7 mmol/l aprotinin, 120 mmol/l phenylmethane-sulfonyl fluoride, 1 mmol/l iodoacetamide, 0.7 mmol/l pepstatin, 1 mmol/l diisopropylfluorophosphate). The homogenate was centrifuged at 41 000 g for 45 min in a Sorvall SS34 rotor. The pellet was suspended in 0.1 mol/l NaCl, 30 mmol/l Imidazole, 8% sucrose, pH 6.8 in the presence of the protease inhibitors. Protein content was determined by the method of Lowry using bovine serum albumin as a standard [38]. The samples of RV were not analysed because all RV tissue was collected for mRNA analysis.

2.9 Western blot analysis
Detection and quantification of {alpha}1, {alpha}2, and {alpha}3 Na+,K+-ATPase isoforms contained in CPP were performed using the Western blot technique [39]. Polyclonal antibodies specific for rat {alpha}1 and {alpha}3 isoforms were kindly provided by Dr. R. Mercer (Washington University, School of Medicine, St. Louis, MO, USA) and monoclonal antibodies McB2 against rat {alpha}2 isoform were kindly provided by Dr. K. Sweadner (Massachusetts General Hospital, Boston, MA, USA). Heart proteins (60 µg for immunological detection of {alpha}1, and 120 µg for {alpha}2 and {alpha}3) and brain proteins (5 µg) from CPP were loaded on a 6% polyacrylamide SDS gel. After electrophoretic separation, proteins were transferred to a nitrocellulose sheet (Hybond-P PVDF membrane, Amersham, Les Ulis, France) at 150 V for 1.5 h. The membranes were then incubated with blocking solution (5% fat-free milk, 0.1% Tween-20 in PBS) for 2 h at room temperature. Specific antibodies were diluted in blocking solution (1:400 for {alpha}1, 1:200 for {alpha}2, and 1:500 for {alpha}3) and applied overnight at 4°C. Incubation with horseradish peroxydase-conjugated anti-rabbit (for {alpha}1 and {alpha}3 Na+,K+-ATPase isoforms) or anti-mouse (for {alpha}2 Na+,K+-ATPase isoform) IgG antibodies (diluted 1:5000, Amersham, Les Ulis, France) was performed for 2 h at room temperature. The reactive proteins were detected by chemiluminescent reaction (ECL+plus kit-Amersham International, UK) followed by exposition of the membranes to Hyperfilm Formula film. The PVDF sheets were washed (5x5 min) with a medium composed of 0.1 mol/l sodium acetate (pH=4) and 0.5 mol/l sodium chloride to eliminate the antibodies. Transferred proteins were then stained with Coomassie Blue R250 (0.1% in 1% acetic acid, 40% methanol) for 5 min. Non-specific staining was then suppressed by a 5 min wash in 10% acetic acid, 40% methanol. Quantification of both specific bands on autoradiography and the transferred protein bands stained with Coomassie blue was performed by densitometric analysis after scanning (Molecular Analysis System, Bio-Rad Laboratories, Hercules, CA, USA). Each individual value represents the mean of three independent determinations for each animal (n=7 in aldosterone-salt group; n=5 in control-salt and control groups).

2.10 Statistical analysis
Results are expressed as mean±SEM. Comparisons between groups were made with the Mann–Whitney test after the Kruskall Wallis test (non parametric analysis of variance). Because comparisons were made among the three groups, the difference was considered significant if P<0.0125 (Bonferroni correction method).


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Chronic treatment of guinea pigs with aldosterone-salt causes hypertension-induced left ventricular hypertrophy
Data were determined after 90 days of treatment. LV diastolic diameter and the diastolic thickness of the anterior and posterior walls were measured using M-mode echocardiography 48 h before sacrifice. These parameters, indexed to body weight (BW), were significantly increased in the treated groups (P<0.01; Table 1). The results of the echocardiographic study agreed with postmortem anatomical data. The ratio of LV weight (LVW) to BW in the aldosterone-salt group was increased by 60±4% compared with the control and control-salt groups (P<0.01). The LVW/RVW ratio, an index of LV hypertrophy which is independent of body weight, was increased by 56±3% in the aldosterone-salt group compared to controls (P<0.01). There was no evidence of RV hypertrophy and no signs of heart failure in the aldosterone-salt group. Table 2 shows non-invasive indices of contractility and invasive hemodynamic data. LV fractional shortening, measured by M-mode echocardiography, did not differ between the three groups. Heart rate and systolic blood pressure were measured by right carotid arterial catheterisation and the values in control animals were in agreement with those reported by Randhawa et al. [40]. There was no difference in heart rate between groups. An increase in systolic blood pressure was observed in the aldosterone-salt group compared with the control and control-salt groups (P<0.01). Therefore, the aldosterone-salt group was characterized by increases in systolic blood pressure and LV hypertrophy indexes measured both in vivo and ex vivo.


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  		Table 1 Left ventricular size assessed using M-mode echocardiography, and anatomical data

 

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  		Table 2 Hemodynamic data

 
Table 3 shows plasma hormone and electrolyte concentrations. Aldosterone, AI and AII plasma concentrations were significantly decreased in the control-saline group compared with the control group. In the aldosterone-salt group, plasma aldosterone and Na+ concentration were significantly increased compared with the control-salt group, while plasma renin activity and angiotensin I and II fell significantly. As catecholamines play a role in systemic adaptation in cardiac hypertrophy and regulate the Na+-K+ pump [14, 20], plasma catecholamine concentrations were also evaluated. Aldosterone-salt treatment was associated with a marked increase in plasma norepinephrine concentrations compared with the control and control-salt groups.


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  		Table 3 Plasma hormone and electrolyte values

 
3.2 Ventricular mRNA levels of Na+,K+-ATPase and Na+/Ca2+ exchanger
3.2.1 Detection of Na+,K+-ATPase subunit mRNAs
Northern blot analysis was performed with total RNA from both ventricles of the control, control-salt and aldosterone groups. Expression of the different subunits in brain, LV and RV samples from the three groups is shown in Fig. 1. The rat {alpha}1 cDNA probe hybridized to a single 3.7-kb mRNA, the rat {alpha}2 cDNA probe gave a faint 5.3-kb signal, and hybridization of the Northern blot with the guinea pig {alpha}3 cDNA probe revealed one band at 3.7 kb. Taken together, these findings confirm that {alpha}1 is the main isoform of the catalytic subunit in guinea pig heart and show that the {alpha}2 and {alpha}3 transcripts are also expressed. The rat β1 subunit cDNA probe hybridized to two mRNA species (2.7 and 2.35 kb), as in the rat heart [3], whereas the β2 subunit mRNA was not detected (data not shown). Aldosterone-salt treatment resulted in an increase in {alpha}2 and {alpha}3 mRNA levels (Fig. 1). However, slot blot analysis was required to accurately measure {alpha} isoform and β subunit mRNA levels.


Figure 1
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  		Fig. 1 Representative Northern blot of {alpha}1, {alpha}2 and {alpha}3 isoforms and β1 subunit of Na+,K+-ATPase, Na+/Ca2+ exchanger, and GAPDH mRNAs in left and right ventricles from control (C), control (CS), and aldosterone-salt treated (AS) guinea pigs (20 µg of total RNA) and guinea pig brain (5 µg of total RNA).

 
3.2.2 Quantitative determination of Na+,K+-ATPase isoform mRNAs in left and right ventricles from hypertensive guinea pigs
In order to study the respective influences of hypertension and hormonal stimulation on mRNA accumulation, both LV and RV were studied. Results from slot blot analysis of the {alpha}1, {alpha}2 and {alpha}3 isoforms and β1 subunit mRNAs from LV and RV of control and hypertensive animals are summarized in Fig. 2Fig. 3. As shown in Fig. 2, the better sensitivity of the phosphoscreen, compared to autoradiography, allowed us to quantify the signals, even though the {alpha}2 and {alpha}3 mRNAs were difficult to detect by Northern blotting (Fig. 1). The values were normalized to GAPDH mRNA and expressed in arbitrary units.


Figure 2
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  		Fig. 2 Representative slot blot analysis of {alpha}1, {alpha}2 and {alpha}3 isoforms and β1 subunit of Na+,K+-ATPase, Na+/Ca2+ exchanger, and GAPDH mRNAs in left and right ventricles from control (C), control-salt (CS), and aldosterone-salt treated (AS) guinea pigs. Results with 5, 10 and 15 µg of total RNA (a, b and c, respectively) are shown.

 

Figure 3
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  		Fig. 3 Bar graphs showing mRNA levels (relative to GAPDH mRNA) of Na+,K+-ATPase {alpha}1, {alpha}2 and {alpha}3 isoforms and the β1 subunit in left (Panel A) and right (Panel B) ventricles: control (left clear block, n=5), control-salt (middle hatched block, n=5), and aldosterone-sale (right hatched block, n=7) treated guinea pigs. Each data point per animal is the result of two independent experiments performed with 2.5, 5, 10 and 15 µg of total RNA. Values are mean±SEM.

 
In the LV of the aldosterone-salt group, relative mRNA levels of the {alpha}1 isoform and β1 subunit were unchanged compared with the control group, whereas {alpha}2 and {alpha}3 mRNA levels were significantly increased (values for {alpha}2 were 1.0±0.1 and 1.30±0.03 in control and aldosterone-salt groups respectively, and values for {alpha}3 were 1.0±0.3 and 2.3±0.2 in control and aldosterone-salt groups respectively). Therefore, a 30% increase for {alpha}2 was found in the aldosterone-salt group, and a greater than 100% increase for {alpha}3 mRNA (Fig. 3A). As in the LV, {alpha}1 and β1 isoform mRNA levels were similar in the RV of control and hypertensive animals, and {alpha}2 mRNA levels were significantly higher in the aldosterone-salt group. In contrast, the {alpha}3 mRNA level remained similar in RV from control and hypertensive guinea pigs (Fig. 3B).

No significant difference in mRNA levels was found between the control-salt group and the control group. The differences observed in this study were only significant when the aldosterone-salt group was compared to the control group, probably because of a trend towards higher values of {alpha}2 and {alpha}3 accumulation in the control-salt vs. the control group. There was a parallel increase in LV an RV from aldosterone-salt group between {alpha}2 mRNA levels and plasma aldosterone level whereas {alpha}3 mRNA was increased only in the hypertrophied LV, which suggest an {alpha}3 regulation independent from the hormonal changes.

3.2.3 Cardiac Na+/Ca2+ exchanger mRNA levels in aldosterone-salt-induced hypertension
The Northern blot of guinea pig ventricular RNA probed with the rat Na+/Ca2+ exchanger cDNA is shown in Fig. 1. A major band was detected at 7 kb, as previously reported for the rat [23]and canine [41]exchanger. Slot blot analyses showed that Na+/Ca2+ exchanger mRNA levels were lower in hypertrophied LV and non hypertrophied RV of animals treated for 90 days with aldosterone-salt, than in the LV and RV of control animals (0.75±0.1- and 0.54±0.2-fold, respectively) (Figs. 2 and 4Go). Therefore, regulation of Na+/Ca2+ exchanger mRNA like that of {alpha}2 mRNA occurs in both LV and RV but in opposite mode.


Figure 4
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  		Fig. 4 Bar graphs showing mRNA levels (relative to GAPDH mRNA) of the Na+,Ca2+ exchanger in left and right ventricles: control (left clear block, n=5), control-salt (middle hatched block, n=5) and aldosterone-sale (right hatched block, n=7) treated guinea pigs. Each data point per animal is the result of two independent experiments performed with 2.5, 5, 10 and 15 µg of total RNA Values are mean±SEM.

 
3.3 Western blot analysis of Na+,K+-ATPase {alpha}-isoforms in left ventricles from hypertensive guinea pigs
Fig. 5A shows representative results of Western blot analysis of Na+,K+-ATPase {alpha} isoforms in LV from control and hypertensive guinea pigs. Guinea pig brain CPP was used as a positive control and each {alpha} isoform was detected as a unique specific band at Mr 100 kDa. In contrast, only {alpha}1 and {alpha}2 isoforms were detected in LV from all groups. The {alpha}3 isoform was not detected despite the high amount of total protein per lane (120 µg). After normalization of the data for the amount of transferred proteins per lane, the results are expressed as the relative amounts of {alpha}1 and {alpha}2 isoforms in CPP compared to an arbitrary value of 100% obtained in control animals. Fig. 5B shows that the amount of {alpha}1 isoform did not vary between control, control-salt, and aldosterone-salt groups, while the amount of {alpha}2 isoform was increased by 150% in LV from aldosterone-salt guinea pigs. As the protein yield expressed in mg of protein per g of LV was not significantly different between groups (control LV: 53±6, n=5; control-salt LV: 54±7, n=5; aldosterone-salt LV: 62±9, n=7), results from Fig. 5B are representative of the changes at tissue level. As reported for {alpha}2 mRNA, {alpha}2 isoform increased in both LV and RV suggesting that the regulation is dependent of hormonal changes and independent from hypertrophy.


Figure 5
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  		Fig. 5 Western blot analysis of Na+,K+-ATPase {alpha}1, {alpha}2 and {alpha}3 isoforms in left ventricle. A: immunoblot analysis in adult guinea pig brain (5 µg for each isoform) and left ventricle from control (C), control-salt (CS), and aldosterone-salt (AS) treated guinea pigs. {alpha}1 isoform detection was performed with 60 µg protein. {alpha}2 and {alpha}3 were performed with 120 µg protein. B: Bar graph showing protein levels of Na+,K+-ATPase {alpha} isoforms. Densitometric score normalized to total proteins. Each individual value represents the mean of three independent determinations for each animal: control (left clear block, n=5), control-salt (middle hatched block, n=5) and aldosterone-salt (right hatched block, n=7) treated guinea pigs. Values are mean±SEM, expressed as % relative to the control group.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The major findings of this study are that long-term aldosterone-salt treatment of guinea pigs increases systolic blood pressure, left ventricular weight, and {alpha}2 mRNA and protein levels of Na+,K+-ATPase in both ventricles. Such increase in {alpha}2 mRNA in the aldosterone-salt group was associated with the decrease in Na+/Ca2+ exchanger mRNA. The {alpha}3 mRNA level was increased in hypertrophied LV but the protein was not detected.

Aldosterone-salt administration to guinea pigs for 90 days had a hypertensive effect and induced compensatory LV hypertrophy, as previously described in the rat [24, 26], but had no effect on LV fractional shortening, an index of heart contractility. Mineralocorticoid administration has been reported to modify the activity of the renin–angiotensin system in the rat. Brilla et al. [26]reported that plasma levels of angiotensin II were depressed then returned to normal values after 8 weeks of aldosterone-salt treatment, and Karam et al. [42]reported a decrease in plasma renin activity in the DOCA-salt hypertensive rat. Consistent with previous reports, decreases in plasma renin activity and plasma levels of angiotensin I and angiotensin II were observed in the present study. However, the mechanism of the 3- to 4-fold increase in norepinephrine plasma concentrations observed in this study is not clear. It could be a consequence of aldosterone administration by inhibition of direct norepinephrine uptake by aldosterone [43]. Such an increase, which correlated closely with LV hypertrophy (LVW/RVW ratio), could play a compensatory role by restoring stress on myocardial fibers and then maintaining contractility. This would not be the case at a more advanced stage with LV dysfunction, where catecholamines could participate in the auto-aggravation of heart failure [44, 45]. Similarly, hyperaldosteronism in humans induces hypertension associated with secondary suppression of plasma renin activity [46]and enhanced sympathetic tone [47].

In this study, mRNAs encoding the three {alpha} isoforms and the β1 subunit of Na+,K+-ATPase were detected in the guinea pig heart, whereas the β2-subunit was not detected. At protein level, the presence of {alpha}1 and {alpha}2 isoforms was demonstrated. In keeping with the expression of multiple isoforms of the {alpha} catalytic subunit of Na+,K+-ATPase in the guinea pig heart, the presence of at least two components with different affinities for digitalis has been demonstrated [29, 30]and evidence for two {alpha} subunit proteins has been obtained by immunological methods [30]. However, Sweadner et al. showed the presence of the {alpha}1 isoform but failed to detect the {alpha}2 or {alpha}3 subunits in microsomal membranes from guinea pig hearts [48]. It is likely that differences in {alpha}2 detection are related to the preparation (crude preparation vs. microsomal membranes) or the amount of protein used in Western blot assays. In guinea pig myocardium, the detection of {alpha}3 isoform mRNA has required the use of a specific guinea pig probe but {alpha}3 mRNA expression was not accompanied by protein detection. The discrepancy between the expression of {alpha}3 mRNA and the absence of detection of the related protein could be due to translational regulation, but we cannot exclude the possibility that the protein is present at very low level. In the context of previous reports, our results support the conclusion that the {alpha}1 isoform is the predominant transcript of the {alpha} catalytic subunit of Na+,K+-ATPase and that the {alpha}2 isoform is coexpressed in the guinea pig heart.

{alpha}1 mRNA and protein levels were not significantly altered in hypertensive guinea pigs, in agreement with results reported by Robert et al. after 2 weeks of aldosterone administration to rats [24]. In contrast, aldosterone increases {alpha}1 expression in isolated rat cardiomyocytes exposed to aldosterone for a short period [19]and in renal tubules [27]. This discrepancy points to the fact that the results obtained in vitro may differ from results in vivo, but suggests that the resultant effect of chronic administration in vivo is an unchanged expression of the cardiac {alpha}1 isoform. Moreover, unchanged {alpha}1 mRNA and/or protein levels have been reported in different animal models of hypertension [16, 22, 24]. The β1 subunit mRNA level was unchanged in both guinea pig ventricles, as also reported in different models of hypertension in the rats [12, 24].

In this study, expression of {alpha}2 and {alpha}3 isoforms in aldosterone-treated guinea pig hearts contrasted strongly with that of the {alpha}1 isoform. Enhanced expression of {alpha}2 mRNA and protein was observed in both ventricles, suggesting hormonal regulation of the isoform. However, this increase in {alpha}2 mRNA was not observed during the pre-hypertensive period of aldosterone administration in rats [24]; furthermore, a decrease in {alpha}2 mRNA and/or protein accumulation has been reported in hypertensive rats with high levels of angiotensin II [12, 22, 48]. Whether the discrepancies between these rat models and our guinea pig model are related to plasma levels of aldosterone and/or angiotensin II, or to the species, is difficult to determine.

Contrary to the parallel increase in LV and RV from aldosterone-salt group between {alpha}2 mRNA levels and plasma aldosterone level, {alpha}3 mRNA was increased only in the hypertrophied LV. It suggests that {alpha}3 regulation is modulated by an increase in systolic blood pressure (Table 2) and is independent from the hormonal changes. These results are in agreement with previous studies of severe LV hypertrophy in the rat [22]and suggest that {alpha}3 regulation is modulated by an increase in systolic blood pressure (cf. Table 2). Consistent with this assumption, unchanged {alpha}3 mRNA levels were reported during aldosterone administration to rats, before the onset of hypertension and hypertrophy [24]. Although {alpha}3 mRNA was easily observed in hypertrophied LV (Figs. 1 and 2Go), the corresponding protein was not detected. The reason for this is unknown. It might be related to a low amount of protein, increased protein degradation or translational regulation.

The mRNA levels of the cardiac Na+/Ca2+ exchanger were significantly depressed in both ventricles. Simultaneously, {alpha}2 mRNA levels increased. This is in agreement with the reciprocal regulation of the Na+/Ca2+ exchanger and {alpha}2 isoform reported by Magyar et al. [23], who suggested that this might be effective in maintaining intracellular calcium concentrations. Although mRNA or protein expression does not reflect enzyme activity, it is likely that an increase in {alpha}2 isoform expression would increase Na+,K+-ATPase activity and maintain the Na+ gradient. Reciprocal expression of the Na+/Ca2+ exchanger and Na+,K+-ATPase may be a cardiac adaptive response to prevent any reduction in intracellular calcium concentration and thus prevent any decrease in myocardial contractility, as observed in this model of compensatory hypertrophy.

In conclusion, we show that the three cardiac {alpha} isoforms of Na+,K+-ATPase are independently regulated during administration of aldosterone to guinea pigs. The aldosterone effect on Na+,K+-ATPase was restricted to the {alpha}2 isoform, and was coupled to a decrease in mRNA levels of the Na+/Ca2+ exchanger. These reciprocal changes suggest an adaptational mechanism to maintain an appropriate sodium gradient and calcium concentration in hypertensive myocardium. The guinea pig model of aldosterone-induced cardiac overload could provide an interesting system for studying neuro-hormonal interactions in cardiac hypertrophy and failure.

Time for primary review 39 days.


   Acknowledgements
 
This work was supported in part by a doctoral fellowship grant from Ministère Français des Affaires Etrangères and Colombian Institute of Science and Techology (COLCIENCIAS) (JFRG). The authors thank Dr. L. Rappaport and Dr. C. Delcayre for extremely helpful and constructive discussions, and Dr. C. Landault for plasma catecholamine assays.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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P. Fransen, J. Hendrickx, D. L Brutsaert, and S. U Sys
Distribution and role of Na+/K+ ATPase in endocardial endothelium
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