Calcium channels and cation transport ATPases in cardiac hypertrophy induced by aortic constriction in newborn rats. Lei Zheng, 1 Maurice Wibo, 1 Frantigek ...
Molecularand CellularBiochemistry163/164: 23-29, 1996. © 1996KluwerAcademicPublishers.Printedin the Netherlands.
Calcium channels and cation transport ATPases in cardiac hypertrophy induced by aortic constriction in newborn rats Lei Zheng, 1 Maurice Wibo, 1 Frantigek Kol/tf 2 and Th60phile Godfraind 1 tLaboratoire de Pharmacologie, Universitg Catholique de Louvain, B-1200 Brussels, Belgium and 2Institute of Physiology, Academy of Sciences of the Czech Republic, CZ-142 20 Prague, Czech Republic
Abstract Cardiac enlargement due to gradual pressure overload was induced by abdominal aortic constriction in 2-day-old rats. On day 90, the functional performance of the left ventricle was assessed by acute load test (ligation of ascending aorta) in open-chest anaesthetized animals. Two subgroups, designated compensated and decompensated hypertrophy (CH and DH), were distinguished on the basis of the functional reserve of left ventricle, which was significantly impaired in DH but not in CH, and of right ventricle weight, which was markedly increased in DH but not significantly modified in CH. In total particulate fractions prepared from hypertrophied left ventricles, the levels (per g tissue) of sarcoplasmic reticulum Ca2+-transport systems were decreased, either slightly (by 13-16%: [3H]ryanodine binding) or moderately (by 28%: thapsigargin-sensitive Ca2+-AYPase activity). The number of sarcolemmal L-type Ca 2÷ channels ([3H]PN200-110 binding) was not modified significantly, while that of 131-adrenoceptors ([3H]CGP-12177 binding) was reduced, especially in the DH group (by 39%). Na+,K+-ATPase activity was reduced by 28% in CH and 41% in DH. [3H]Ouabain binding experiments (saturation and dissociation) indicated the existence of two high-affinity binding sites, attributable to the Na÷,K+-ATPase ct3 and ct2 subunit isoforms; while the relatively minor cz3 component did not change significantly in hypertrophied ventricles, the (z2 component was markedly downregulated, decreasing by 57% in CH and 82% in DH. (Mol Cell Biochem 163/164: 23-29, 1996) Key words: Ca 2+ channel, ryanodine, 13-adrenoceptor, Na+,K+-ATPase, Ca2*-ATPase, cardiac hypertrophy
Introduction The early postnatal period is of critical importance for the development of the heart. After birth, the heart has to cope with dramatic increase of functional load, which results in rapid cardiac growth and differentiation. One of the key events of this period is a loss of proliferative capacity ofventricular myocytes and thus a termination ofhyperplastic heart growth. In the rat, this ability decreases rapidly already during the first week after birth; thereafter, growth of the ventricular myocardium occurs by myocyte hypertrophy [ 1]. It is apparent that any alteration in load imposed early after birth may have different consequences for the development of cardiac structure and function than the same intervention at later stage. We elaborated an experimental model allowing to in-
crease cardiac workload by abdominal aortic constriction already in 2-day-old rats. This additional growth stimulus results in considerable cardiomegaly at adult stage, with low neonatal mortality [2]. Moreover, after aortic constriction at 2 days of age, a significant proportion of adult rats develop cardiac failure, thus allowing to compare compensated and decompensated stages ofcardiomegaly in age-matched animals. With the aim to getting better insight into the mechanisms responsible for transition from compensated cardiomegaly to failure, we decided to investigate in this model the properties of several channels (L-type Ca2+ channel, sarcoplasmic reticulum Ca2+ channel), pumps (CaZ+-ATPase,Na+,K+-ATPase) and receptors (13-adrenoceptor) involved directly or indirectly in excitationcontraction coupling. Some of our results have been reported previously in abstract form [3].
Addressfor offprints: M. Wibo,Laboratoirede Pharmacologic,UCL 54.10, AvenueHippocrate54, B-1200 Brussels,Belgium
24
Materials and methods Animal model Newborn male Wistar rats born in our animal care facility (Institute of Physiology, Prague) were used. Pressure overload was induced by aortic constriction in 2-day-old rats. Under light ether anaesthesia, the abdominal aorta was exposed from the dorsolateral side in the subdiaphragmatic suprarenal region. A silk ligature was tied around abdominal aorta, with a hypodermic needle of 0.25 mm in diameter serving as a template. In sham operated littermates which were used as age-matched controls, the aorta was exposed but not constricted; the number per litter was kept at eight.
Evaluation of left ventricular function On day 90, the functional performance of the left ventricle was assessed in open-chest anaesthetized animals as previously described [4]. Briefly, after stemotomy, a 21 gauge needle was introduced transmurally, via the apex, into the left ventricular cavity. Pressure changes transmitted to a pressure transducer were analyzed after amplification by our computer program. Acute ligation of the ascending aorta served as a loading test for left ventricle function. Left ventricular systolic pressure (LVSP) and maximum rate of pressure development (dP/dtmax) were derived from pressure curve before and after loading. The difference in LVSP or dP/dtm,x after and before loading was considered as the functional reserve. This study conforms with the Guide for the care and use of laboratory animals published by the US National Institute of Health (NIH publication N ° 85-23, 1985).
Membrane fractions After evaluation of left ventricular function in vivo, the hearts were removed and dissected into right (RV) and left (LV) ventricle (including septum). The ventricles were quickly rinsed in cold (5°C) saline, weighed, frozen in liquid nitrogen, and stored frozen at -80°C. After thawing, finely minced ventricular tissue (1-2 g) was homogenized with 10-12 ml of a chilled solution (pH 7.3) containing (mM): sucrose 250, imidazole 5, dithioerythritol 2, phenylmethylsulfonyl fluoride 0.2 and EDTA 0.2, by means of an Ultra-Turrax homogenizer (three 10 sec bursts at 13500 r/min). The suspension was filtered on a nylon sieve (400 lam) to remove the largest pieces of unhomogenized material. An aliquot was taken for DNA assay (see below) and the filtered suspension was centrifuged at 110,000 g for 35 min (at 2°C) in a TFT 50.38 rotor (Kontron AG, Zurich, Switzerland). The pellet was resus-
pended in a solution (pH 7.4)containing (raM): sucrose 250, imidazole 3 and phenylmethylsulfonyl fluoride 0.1, bymeans of a Dounce-type homogenizer, and this suspension was designated total particulate fraction. In some experiments, microsomal fractions were prepared from ventricular homogenates and subfractionated by isopycnic density gradient centrifugation, as previously described [5].
Protein, DNA, enzyme activity and radioligand binding assays Protein was assayed by the Lowry method, with bovine serum albumin as standard [6]. DNA determinations were performed with compound Hoechst 33258 (bisbenzimidazole) [7]. Samples (0.1 ml) from homogenates were quickly diluted in 1.9 ml of a chilled solution (pH 7.4) containing (mM): NaH2PO 450, NaC1 2000, EDTA 2, and stored at -20°C. Aliquots of diluted homogenates were then mixed with a solution (pH 7.4) containing 50 mM NaH2P04, 2 M NaCI and Hoechst 33258 at a final concentration of 1 lag/ml. Fluorescence measurements (excitation, 350 nm; emission, 455 nm) were made with an Aminco spectrofluorometer (SPF-500). Calf thymus DNA was used as a standard; its concentration was determined spectrophotometrically [8]. CaE÷-ATPase and Na+,K+-ATPase were assayed by coupled optical assays as reported previously [5]. The thapsigarginsensitive CaZ*-ATPase activity (25°C) was defined as the activity inhibited by 50 nM thapsigargin. Na+,K+-ATPase activity (37°C), defined as the activity inhibited by 4 mM ouabain, was determined on tissue fractions that had been preincubated for 2 min at room temperature in the presence of sodium dodecyl sulfate at a concentration of 0.1 mg/ml. Specific binding of [3H](+)-PN200-110 and [3H]ryanodine was measured exactly as reported previously [5]. [3H]Ouabain binding was determined as described [5], except that membrane fractions were preincubated with saponin (0.5 mg/ ml) at 2°C, instead of room temperature. [3H](-)-CGP- 12177 binding was determined as described [5]; binding to 131adrenoceptors was estimated by subtracting from total binding the binding obtained in the presence of 0.5 laM CGP20712A, a highly selective 131 ligand [9].
Drugs [3H](+)-PN200-110 [isopropyl-4-(2,1,3-benzoxadiazol-4-yl)1,4-dihydro-5-methoxycarbonyl-2,6-dimethyl-3-pyridinecarboxylate] (70 Ci/mmol), [3H]ryanodine (87 Ci/mmol) and [3H]ouabain (23 Ci/mmol) were from NEN Research Products (Boston, MA, U.S.A.). [3H](-)-CGP-12177 [(-)-4-(3-tbutylamino-2-hydroxypropoxy)-benzimidazol-2-one](53 Ci/ mmol) was from The Radiochemical Centre (Amersham,
25 U.K.). CGP-20712A was kindly provided by Ciba-Geigy AG (Basel, Switzerland). Bisbenzimidazole (Hoechst 33258) and calf thymus DNA were from Sigma (St Louis, MO, U.S.A.). The origin of the other reagents used has been reported [5]. Experiments with 1,4-dihydropyridines were carried out under yellow light.
Statistical analysis Differences between groups were assessed by one-way analysis of variance and Dunnett t test. In equilibrium binding studies (Table 4), parameters were estimated by a non-linear curve fitting program (Ligand [ 10]). As for [3H]ouabain binding, data were best fitted by a two-site model (as indicated by an approximate F-test) and reliable K a values could only be estimated from the simultaneous analysis of several experiments (4 different membrane preparations); 95% confidence limits were first calculated for log K dvalues and then converted back [10]. K d values estimated by this method for the two [3H]ouabain binding sites were then introduced as constant parameters in Ligand to calculate the corresponding Bmax values of individual membrane preparations. [3H]Ouabain dissociation curves were analyzed by the Kinetic program [10].
Results Gradual pressure overload induced by aortic banding in 2day-old rats evoked marked cardiac enlargement at 90 days (Table 1). Two subgroups, designated compensated and decompensated hypertrophy (CH and DH), could be distinguished on the basis of right ventricle weight divided by tibial length (RV/TL): when this ratio was unchanged or increased by less than 30% as compared to age-matched controls, animals were included in the CH group; when it was increased by at least 100%, they were included in the DH group. Left ventricular hypertrophy was significantlymore pronounced in
DH than in CH: the ratio of left ventricle weight to tibia1 length (LV/TL) was increased by 66% in CH and 112% in Dtt. In vivo evaluation of left ventricular function yielded the data listed in Table 2. Left ventricular systolic pressure (LVSP) in CH and DH rats was significantly higher than in control rats before as well as after acute aortic clamping. Maximum rate of pressure development (dP/dtmax) was significantly increased at rest, but not after aortic clamping. For both parameters, the functional reserve was not affected in CH rats, but was impaired in DH rats, which confirmed that our criterion based on RV/TL for sorting out failing left ventricles was correct. As shown in Table 3, the amount of DNA per g tissue was moderately, but significantly reduced in CH and DH left ventricles (by 10-15%). The thapsigargin-sensitive Ca2+-ATPase activity per g tissure, measured at optimal free Ca 2+concentration (20 ~tM), was reduced by 28% in both CH and DH groups, but when the activity was expressed per mg DNA, no significant modification was observed. (Under our assay conditions, almost no thapsigargin-resistant Ca2+-ATPase activity could be detected in total particulate fractions.) The Na+,K÷-ATPase activity per g tissue was decreased by 29% in CH, and 41% in DH; when expressed per mg DNA, reductions were less but still significant. Saturation experiments were carried out with four radioligands in order to characterize equilibrium binding parameters in total particulate fractions from left ventricles, that is dissociation constants (Kd) and maximal binding capacities (Bmax). Except for [3H]ouabain, equilibrium binding data were best fitted by a single-site model when analyzed by the Ligand program [10]. As shown in Table 4, K d values were not significantly affected after aortic constriction. As for [3H] PN200-110 binding (sarcolemmal L-type channels), Bma x values did not differ significantly among the three groups. Table 2. Functional parameters
Con~ol (14)
Table 1. Weight parameters
Control
Body (g) LV (mg) RV (rag) LV / TL (mg/mm) RV/TL (mg/mm)
(14)
Compensated hypearophy (6)
Decompensated hype~rophy (6)
330.00 ± 10 648.00±20 194.00 ± 4 17.30 ± 0.4 5.19 + 0.12
320.00 ± 17 1057.00+62" 215.00 ± 9 28,70 ± 1.3" 5.82 ± 0.20
262.0 ± 17'* 1237.0+ 17"* 385.0 ± 21":~ 36.7 + 0.8"* 11.4 ± 0.6**
Values are mean ± S.E.M. from n rats (between parentheses). LV, left ventricle including septum; RV, right ventricle; TL, tibial length; *Significantly different from control (p < 0.01); *Significantly different from compensated hypertrophy (p < 0.05); *Significantly different from compensated hypertrophy (p < 0.01)
LVSP (mmHg) before clamp after clamp reserve (dP/dt)m.x (mm Hg/s) before clamp after clamp reserve
92 ± 4 290 + 6 198 ± 5 4529 + 331 9006 + 278 4477 + 291
Compensated Decompensated hypertrophy hypertrophy (5) (5) 160 ± 10"* 374 ± 20** 214 ± 20
192 ± 13"** 345 ± 12"* 153 ± 12'*~
6448 ± 579* 6893 + 690** 10716 + 844 9453 + 714 4268 + 697 2560 + 640***
Values are mean ± S.E.M. from n rats (between parentheses). LVSP, left ventricular systolic pressure; (dP/dt)ma~, maximum rate o f pressure development; reserve, difference between value after and before clamp; *Significantly different from control (p < 0.05); **Significantly different from control (p < 0.01); ?Significantly different from compensated hypertrophy (p < 0.05); :~Significantly different from compensated hypertrophy (p < 0.01)
26 Table 3. Protein, DNA and ATPase activities in total particulate fractions from left ventricular tissue
Control
Protein (mg/g) DNA (mg/g) Ca2+-AYPase (~tmol/min/g) (~mol/min/mg DNA) Na +, K+-ATPase (lamol/min/g) (~tmol/min/mg DNA)
121.80 + 1.75 ± 6.14 + 3.56 ± 10.63 + 6.17 ±
4.5 (5) 0.03 (6) 0.33 (4) 0.49 (4) 0.61 (4) 0.90 (4)
Compensated hypertrophy
Decompensated hypertrophy
120.30 + 1.58 ± 4.67 ± 3.01 ± 7.59 + 4.91 +
112.90 1.48 4.68 3.27 6.30 4.43
4.9 (6) 0.06* (6) 0.33** (5) 0.46 (5) 0.49** (5) 0.81" (5)
+ ± ± ± + +
2.6 (4) 0.06** (5) 0.28* (4) 0.33 (4) 0.24** (4) 0.50** (4)
Values are mean ± S,E.M. from n preparations (between parentheses). Protein and DNA amounts are referred to 1 g of tissue wet weight; ATPase activities are referred to 1 g of tissue and to 1 mg DNA. Ca2+-ATPase refers to the thapsigargin-sensitive activity. * Significantly different from control (p < 0.05); • * Significantly different from control (p < 0.01) Table 4. Binding parameters in total particulate fractions from left ventricular tissue
Control
[3H](+)-PN200-110 K d (pM) B a~ (pmol/g tissue) B x (pmol/mg DNA [3H]Ryanodine K d (pM) Bm,~(pmol/g tissue) Bm~x (pmol/mg DNA
88.20 ± 7.5 15.50 + 0.7 8.86 ± 0.34
593.00 ± 16 87.10 + 3,2 49.80 ± 2.2
[3H](-)-CGP-12177 (131) K d (pM) 105.00 ± 6.2 Bm~~ (pmol/g tissue) 2.00 ± 0.13 B (pmol/mg DNA) 1.14:1:0.07 [3H]ouabain ct3 K d (nM) Bm~x (pmol/g tissue) Bm~x (pmol/mg DNA) ct2 K d (nM) Bin,~(pmol/g tissue) Bm~~ (pmol/mg DNA)
Compensated Decompensated hypertrophy hypertrophy
72.30 ± 11.3 14.20 ± 0.6 9.22 + 0.26
70.60 ± 7.8 14.30 ± 1.0 9.60 + 0.30
559.00 ± 20 557.00 ± 14 76.00 ± 1.7" 73.10 ± 2.7** 49.40 ± 1.4 49.70 + 2.8
87.50 ± 5.6 93.60 ± 6.1 1.52 + 0.15" 1.22 ± 0.10** 0.98 ± 0.08 0.83 ± 0.07*
6.4 (0.7-59.1) 7.2 (0.9-57.1) 6.9 (1.2-40.8) 3.93 + 1.36 2.44 ± 0.66 2.97 + 0.63 2.55 + 0.83 1.68 ± 0.44 1.73 ± 0.39 66 (32-134) 173 (99-300) 114 (53-244) 92.40 ± 10.3 39.80 ± 4.9** 17.00 ± 2.8**; 2 6 . 7 0 2 3 . 5 * * 11.80+ 1.7 *** 53.20 ± 5.3
Values are mean + S.E.M. from 5 ~ preparations, except for [3H]ouabain binding (n = 4). K~ values for [3H]ouabain binding were estimated from the simultaneous analysis of7-10 experiments according to the Ligand program [10], as described in Materials and methods; 95% confidence limits are given between parentheses. * Significantly different from control (p < 0,05); • *Significantly different from control (p < 0.01); ~Significantly different from compensated hypertrophy (p < 0.01)
Using density gradient centrifugation [5], we found no difference in the subsarcolemmal distribution of [3H]PN200-110 binding sites between the control and DH groups (data not shown). As for [3H]ryanodine binding (SR calcium release channels), Bin,x values were slightly lower (by 13-16%) in hypertrophied ventricles, but only when expressed in pmol per g tissue. [3H]CGP- 12177 binding was essentially attributable to 131-adrenoceptors under our assay conditions (see
Materials and methods); Bmax values were distinctly lower after aortic banding, especially in the DH group (-39 or -27%, depending on the reference used). Scatchard plots of [3H]ouabain equilibrium binding data were concave upwards, as illustrated in Fig. 1 for typical experiments. In each group, data were best fitted by a twosite model when 7-10 experiments on 4 different membrane preparations were analyzed simultaneously by the Ligand program (see Materials and methods). The site with the lowest K d (6-7 nM) accounted for only 3% of the total number of sites in control ventricles; this component, which is likely to be the or3 isoform (see Discussion), showed comparable Bin,x values in the three groups. In contrast, the second highaffinity component (Kd 70-170 nM), which is presumably the a2 isoform, was markedly reduced in hypertrophied ventricles (CH, -57 t o - 5 0 % and DH, -82 to -78%, depending on the reference used). In kinetic experiments carried out on total particulate fractions from control and DH hearts (Fig. 2), we observed that dissociation of bound [3H]ouabain was best fitted by two exponentials in line with the presence of two high-affinity binding sites. The amount of bound [3H]ouabain that followed fast dissociation kinetics (dissociation rate constant, k t, 0.494).67 min -~) was 4-fold higher in control than in DH (24.0 + 2.2 vs 6.0 + 1.1 pmol g tissue, at a [3H]ouabain concentration of 43 nM), while the amount of bound [3H]ouabain that followed slow dissociation kinetics (k l, 0.035-0.037 min-') did not differ significantly between the two groups, in agreement with the Bmax values ascribed to the a2 and ct3 isoform, respectively.
Discussion Banding of abdominal aorta applied early after birth induced gradual pressure overload and the consequent development of severe cardiomegaly in adult rats. This model is particularly attractive, since it allows to analyze two age-matched groups differing not only by the degree of left ventricular enlargement, but also by the presence or absence of signs of
27 0.010 l
0.008v m
i.l_
~
0.0060.004-
~
~
cO
0.0020 0
0.5 1.0 Bound[3H]ouabain(prnol/mg)
!
1.5
Fig. 1. Scatchard plot of [3H]ouabain binding to total particulate fractions from rat ventricles. Each curve was obtained from a single membrane preparation (control, filled squares; compensated hypertrophy, open squares; decompensated hypertrophy, filled triangles). Bound and free radioligand were expressed as pmol per mg protein and pmol per ml, respectively. Each point is the mean of triplicate or quadruplicate determinations.
100
g 55 e--
0 1
0
i
I0
i
20 Time(rain)
i
30
Fig. 2. Dissociation kinetics of [3H]ouabain bound to total particulate fractions from rat ventricles. Each curve was obtained from 3-4 membrane preparations (control, filled squares; decompensated hypertrophy, open squares); S.E.M. are indicated by vertical bars. Membranes were first incubated for 50 min with 43 nM [3H]ouabain as indicated in Materials and methods, and, thereafter, dissociation was induced by adding 0.1 mM ouabain.
cardiac failure (as judged from right ventricular mass and functional reserve of left ventricle evaluated in vivo). Although hypertrophy of left ventricular myocytes was indeed present in this model (as indicated by a 20% reduction inthe number of myocytes per unit volume at 60 days, without concomitant change in the proportion of cardiac volume occupied by myocytes [unpublished data]), it is likely that hyper-plastic growth also contributed to increased cardiac mass. This is supported by the observation that proliferation of myocytes can be stimulated by aortic constriction appliedyet in 5-day-old rats [11]. In several models of cardiac hypertrophy and failure, alterations of excitation-contraction coupling have been re-
ported, including prolongation of the action potential, prolongation of the Ca 2+transient and decrease of its peak value. Therefore, several groups have been involved in the analysis of Ca 2÷ currents (reviewed in [12]) and 1,4- dihydropyridine binding sites on the one hand, and Ca2+-transport systems of sarcoplasmic reticulum on the other hand. After banding of abdominal aorta in adult rats, severe cardiac hypertrophy, but without evidence of failure, did not evoke modifications in Ca 2+current density [13] or in the Bm~X(per g tissue) of [3H]PN200-110 binding [14]. We show here that even in rats showing signs of cardiac failure, the properties of [3H]PN200-110 binding were not modified. This is in line with the absence of modification of [3H]PN200-110 binding reported in dog cardiac failure induced by rapidpacing [15], but in contrast with the decrease of [3H]PN200-110 binding observed in models of Cardiac failure due to cardiac infarction [16-18]. Reports on 1,4-dihydropyridine receptors in human cardiac failure are contradictory [19, 20]. It has been suggested that alterations in Ca 2÷ channel density might be restricted to end-stage cardiac failure [12]. Our data indicating that the SR CaZ÷-ATPase activity and the number ofryanodine receptors per g tissue were reduced after aortic banding in newborn rats are in agreement with previous findings in human and experimental cardiac hypertrophy and failure at the level of mRNA expression, immunoreactive protein or enzyme activity (reviewed in [21]). These alterations of critical components of Ca 2+cycling system may be responsible in part for the decreased function of SR and impaired left ventricular contraction and relaxation reported in some studies. However, the decrease in the myocardial density of ryanodine receptors was rather limited in our model, in agreement with data obtained after aortic banding in adult rats [14]. In contrast to L-type Ca 2÷channels, some other sarcolemmal
28 constituents, that is /31-adrenoceptors and Na+,K+-ATPase, were markedly downregulated in the CH and DH groups. Our data on 13-adrenoceptors confirm those obtained previously in compensatory hypertrophy evoked by aortic banding in adult rats [22], where reduced gene expression o f the left ventricular [31-adrenoceptor has also been recently reported [23]. A reduction o f Na+,K+-ATPase activity has been observed in several studies on cardiac hypertrophy and failure (for references, see [24, 25]). Na+,K÷-ATPase consists of a catalytic, ouabain-binding a subunit and a glycosylated I] subunit. Three a subunit isoforms have been found in rat ventricular tissue, among which the al isoform, with low affinity for ouabain, is the most abundant, accounting for up to 90% o f the total enzyme activity [26]. Under our assay conditions, m o s t o f the Na÷,K+-ATPase activity in total particulate fractions from control and hypertrophied ventricles displayed low sensitivity to ouabain (data not shown), which suggests that the decrease in Na÷,K+-ATPase activity in the DH and CH groups is likely to reflect mainly a corresponding decrease in the level o f the al isoform. This possibility could not be confirmed by binding studies, which are not suitable to measure the concentration o f a l in rat tissues, in view of its especially low affinity for ouabain. Interestingly, the level ofimmunoreactive al protein did not change in compensatory cardiac hypertrophy produced by aortic constriction in adult rats [27]. In both equilibrium and kinetic binding experiments, we found that [3H]ouabain bound to two specific sites, a major one with a K a of 70-170 nM and a k~ o f 0.5-0.7 min -1, and a minor one with a K d of 5-10 nM and a kq o f 0.03~3.04 min -1. These features indicate that the major and minor components are attributable, respectively, to the eL2 and c~3 isoforms of Na+,K÷-ATPase [5]. Aortic constriction induced a selective loss of high-affinity binding corresponding to ct2, leading to a decrease in the global rate of[3H]ouabain dissociation from high-affinity binding sites. In agreement with this interpretation, a marked reduction o f or2 at the level o f m R N A and immunoreactive protein has been reported in most models o f cardiac hypertrophy [25, 27-29]. We found that binding attributable to ct2 was significantly more reduced in the DH than in the CH group. This quasi-disappearance ofet2 in the DH group might be interpreted as a return to the neonatal phenotype, but there was no concomitant increase of the binding attributable to ct3, which is the predominant high-affinity isoform in neonatal ventricles. However, Charlemagne et al. [27] did find some up-regulation o f et3 in their model of hypertrophic heart. What could be the functional significance o f the downregulation o f ~ 2 in rat cardiac hypertrophy? The selective loss o f this high-affinity isoform might have physiological effect similar to that o f inhibition of~t2 by ouabain in control heart. As shown by Finet et al. [30], selective inhibition of cx2 by low ouabain concentrations induces a marked positive ino-
tropic effect in rat ventricular tissue. Thus, down-regulation of Na÷,K+-ATPase (especially cx2) may be viewed as an adaptive change in pressure overload. Excessive down-regulation, however, which could be in relation with the heightened sympathetic activity [31], might be a contributing factor in the transition from compensatory hypertrophy to cardiac failure.
Acknowledgments This w o r k was supported by Fonds de D r v e l o p p e m e n t Scientifique U.C.L., Fonds de la Recherche Scientifique Mrdicale (grant FRSM n ° 3-4546-92), Ministrre de l'Education et de la Recherche Scientifique (grant ARC n ° 89/95135) and Grant A g e n c y o f the Czech Ministry o f Health (grant Z192). The authors thank Anne Lebbe and MarieChristine Hamaide for their skillful technical assistance.
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