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2-isoform expression in guinea pig hearts during
transition from compensation to decompensation
1 Institut National de la Santé et de la Recherche Médicale, Unité 127, Institut Fédératif de Recherche Circulation Lariboisière, Université Denis Diderot, 75475 Paris; and 2 Centre Hospitalier Régional et Universitaire de Rennes, Pontchaillou, 35033 Rennes, France
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Disturbance in ionic gradient across sarcolemma
may lead to arrhythmias. Because Na+-K+-ATPase
regulates intracellular Na+ and K+
concentrations, and therefore intracellular Ca2+
concentration homeostasis, our aim was to determine whether changes in
the Na+-K+-ATPase 
-isoforms in guinea pigs
during transition from compensated (CLVH) to decompensated left
ventricular hypertrophy (DLVH) were concomitant with arrhythmias. After
12- and 20-mo aortic stenosis, CLVH and DLVH were characterized by
increased mean arterial pressure (30% and 52.7%, respectively). DLVH
differed from CLVH by significantly increased end-diastolic pressure
(34%), decreased sarco(endo)plasmic reticulum Ca2+-ATPase
(
75%), and increased Na+/Ca2+ exchanger
(25%) mRNA levels and by the occurrence of ventricular arrhythmias.
The -isoform (mRNA and protein levels) was significantly lower in
DLVH (2.2 ± 0.2- and 1.4 ± 0.15-fold, respectively, vs. control) than in CLVH (3.5 ± 0.4- and 2.2 ± 0.13-fold,
respectively) and was present in sarcolemma and T tubules. Changes in
the levels of 1- and 3-isoform in CLVH
and DLVH appear physiologically irrelevant. We suggest that the
increased level of 2-isoform in CLVH may participate in
compensation, whereas its relative decrease in DLVH may enhance
decompensation and arrhythmias.
cardiac hypertrophy; sarco(endo)plasmic reticulum calcium-adenosine 5'-triphosphatase; sodium-calcium exchanger; arrhythmias; guinea pig
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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PHENOTYPIC (1, 16) and physiological alterations, including arrhythmias (2, 30), have been reported during hypertrophy and the transition phase from compensated left ventricular hypertrophy (CLVH) to heart failure. Most of the reports concern the expression of proteins directly involved in excitation-contraction coupling, Ca2+ cycling (1, 16, 17, 32), and alterations in the Ca2+ transient (2, 7, 10, 41, 47), but very few deal with Na+-K+-ATPase expression. However, cardiac Na+-K+-ATPase plays a key role in Na+ and K+ homeostasis, membrane repolarization, inotropic response to cardiac glycosides, and, indirectly, intracellular Ca2+ movements by its functional coupling to Na+/Ca2+ exchanger (27, 46). In addition, decreased Na+-K+-ATPase activity (39) has been shown to participate in the alteration of cellular electrical properties occurring in human heart failure, which may trigger arrhythmias (34). Therefore, the question arises as to whether alterations of Na+-K+-ATPase may participate together with sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) and Na+/Ca2+ exchanger to the transition from CLVH to decompensation and to the occurrence of arrhythmias.
Na+-K+-ATPase is a heterodimer composed
of a catalytic -subunit and a glycoproteic 
-subunit (31,
43). There are four isoforms of the catalytic -subunit
(1-4) that differ in their affinity for cardiac glycosides, Na+, and K+ (43,
48, 49). Their expression is species and tissue specific and
varies during ontogenic development (31). The
1-isoform is the "housekeeping" form of the
catalytic subunits. It is ubiquitous in the mammalian heart and shows a
low affinity for ouabain in the rat and guinea pig heart (5, 26,
31, 43) but a high affinity in the human heart
(39). The presence of 2-isoform has been
reported in adult rat (11, 31, 43), human (39, 46), and guinea pig (35) myocardium. According to
previous results obtained by enzymology, 2-isoform has a
high affinity for ouabain in guinea pigs (5, 26), as in
rats (44). It has been very recently identified as the
Na+-K+-ATPase isoform specifically involved in
the regulation of cardiac Ca2+ handling (22).
The 3-isoform is expressed in human (39, 46), neonatal rat (31), and dog hearts
(6).
Expression of the -isoforms has been reported to be regulated by
hormonal stimulation (20, 28, 44) and during hypertension (19), LVH (9, 12), and heart failure
(23, 39, 46). In the hypertrophied or failing heart, the
most common feature is a decrease in 2-isoform level
(12, 19, 20, 39, 44) that is associated with a shift from
the 2-isoform to the neonatal 3-isoform
in the rat (12) or accompanied by a parallel decrease of
both 1- and 3-isoform levels in human
heart failure (39). However, an increase in
1-isoform expression has been reported in the abdominal
aortic constriction in the ferret (28), and a recent work
from Ramirez-Gil et al. (35) showed an increased level of
2-isoform in the hypertensive guinea pig left ventricle (LV). Besides the differences due to species, the inconsistency of
previous results may be accounted for by the severity of overload. Accordingly, it was the goal of the present study to determine the
expression of 1-, 2-, and
3-isoforms and 1-subunit of Na+-K+-ATPase in the same species, with the
assumption that CLVH would turn into decompensated LVH (DLVH) with
longer periods of overload. The underlying hypothesis was that
arrhythmias triggered by ionic disturbance and linked to changes in the
expression of -isoforms would occur during the transition phase from
compensated hypertrophy to heart failure.
The guinea pig was chosen because of the similitude shared with humans
regarding the regulation of cardiac Ca2+ movements
(45), and LVH was induced by aortic stenosis. Anatomic measurements, hemodynamic and Holter recordings, and collagen analysis
were performed to characterize CLVH and DLVH. SERCA2 and
Na+/Ca2+ exchanger expression were studied as
markers of DLVH (1, 16, 17, 42). Expression and
localization of Na+-K+-ATPase subunits were
studied with the use of Northern blot, Western blot, and
immunohistofluorescence. Our results support the hypothesis that the
increase in the expression of the 2-isoform of
Na+-K+-ATPase in CLVH is adaptive, whereas the
relative decrease observed in DLVH may participate in the occurrence of
arrhythmias and decompensation.
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MATERIALS AND METHODS |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Induction of cardiac hypertrophy. The studies were in accordance with the requirements of the French Ministry of Agriculture (Executive Order no. 87-848, authorization 02362).
Female guinea pigs (350 ± 50 g) were fasted overnight before surgery. They were anesthetized by intraperitoneal injections of ketamine hydrochloride (20 mg/kg) and then 2% xylazine (0.25 ml/kg) 10 min later. LVH was gradually produced by a mild stenosis of the abdominal aorta, above the renal arteries, with a titanium clip [aortic stenosis (AS) groups]. Sham-operated (SH) animals underwent an identical procedure except that the hemoclip was not placed around the aorta. Guinea pigs were killed 6, 12, and 20 mo after surgery. A high mortality rate in the AS group occurred after 12 mo, and only 10 of 30 operated guinea pigs survived after 20 mo.Electrocardiographic recordings. The 24-h Holter monitoring was performed by telemetry (Physiotel Receiver, model RLA 1020; Data Sciences) with the use of an abdominal subcutaneous transmitter and a receiver placed under the cage. During recording, animals had free and normal activity. Heart rate was measured every 30 min, and each measurement was made on eight consecutive beats.
The total numbers of supraventricular (SVPB) and ventricular premature beats (VPB) and atrioventricular blocks (AVB) were counted during the whole recording period. The classic definition of arrhythmias in humans was used in this study (15).In vivo hemodynamic measurements. Hemodynamic parameters were measured 12 and 20 mo after surgery in animals already submitted to Holter monitoring. Animals were anesthetized as described above. A miniature pressure transducer catheter (model TC 50; Millar Instruments) was inserted into the right carotid artery and advanced into the LV. Arterial pressures (AP), left ventricular systolic (LVSP), diastolic (LVDP), and end-diastolic pressures (LVEDP), and the maximal rate of rise and fall of left ventricular pressure (positive and negative dP/dt) were measured using a precalibrated dynograph (Gould Electronic).
Tissues.
After hemodynamic measurements were completed, the heart was removed,
rinsed in ice-cold saline solution, and blotted dry. The LV with septum
and right ventricle (RV) was separated, weighed, frozen in liquid
nitrogen, and stored at 80°C until use. Wet weights of lungs were measured.
Synthesis of cDNA probes.
Rat-specific cDNA probes for 1- (nucleotides
89-491) and 2-isoforms (nucleotides 121-502)
(40) and 1-subunits (nucleotides 914-1,184) (31) of the
Na+-K+-ATPase were cloned into the
Pst I site of pBluescript. 1- and 2-isoform cDNA were then obtained by digestion with
EcoR I and Hind III, and 1-subunit
was obtained with Hind III and Pst I restriction endonucleases. Guinea pig-specific cDNA probe for the
3-isoform was prepared by nested PCR according to the
method of Jaisser et al. (21). A first-strand cDNA was
transcribed from guinea pig brain poly(A+) mRNA. A 618-base
pair cDNA was obtained by PCR with the use of specific primers common
to the three -isoforms (5'-sense, 5'-CGGATCGATGAA/GATIGAA/GCAC/TTT-3' and 3'-antisense,
3'-GCGTCTAGATCIGGIGGICCT/CTTCAT-5'). The product of the first PCR was
then amplified with the 5'-sense primer and an
3-isoform-specific degenerated 3'-antisense primer (3'-CGGAATTCCAT/CGAA/GGCIGAT/CACIACICAA/GGAT/C). Annealing temperature was 50°C because of the use of degenerated primers. PCR products of
320 base pairs (nucleotides 1,212-1,524) were subcloned into pBluescript and extracted by BamH I and EcoR I
enzymes. Sequencing of the cDNA fragment showed 80% homology with rat
3-isoform, between H4 and H5 segments of the
Na+-K+-ATPase, in the intracytoplasmic domain
of the molecule.
Northern and slot blot assays. Total RNA from 200 mg of LV tissue were prepared according to the method of Chirgwin (14). For Northern blots, 20 µg of total RNA were denatured in 50% formamide, 2.2 M formaldehyde, and 1× MOPS buffer (pH 7.4) and electrophoresed in a 1% agarose gel. Total RNA was then transferred to a nylon Hybond-N membrane (Amersham). For slot blot analysis, 2, 5, 10 and 15 µg of RNA of each sample were denatured and applied directly to nylon Hybond-N membranes. All blots were submitted to ultraviolet irradiation to covalently link the RNA samples.
Hybridizations with cDNA probes were carried out in rapid hybridization buffer from Amersham. cDNA probes were radiolabeled by random primer extension (Amersham Megaprime DNA labeling system) with [-32P]dCTP (specific activity 3,000 Ci/mmol; NEN,
Boston, MA). Northern and slot blots were exposed to intensifying
screens and analyzed in a Bio-Imaging Analyser System (BAS; 1,000 Mac
Bas; Fuji, Clichy, France). Normalization was carried out after
exposure of the same blots to GAPDH cDNA probe and quantification.
RNase protection assay.
RPA was performed using an RPA kit (Ambion) to detect
3-isoform mRNA. The antisense 3 riboprobe
of 402 base pairs was transcribed with [-32P]dUTP
(specific activity 3,000 Ci/mmol) and purified on 5% polyacrylamide-8 M urea gel. The 3 probe [specific activity 6 × 105 counts/min (cpm) per ng] and GAPDH probe (specific
activity 3 × 105 cpm/ng) were hybridized with 20 µg
of total RNA and then hydrolyzed with 0.2 units of RNase A and 10 units
of RNase T1. The 3 (320 base pairs) and GAPDH (194 base
pairs) protected fragments were separated on 5% acrylamide-8 M urea
gels. The dried gel was exposed to an intensifying screen and then
analyzed with the use of a computer-based imaging system (Bas 100, Fuji).
Western blot analysis. Crude particulate preparations (CPP) were obtained according to the method of Rannou et al. (37). Briefly, tissue samples (200 mg) were thawed and homogenized in 10 ml of buffer (200 mM sucrose and 20 mM HEPES, pH 7.4 ) containing protease inhibitors (1.1 µM leupeptin, 0.7 µM aprotinin, 120 µM phenylmethylsulfonyl fluoride, 1 mM iodoacetamide, 0.7 µM pepstatin, and 1 mM diisopropyl fluorophosphate). The homogenate was centrifuged at 41,000 g for 45 min in a Sorvall SS34 rotor. The pellet was suspended in 0.1 M NaCl, 30 mM imidazole, and 200 mM sucrose, pH 6.8, in the presence of the protease inhibitors. Protein content was determined by the method of Lowry with bovine serum albumin (BSA) as a standard (24).
Heart proteins from CPP (60 µg for-1 and
2 and 100 µg for 3) were
electrophoresed on a 6% polyacrylamide SDS gel and transferred to a
nitrocellulose sheet (Hybond ECL, Amersham) at 150 V for 1.5 h,
saturated in fat-free milk, and incubated with specific antibodies
(1:200 for 1 and 2 antibodies, 1:500 for
3 antibodies) and then horseradish peroxidase-conjugated
anti-rabbit antibodies. The specific proteins were detected by enhanced
chemiluminescence reaction (ECL+ kit, Amersham
International). The total proteins were then stained with Coomassie
blue R250 to normalize the specific signal of each lane. Polyclonal
antibodies against rat 1- and 3-isoforms
were gifts from Dr. R. Mercer (Washington University, School of
Medicine, St. Louis, MO) and were previously used by Charlemagne et al. (12). Monoclonal antibodies McB2 against rat
2-isoform were kindly provided by Dr. K. Sweadner
(Massachusetts General Hospital, Boston, MA) (3).
Collagen morphometry. Collagen morphometry was performed on hearts from eight AS and four SH animals after 12 and 20 mo. A sectional part of the LV taken in the inferior third of the heart was embedded in medium for frozen tissue specimen from Miles. Cryosections (7-µm thick) were stained with the collagen-specific Sirius red stain (0.5% in saturated picric acid) and studied by a single examiner. At least four fields of each section were digitized on a Macintosh IIfx by a gray-level camera (Hamamatsu) mounted on a light microscope (Leitz) at ×100 magnification, and collagen was quantified with the use of image analysis software (Optilab, Graftek, France). Interstitial collagen fraction and perivascular collagen of the stained tissue were determined separately.
Immunohistofluorescence studies.
Na+-K+-ATPase localization was determined by
using an anti-1 antibody antiserum [gift from Dr. J. Ball (4); 1:50 in 2% BSA-PBS for 30 min at 37°C] and
the McB2 monoclonal antibody (3). Samples of left
ventricular myocardium from SH and from 12- and 20-mo AS animals were
placed in embedding medium (Tissue-Tek; Miles-Diagnostics, Elkhart, IN)
and frozen in isopentane at 155°C. Cryosections (7-8 µm)
were fixed with acetone:methanol (1:1 for 20 min at 20°C), saturated with 5% BSA in PBS (30 min at room temperature), incubated with anti--1 Na+-K+-ATPase or
McB2 monoclonal antibody. After being washed three times in
PBS, the sections were successively incubated for 30 min at room
temperature with either a 1:50 dilution of anti-rabbit immunoglobulins
conjugated with fluorescein isothiocyanate (FITC) fluorochrome for
1-subunit labeling or a 1:100 dilution of biotinylated anti-mouse second antibody and streptavidin-FITC (1:50 in PBS for 30 min at room temperature) for 2-isoform. Secondary
antibodies alone did not reveal any significant fluorescence signal
after the incubation with the tissue slices. Fluorescence was
visualized using a Leica DM RD microscope equipped with epifluorescence optics.
Statistical analysis. Results are expressed as means ± SE. The statistical significance of differences among the groups was determined by one-way ANOVA, and comparisons between groups were performed by using the Scheffé's test. A value of P < 0.05 was considered to be statistically significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Compensated hypertrophy has been characterized by increased LV weight and LV weight-to-body weight ratio and increased LVSP and LVDP, whereas heart failure has been characterized by increases in RV weight- and lung weight-to-body weight ratios and increased LVEDP (8, 16). Furthermore, a decrease in SERCA2 expression has been claimed to be a marker of heart failure at the molecular level (1, 16, 17). These various criteria were taken into account to characterize and differentiate CLVH and DLVH.
Compensated left ventricular hypertrophy.
The anatomic data are shown in Table 1.
After 6 and 12 mo, the degree of LVH assessed by LV weight and LV
weight-to-body weight ratio were significantly higher in the AS groups
than in the SH groups. Compared with data from other laboratories, this increase was consistent with a mild to moderate hypertrophy (1, 10, 16).
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Decompensated left ventricular hypertrophy. After 20 mo of overload, the degree of LVH as assessed by LV weight and LV weight-to-body weight ratio was significantly higher in the AS group than in the SH group (Table 1). The lung weight and lung weight-to-body weight ratio were significantly increased, whereas the RV weight and RV weight-to-body weight ratio were not significantly different, although 4 of 10 animals from the AS group showed increased RV weights.
A representative recording of Holter monitoring is shown in Fig. 2. Normal sinus rhythm was recorded in both SH (Fig. 2A) and AS (Fig. 2B) guinea pigs. Arrhythmias were frequent in the AS group, and illustrations of recorded VPB and tachycardia as well as AVB are shown in Fig. 2B. Meaningful recordings were only obtained from five AS guinea pigs. Examination of the 24-h Holter recordings showed that AVB were observed in 4 of 5 AS animals: 3 animals had AVB 2:1 and 1 animal had AVB of third grade, whereas only 2 of 10 SH animals showed AVB 2:1. An increased number of VPB per 24 h was seen in 4 of 5 AS animals compared with SH animals (4.7 ± 1.0 and 1.8 ± 0.4, respectively, per 24 h; P < 0.05). SVPB were detected, but the difference in the number of incidents per 24 h in AS compared with SH animals (21.2 ± 10.1 and 27.8 ± 0.3, respectively) was not significant. The mean 24-h heart rate was unchanged (Table 2).
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Left ventricular expression and localization of
Na+-K+-ATPase -isoforms and
1-subunit in compensated and decompensated hypertrophy.
Expression of 1-isoform and -subunit mRNAs was
studied by Northern (Fig. 3A)
and Western blot analysis (Fig.
4A). In both SH and AS
animals, 1-isoform and 1-subunit mRNAs
were easily detected at 3.7 and 2.7 kb, respectively, whereas the
2-subunit mRNA was not detected (data not shown).
Quantification by slot blot of 1-isoform and
1-subunit mRNA levels did not show a significant difference between the SH and AS groups. Similarly,
1-isoform protein level (Fig. 4) was unchanged in SH,
CLVH, and DLVH.
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2-isoform mRNA level at 5.3 kb was
increased in AS compared with SH groups (Fig. 3A).
Quantification was carried out by slot blot analysis after
normalization to GAPDH mRNA level (Fig. 3C). It demonstrated
a significant increase in 2-isoform mRNA level after 6, 12, and 20 mo of overload. Moreover, the 2-isoform level
was significantly reduced in DLVH compared with CLVH. Finally,
expression of the 2-isoform of
Na+-K+-ATPase was studied by Western blot
analysis in CLVH and DLVH (Fig. 4A). Quantification was
performed by normalization of the specific band to the total amount of
transferred proteins stained by Coomassie blue. It revealed a
significant increase in the level of the 2-isoform in
CLVH and DLVH (Fig. 4B). In addition, the level of the
2-isoform was significantly lower in DLVH than in CLVH.
Under the above conditions, 3-isoform mRNA was barely
detected by Northern blot in SH animals. Therefore, the detection
sensitivity was increased by using RPA performed with a specific guinea
pig probe (Fig. 3B). The protected fragment was 308 base
pairs long. It was easily detected in guinea pig brain and in LV
samples from AS guinea pigs after 12 and 20 mo, whereas in SH animals
and after 6 mo in AS animals, a weaker signal could only be detected by a longer exposure time (not shown). Normalized to GAPDH mRNA levels, 3-isoform mRNA levels were significantly increased
1.6 ± 0.4- (P < 0.05), 2.8 ± 0.4- (P < 0.01), and 2.2 ± 0.1-fold
(P < 0.01) after 6, 12, and 20 mo of stenosis,
respectively. At the protein level, the 3-isoform was
undetectable by anti-rat antibodies in guinea pig LV from both groups,
even though 100 µg of proteins had been used for the detection.
However, the 3-isoform was detected by the same antibody
in positive controls from rat LV and from rat and guinea pig brains,
but the signal was weaker in guinea pig brain.
To document the whole Na+-K+-ATPase and the
2-isoform localization in CLVH and DLVH, we performed
immunofluorescence studies. Our results (Fig.
5) show that
Na+-K+-ATPase, by means of
1-subunit labeling, was mainly present in sarcolemma and
T tubules of cardiomyocytes, as previously reported in other species
and in guinea pigs (25, 46). There was no difference in
the labeling between SH and either CLVH (data not shown) or DLVH,
although a trend toward an increased fluorescence was observed in DLVH.
In SH animals, the specific labeling of the 2-isoform
was detected mainly in vessels and barely in myocytes, whereas in CLVH
and DLVH, the labeling of the 2-isoform was maintained in vessels and highly enhanced in cardiomyocytes at the sarcolemmal and T-tubule levels (Fig. 6).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The major finding of this study is that the level of the
2-isoform of Na+-K+-ATPase is
increased during CLVH and DLVH in the guinea pig and that the
2-isoform is present in T tubules in CLVH and DLVH. In
addition, we have shown, for the first time, that the transition from
compensation to decompensation is characterized by a lower expression
of the 2-isoform (mRNA and protein) in DLVH than in CLVH
and by an increased incidence of VPB and AVB.
According to physiological (Tables 1 and 2) and molecular (Fig. 1) criteria, we have shown that hypertrophy remained moderate and compensated for 12 mo and that anatomic, hemodynamic, and phenotypic changes were consistent with a transition from compensation to decompensation at 20 mo (8, 16). Furthermore, arrhythmias were recorded after 20 mo with no modification of the heart rate. The high mortality rate between 12 and 20 mo (1 of 3) suggested that decompensation and heart failure was progressively occurring. After 20 mo, the surviving animals were likely constituting a heterogeneous group at different states of decompensation, but none of the animals exhibited severe heart failure. Previous studies on guinea pigs reported that banding of the suprarenal abdominal aorta produced hypertrophy without heart failure within 8 wk and then led to heart failure with an abnormal Ca2+ transient (41). Severe hypertrophy and heart failure were associated to fibrosis development after 4 mo of AS (36). Our results partly agree with these two studies, but we found that a longer period of time was necessary to reach decompensation, probably because of a weaker overload. Furthermore, during the transition from CLVH to DLVH in this study, LVH remained moderate without the development of fibrosis, in contrast to the models in which decompensation was accompanied by a more severe LV hypertrophy and fibrosis development (36). Therefore, we showed for the first time that the decreased level of the SERCA2 mRNA occurred during decompensation, even if the hypertrophy remained moderate. This result confirms and further extends the finding that a decrease in SERCA2 expression (16) can be considered as an early sign of heart failure.
In the guinea pig, we confirmed that the 1- and
2-isoforms of the catalytic subunit were expressed in
the LV (35). These results were consistent with the
presence of at least two ouabain components: one of high affinity and
one of low affinity (5, 26). Whether the
3-isoform is expressed in the LV is still a matter of
debate: with a specific guinea pig cDNA probe, the 3-isoform mRNA was hardly detected in control LV, and at
the protein level the 3-isoform was undetectable.
Although the 3-isoform mRNA level was increased in CLVH
and DLVH, 3-isoform protein was still undetectable.
Therefore, we assumed that 3-isoform is a minor
-isoform in adult guinea pig heart and that the changes observed in
both CLVH and DLVH are of questionable physiological relevance.
In both CLVH and DLVH, the 1-isoform of
Na+-K+-ATPase and the 1-subunit
remained unchanged, as reported in other studies (12, 35).
The increase in 2-isoform in CLVH and DLVH [in this
study and after aldosterone-salt treatment (35)] differs
from previous reports, which showed a decrease in
2-isoform in severe hypertrophy in rats after abdominal
AS or induced hypertension (12, 19). This discrepancy
might be due to the difference in species or to a possible undocumented
onset of heart failure associated with severe hypertrophy. In fact, it
is important to note that the 2-isoform showed a
subsequent decay during the transition from compensation to
decompensation. Such a bimodal regulation has also been reported for
SERCA2 in the rat heart (1).
What could be the physiological relevance of the changes in the
expression of 2-isoform during CLVH and DLVH? The
2-isoform represented roughly 55% of the active
Na+-K+-ATPase isoforms in control guinea pig LV
(5, 26). Therefore, it is likely that a decrease in
2-isoform, together with an unchanged level of
1-isoform, should help in maintaining intracellular Na+ concentration
([Na+]i)homeostasis. It is widely accepted
that the induction of arrhythmias is mediated through the combined
effects of Na+-K+-ATPase,
Na+/Ca2+ exchanger, and SERCA2
in contributing to changes in [Na+]i and
[Ca2+]i homeostasis (2, 29, 30,
32) . In this context, it has been shown that a decrease in
Na+-K+-ATPase expression during heart failure
could participate in both alteration in ionic movements and the
occurrence of delayed afterdepolarizations (34) leading to
arrhythmias. Moreover it is important to note that
2-isoform was recently identified in mice with
genetically reduced levels of 2-isoform expression as
the specific isoform able to regulate Ca2+ in the heart
(22).
In CLVH, the increase in 2-isoform would help in
maintaining low [Na+]i, polarization of the
membrane, and prevention of DAD. This would be consistent with the
absence of arrhythmias in CLVH.
In DLVH, the decrease in SERCA2, the increase in
Na+/Ca2+ exchanger, and the lower expression of
2-isoform than in CLVH must be taken into account. Such
changes have been independently related to alterations in
Ca2+ handling. First, in failing human hearts, it has been
previously shown that the decrease in SERCA2 expression and/or activity
resulted in a reduced capacity of the sarcoplasmic reticulum to
accumulate Ca2+ and an impaired diastolic function
(17, 18, 38). These changes in SERCA2 expression could
contribute to arrhythmias by increasing levels of cytosolic
Ca2+ (1, 29). Second, the increased
Na+/Ca2+ exchanger level that was observed in
some failing hearts has been suggested to compensate for the decreased
SERCA2 activity and preserve Ca2+ homeostasis and diastolic
function (42). However, the increased expression of the
Na+/Ca2+ exchanger would also result in an
increased [Na]i, and Pogwid et al. (33) have
recently shown in a rabbit model of heart failure that the enhanced
Na+/Ca2+ exchanger activity may contribute to
DAD and mechanical dysfunction in this arrhythmogenic model of heart
failure. Third, James et al. (22) described the
2-isoform as a regulator of Ca2+ in the
heart. This is an important concept regarding isoform function.
Although the genetically modified mouse model cannot be directly
related to our model, we suggest that the lower level of
2-isoform in DLVH than in CLVH and the main distribution
of the 2-isoform in the sarcolemma and T tubules
(Fig. 6) might account for an [Na+]i
increase, leading to a subsequent Ca2+ increase in
agreement with the occurrence of arrhythmias. In our model it is likely
that the changes in the expression of the 2-isoform,
Na+/Ca2+ exchanger, and SERCA2 might
individually or in combination lead to altered Ca2+
handling and arrhythmogenesis, provided that the protein levels reflect
transport function.
In conclusion, abdominal aortic stenosis in guinea pigs may be used as
an experimental model to study the transition phase from compensated
hypertrophy to heart failure. Our study clearly shows that the gene
expression of the 2-isoform of
Na+-K+-ATPase, SERCA2, and the
Na+/Ca2+ exchanger was modified in
decompensated hearts independently of the hypertrophy and, therefore,
may participate in altered Ca2+ handling. The upregulation
of the 2-isoform during compensated hypertrophy, which
could allow better [Na]i homeostasis and membrane repolarization, would participate in an adaptive mechanism, whereas the
subsequent decrease of 2-isoform in DLVH would favor
alteration in ionic movements and the occurrence of DAD and arrhythmias.
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ACKNOWLEDGEMENTS |
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We thank L. Rappaport, J. L. Samuel, and C. Delcayre for helpful and constructive discussions and G. Buttler-Brown for the correction of English language.
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FOOTNOTES |
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This work was supported by grants from Association Française Contre la Myopathie and from Fondation de France. I. Belikova received support from Region Ile de France (INSERM "poste vert"), and D. Charlemagne received support from Centre National de la Recherche Scientifique.
Address for reprint requests and other correspondence: D. Charlemagne, INSERM, Unité 127, IFR Circulation Lariboisière, Université D. Diderot, 41 boulevard de la Chapelle, 75475 Paris, France (E-mail: d.charlemagne{at}inserm.lrb.ap-hop-paris.fr).
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.
Received 6 July 1999; accepted in final form 19 April 2000.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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