INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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).

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.

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.

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).

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.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) and Na+/Ca2+ exchanger (NCX) mRNA levels in left ventricles (LV) from sham-operated (SH) and aortic stenosis (AS) guinea pigs. A: representative Northern blot analysis of SERCA2 and NCX mRNAs. Total RNA (20 µg) from 2 samples of 20-mo SH and 6-, 12-, and 20-mo AS guinea pigs is represented. M, month. B: densitometric quantification of SERCA2 and NCX mRNA levels analyzed by slot blots and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels in SH (solid bars) and AS (open bars) guinea pigs. Analysis was performed on all animals described in Table 1. All values are means ± SE. The value obtained for 6-mo SH represents the 100% value. *P < 0.05; **P < 0.01, AS vs. SH guinea pigs of the same group.

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.

View larger version (35K): [in this window] [in a new window]   Fig. 2.   Typical Holter recording from SH (A) and AS (B) guinea pigs after 20 mo. A: normal sinus rhythm was recorded in SH guinea pigs. B. normal sinus rhythm (top recording), ventricular premature beats (VPB), and ventricular tachycardia (second recording), as well as atrioventricular blocks (AVB; bottom recordings) were examples of recordings in a 20-mo AS guinea pig.

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.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Na+-K+-ATPase isoform mRNA levels in LV from SH and AS guinea pigs. A: a representative Northern blot analysis of 1- and 2-isoforms and 1-subunit. Northern blots were performed by using 20 µg of total RNA from 2 samples of 20-mo SH and 6-, 12-, and 20-mo AS guinea pigs. B: RNase protection assay (RPA) of Na+-K+-ATPase 3-isoform mRNA. RPA was performed by using 25 µg of total RNA for LV and 5 µg for brain (B). Results represent 1 sample from 20-mo SH and 12- and 20-mo AS guinea pigs. MW, molecular weight standards; P, cRNA probes for 3-isoform and GAPDH. C: densitometric quantification of 1-, 2-, and 3-isoform mRNA levels analyzed by slot blot and normalized to GAPDH mRNA levels in SH (solid bars) and AS (open bars) guinea pigs. This analysis was performed on all animals described in Table 1. All values are means ± SE. The value obtained for 6-mo SH represents the 100% value. *P < 0.05; **P < 0.01, AS vs. SH guinea pigs of the same group.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Western blot analysis of Na+-K+-ATPase -isoforms in compensated (CLVH; 12 mo) and decompensated LV hypertrophy (DLVH; 20 mo) SH and AS guinea pigs. A: representative Western blot. 1- and 2-isoform analyses were performed with 5 µg of protein of crude particulate preparations (CPP) from rat and guinea pig brain and 60 µg of CPP from control rat, SH, and AS guinea pig LV; 3-isoform analysis was performed with 5 µg of CPP from rat and guinea pig brain and 100 µg of CPP from control rat, SH, and AS guinea pig LV. GP, guinea pig. B: densitometric quantification of 2- and 3-isoform protein levels in SH (solid bars) and AS (open bars) guinea pig LV. Densitometric values of each specific band were normalized to the total amount of protein stained by Coomassie blue. This analysis was performed on SH (n = 5) and AS (n = 10) guinea pigs after 12 mo and in SH (n = 5) and AS (n = 10) guinea pigs after 20 mo. All values are means ± SE. The value obtained for 6-mo SH represents the 100% value. *P < 0.05, AS vs. SH guinea pigs of the same group.

View larger version (142K): [in this window] [in a new window]   Fig. 5.   Immunofluorescent staining of 1-subunit of Na+-K+-ATPase in frozen left ventricular sections from SH (A and B) and AS (C and D) guinea pigs. Typical labeling of T-tubular sarcolemma are shown in B and D (arrows). Bars represent 25 µm (A and B) and 6.5 µm (C and D).

View larger version (78K): [in this window] [in a new window]   Fig. 6.   Immunofluorescent staining of 2-isoform of Na+-K+-ATPase in frozen left ventricular sections from SH guinea pigs (A) and from AS guinea pigs after 12 (B) and 20 mo (C). Transverse sections show typical labeling of T tubules in B and C (arrows) and vessels (stars). Bars represent 25 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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.