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(Circulation. 1998;98:2065-2073.)
© 1998 American Heart Association, Inc.

Basic Science Reports

Cardiac Endothelin-1 Plays a Critical Role in the Functional Deterioration of Left Ventricles During the Transition From Compensatory Hypertrophy to Congestive Heart Failure in Salt-Sensitive Hypertensive Rats

Yoshitaka Iwanaga, MD; Yasuki Kihara, MD, PhD; Koji Hasegawa, MD, PhD; Koichi Inagaki, MD; Takeshi Yoneda, MD; Satoshi Kaburagi, MD; Makoto Araki, MD; ; Shigetake Sasayama, MD, PhD

From the Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan.

Correspondence to Yasuki Kihara, MD, PhD, Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail kihara{at}kuhp.kyoto-u.ac.jp

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—To investigate whether endogenous ET-1 participates in an adaptive process of left ventricular hypertrophy (LVH) or a maladaptive process from LVH to congestive heart failure (CHF), we used a Dahl salt-sensitive (DS) rat model, in which systemic hypertension caused compensated concentric LVH at the age of 11 weeks followed by marked LV dilatation and global hypokinesis at the age of 17 weeks.

Methods and Results—By specific sandwich enzyme immunoassay, serum and myocardial ET-1 levels at the LVH stage were not elevated compared with age-matched Dahl salt-resistant (DR) rats, despite the marked increase of LV/body weight ratio (LV/BW). However, at the CHF stage, serum and LV ET-1 levels increased by 3.8-fold and 5.4-fold, respectively. LV ET-1 contents had close relationships with the fractional shortening (r=0.763) and the systolic wall stress (r=0.858) measured by in vivo transthoracic echocardiography. Immunohistochemistry demonstrated that the remarkably increased ET-1 in LV is located mainly in cardiomyocytes. By competitive reverse transcriptase–polymerase chain reaction, LV prepro-ET-1 mRNA levels increased by 4.1-fold in CHF rats. We randomized 11-week-old LVH rats to chronic treatment with the endothelin receptor antagonist bosentan (Bos, 100 mg · kg^-1 · d^-1, n=14), the ₁-receptor antagonist doxazosin (Dox, 1 mg · kg^-1 · d^-1, n=12), or vehicle (Cont, n=14). Bos treatment did not alter the LV geometry and function at 15 weeks; however, it attenuated the decrease of LV fractional shortening by 51% (P<0.01) without reducing the LV/BW at 17 weeks. Conversely, Dox, which decreased the blood pressure to the same extent as Bos, did not affect the progression of LV dysfunction. Bos (93%; P<0.0001 versus Cont) but not Dox (42%; P=0.8465 versus Cont) ameliorated the survival rate at 17 weeks (Cont; 36%).

Conclusions—The accelerated myocardial synthesis of ET-1 contributes directly to LV contractile dysfunction during the transition from LVH to CHF. Unelevated levels of LV ET-1 at the established LVH stage and lack of effects on LV mass by chronic bosentan treatment suggest that myocardial growth is mediated through alternative pathways. These studies indicate that chronic ET antagonism may provide an additional strategy for heart failure therapy in humans.

Key Words: endothelin • heart failure • hypertrophy • remodeling • hypertension

	Introduction

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Endothelin-1 (ET-1) is a potent vasoconstrictor peptide originally isolated from the supernatants of cultured endothelial cells.^{1 2} It exerts its effects through stimulation of specific receptors, ET_A and ET_B receptors, widely distributed in the cardiovascular system.^{3 4} The plasma levels of ET-1 are elevated in various pathophysiological states, ie, hypertension, atherosclerosis, myocardial infarction, and congestive heart failure (CHF).⁵ The elevated levels of ET-1 in heart failure are closely associated with the extent of pulmonary hypertension.⁶ In this state, acute administration of endothelin receptor blocker reduced vascular resistance and improved overall left ventricular (LV) performance.^{7 8} These findings suggest that ET-1 plays a pathophysiological role in heart failure as a circulating hormone.

In addition to its action as a systemic hormone, ET-1 has a number of actions as a local factor. ET-1 mediates load-induced hypertrophy in cultured neonatal cardiac myocytes as an autocrine factor. Previous studies reported that the expression of ET-1 in the heart is accelerated after pressure overload and myocardial infarction and that chronic administration of an ET receptor antagonist improved the survival and hemodynamics in heart failure.^{9 10 11} However, several questions remain to be defined with regard to the local roles of ET-1. First, because no precise serial evaluation of the LV function has been performed, it is unclear at present whether the elevation of cardiac ET-1 levels occurs even in the hypertrophied heart with normal systolic function or in association with the deterioration of systolic function. Second, how an ET blocker prevents the development of heart failure is unclear. Specifically, it is unknown whether beneficial effects of ET blockers are mediated by suppressing cardiac growth such as ACE inhibitors or by other mechanisms.

To address these questions, we used the Dahl salt-sensitive (DS) rat. In this rat under a high-salt diet, systemic hypertension induces compensated concentric LV hypertrophy (LVH) at the age of 11 weeks, which is followed by marked LV dilatation and global hypokinesis (CHF) at the age of 16 to 18 weeks. Our previous studies clearly distinguished these 2 states in in vivo as well as in vitro studies.^{12 13} Thus, we investigated the role of endogenous ET-1 in the transition from LVH to CHF using this animal model.

	Methods

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Experimental Animals and In Vivo Assessment of LV Geometry and Function
Male inbred DS and Dahl salt-resistant (DR) rats obtained from Brookhaven National Laboratories, Upton, NY, were further bred and supplied by Eisai Co, Ltd (Tokyo, Japan).^{14 15} After weaning, the rats were fed a 0.3% NaCl (low-salt) diet until the age of 6 weeks, after which they were fed an 8% NaCl (high-salt) diet.

The systolic blood pressure (SBP) of each animal was measured on the day before euthanasia by a tail-cuff method. On the same day, LV dimensions and contractile function were evaluated in vivo by transthoracic echocardiography as previously reported in detail from our laboratory.¹² We determined the LV end-diastolic diameter, end-systolic diameter, and diastolic posterior wall thickness, and the relative wall thickness, LV fractional shortening (FS), and end-systolic meridional wall stress were calculated from these measures.¹²

Measurement of Plasma and Myocardial ET-1 Levels
After pentobarbital anesthesia, blood was collected in a polypropylene tube containing aprotinin (300 kallikrein-inhibiting units/mL) and EDTA (2 mg/mL) and immediately centrifuged at 1800g for 15 minutes at 4°C. Plasma was stored at -80°C until the time of assay. After the heart was excised, the atria were trimmed off, and the right and left ventricles were rinsed in cold physiological saline and homogenized with a Polytron homogenizer for 30 seconds in 9 volumes of 1 mol/L acetic acid containing 0.1% Triton-X, boiled for 7 minutes, and centrifuged at 20 000g for 30 minutes at 4°C. The supernatant was stored at -80°C until use.

ET-1 was extracted from the plasma and the supernatant of homogenized ventricular tissues according to the method of Kitamura et al¹⁶ and measured by means of the sandwich enzyme immunoassay (EIA) kit (Wako Pure Chemical) originally developed by Suzuki et al.¹⁷ This EIA for ET-1 could detect as little as 0.5 pg/mL of ET-1. The cross-reactivity with ET-3 or big ET-1 was <0.1%.

Immunohistochemical Staining for ET-1 in LV
The left ventricles of DS and DR rats were excised and then fixed in 10% phosphate-buffered formalin solution for >24 hours. They were embedded in paraffin and cut into 4-µm-thick sections. After deparaffinization and hydration, they were immunostained for ET-1 by an indirect immunoperoxidase method as described previously.¹⁸ Briefly, the sections were incubated with 0.3% hydrogen peroxide for 20 minutes to block endogenous peroxidase activity, followed by further incubation with normal goat serum for 30 minutes to block nonspecific bindings. They were then incubated with ET-1 antiserum (Peninsula Laboratories) at a final dilution of 1:40 for 16 hours at 4°C. In the second step, they were treated with a 1:200 dilution of peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories) for 45 minutes. Peroxidase activity was visualized by use of diaminobenzidine and hydrogen peroxide. The sections were counterstained with hematoxylin and evaluated microscopically. As a control to check the specificity, the primary antibody was preincubated with 1 µg ET-1 peptide (Peptide Institute) for 3 hours before application to the slides.

Quantitative Reverse Transcriptase–Polymerase Chain Reaction for Prepro-ET-1 mRNA
Total RNA was isolated from left ventricles by the acid guanidinium thiocyanate–phenol-chloroform method. Quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) was carried out essentially as described in detail previously.^{19 20} After the synthesis of first-strand cDNA, a constant amount of cDNA was amplified by PCR with a serially diluted nonhomologous DNA fragment containing primer template sequences as an internal control (PCR MIMIC Construction kit, Clontech). To determine the exact amount of target mRNA species, the internal control was diluted 2-fold. Sense primers (A) and antisense primers (AS) for rat prepro-ET-1 and GAPDH were synthesized according to the published cDNA sequences.^{21 22} The sequences of the primers were as follows: prepro-ET-1 (A), 5'-GCTCCTGCTCCTCCTTGATG-3' (position: 158 to 177); prepro-ET-1 (AS), 5'-CTGGCTCTATGTAAGTCATGG-3' (637 to 657); GAPDH (A), 5'-TTGCCATCAACGACCCCTTC-3' (169 to 188); and GAPDH (AS), 5'-TTGTCATGGATGACCTTGGC-3' (558 to 577). These primers were designed to cross introns to avoid the amplification of genomic DNA. PCR was performed in a 50-µL reaction volume containing 200 µmol/L dNTP, 40 µmol/L of each specific primer, 10 mmol/L Tris-HCl, 50 mmol/L KCl, 1.5 mmol/L MgCl₂, 0.001% gelatin, 1.5 U Taq polymerase (Takara), and 1 µCi [³²P]dCTP (Amersham). PCRs using ET-1 and GAPDH primers were carried out for 40 and 30 cycles (45 seconds at 94°C, 45 seconds at 55°C, 90 seconds at 72°C), respectively. Under these conditions, linearity of the amplification was confirmed. A portion of the PCR reaction product was then resolved by electrophoresis on a 5% polyacrylamide gel and analyzed with a FUJIX bioimaging analyzer BAS 2000. The molar ratio between the internal control and target was calculated according to the formula target/internal control=(I_T/I_C)x(C_C/C_T), where I_T and I_C represent the intensity of the PCR product from the target and the internal control, respectively, and C_T and C_C represent the dCTP content in the PCR product from the target and the internal control, respectively. The amount of target molecule was determined as the point of an equal molar ratio between the internal control and the target (Figure 1). The amounts of ET-1 were divided by those of GAPDH to correct the efficiency of cDNA synthesis.

View larger version (30K): [in this window] [in a new window]   Figure 1. Competitive PCR analysis of relative change in prepro-ET-1 mRNA levels. A constant amount of cDNA was amplified with 2-fold serial dilutions of internal controls. Amount of target molecule was determined as point of equal molar ratio between internal control and target. There was good correlation between amount of internal control and target/internal control ratio (r=0.988, P<0.001).

Assessment of Chronic Effects by Bosentan and Doxazosin
Eleven-week-old LVH-DS rats with compensatory LV hypertrophy were randomly subjected to treatment with either bosentan, a mixed ET_A and ET_B receptor antagonist (F. Hoffmann-La Roche, Ltd)^{23 24} (Bos group, 100 mg · kg^-1 · d^-1, n=14); doxazosin, an ₁-receptor antagonist (Dox group, 1 mg · kg^-1 · d^-1, n=12); or vehicle (Cont group, n=14). Each drug was suspended in 5% gum arabic and injected into the stomach by gastric gavage once a day. The dosage of bosentan 100 mg · kg^-1 · d^-1 was selected because in our preliminary studies, (1) it reduced the blood pressure of the CHF rats the most effectively among doses between 30 and 200 mg · kg^-1 · d^-1 (n=3 in each group; 30, 100, and 200 mg · kg^-1 · d^-1 groups) and (2) chronic treatment of the LVH rats by this dosage significantly blunted the coronary vasoconstriction by exogenous ET-1 administration up to 10^-10 mol/L in the isolated Langendorff setting (n=4). The dosage of doxazosin 1 mg · kg^-1 · d^-1 was selected because it decreased the SBP to the same extent as bosentan throughout the course of the experiment in a preliminary study. So we evaluated the effects of bosentan on the cardiac remodeling and function independent of its systemic hypotensive effect by comparing them with the effects of doxazosin.

Animals were monitored and deaths were recorded every day. Survival after 15 weeks was analyzed by the standard Kaplan-Meier analysis with log-rank test and ² analysis. Body weights and in vivo blood pressures were measured biweekly. Serial echocardiograms were recorded at 11, 15, and 17 weeks. LV mass was calculated from the following equation²⁵: LV mass (g)=1.05x[(EDD+2xPWT)³-EDD³], where EDD is end-diastolic diameter and PWT is diastolic posterior wall thickness. We previously demonstrated that the calculated LV mass shows a linear correlation with postmortem LV weight (r=0.948, P<0.001, echocardiographic LV mass=0.900xpostmortem LV weight+0.017 g).¹²

Statistical Analysis
The results are expressed as mean±SEM. Statistical comparisons between 2 groups were performed by unpaired Student's t test. Relationships between 2 variables were tested by linear regression analysis. The main effects of the drugs were tested by 2-factor ANOVA for repeated measures, and differences at specific time points between the groups were assessed by 1-factor ANOVA with post hoc comparisons by Fisher's protected least significant difference test. In all tests, a value of P<0.05 was considered statistically significant.

	Results

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We performed 2 series of experiments. In experiment 1, animals were killed at 11 and 17 weeks and the levels of ET-1 peptide and mRNA were determined. In experiment 2, 11-week-old LVH-DS rats with compensatory LV hypertrophy were randomly subjected to oral treatment with either bosentan (Bos, 100 mg · kg^-1 · d^-1), doxazosin (Dox, 1 mg · kg^-1 · d^-1), or vehicle (Cont).

Experiment 1
SBP, Heart Weight, and Echocardiography
SBP and heart weight are presented in Table 1. DS rats fed a high-salt diet developed systemic hypertension (>220 mm Hg) at 11 weeks, which continued until the CHF period. At 11 weeks, DS rats showed marked LVH. The ratio of LV mass to body weight (LV/BW) was 50% greater in DS rats than in age-matched DR rats. At 17 weeks, DS rats showed labored respiration and loss of activity. Autopsy of these rats revealed massive pulmonary congestion. The LV/BW increased further at 17 weeks. The right ventricle (RV)/BW ratio also increased (>0.8), consistent with the existence of pulmonary congestion and elevated LV diastolic pressure.²⁶ Echocardiographic data are summarized in Table 2. In LVH-DS (11 weeks), the wall thickness was 24% greater than in age-matched DR and the relative wall thickness of DS reached 0.61, indicating LV concentric hypertrophy. LV FS tended to be higher in DS than in DR. Thus, the wall stress was within the normal range, implying an established mechanical compensation for the increased afterload. However, in CHF-DS (17 weeks), the FS decreased and the chamber diameter increased. The wall stress was 3.5 times greater in CHF-DS than in age-matched DR. These findings are consistent with our previous report¹² and show that the transition from compensatory pressure-overloaded hypertrophy to heart failure occurs between 11 and 17 weeks in this animal model.

View this table: [in this window] [in a new window]   Table 1. SBP and Heart Weights at the LVH and CHF Stages (n=6)

View this table: [in this window] [in a new window]   Table 2. Echocardiographic Parameters at the LVH and CHF Stages

Plasma and Myocardial Endothelin-1 Concentrations and Echocardiographic Correlates
Plasma, RV, and LV ET-1 levels are illustrated in Figure 2. The levels in LVH-DS did not differ from those in age-matched DR rats. By contrast, plasma ET-1 levels in CHF-DS were 3.8-fold higher than those in 17-week-old DR rats. The RV and LV ET-1 levels at 17 weeks were 2.6-fold (P<0.01) and 5.4-fold (P<0.01) higher, respectively, in DS (RV, 1551±154 pg/g tissue; LV, 2123±411 pg/g tissue) than in DR (RV, 595±29 pg/g tissue; LV, 395±16 pg/g tissue). Figure 3A and 3B illustrates correlations between LV ET-1 concentrations and in vivo measured echocardiographic data. LV ET-1 contents had a significant negative correlation with LV FS (r=0.763, P<0.01) and a strong positive correlation with LV systolic wall stress (r=0.858, P<0.01). Plasma ET-1 levels had a significant but weaker relationship with LV FS (r=0.586, P<0.01) and LV systolic wall stress (r=0.651, P<0.01) than LV ET-1 levels. Figure 3C illustrates correlations between LV ET-1 contents and LV/BW. Despite a 57% increase of LV/BW in LVH-DS rats compared with DR, LV ET-1 had no significant difference between DR and LVH-DS rats (1.3-fold, P=NS). By contrast, LV/BW increased 39% from LVH to CHF-DS, which was associated with a 4.6-fold increase in LV ET-1 levels (P<0.01). Thus, the increase in LV/BW was not proportionate to that in LV ET-1 concentrations.

View larger version (16K): [in this window] [in a new window]   Figure 2. ET-1 concentrations in plasma (A), RVs (B), and LVs (C) of DS and DR rats. ET-1 concentrations were measured by specific sandwich EIA as described in Methods. Values are mean±SEM. n=6 per group. *P<0.01 vs 17-week DR rats.

View larger version (12K): [in this window] [in a new window]   Figure 3. Correlation between LV ET-1 concentration (LV-ET) and echocardiographic functional data (A and B) and LV mass (C). LV-ET had a strong correlation with LV FS (r=0.763, P<0.01) (A) and LV systolic wall stress (r=0.858, P<0.01) (B). C represents relationship between LV-ET and LV/BW. Bar indicates mean±SEM in each group.

Immunohistochemical Detection of ET-1 in the LV Myocardium
Strong positive signals for ET-1 were widely distributed within the LV myocardium of CHF-DS (Figure 4A, brown). The specificity of the immunostaining was confirmed when the signals were completely abolished by preincubation of the primary antibody with the excess of synthetic ET-1 (Figure 4B). The ET-1 staining intensity in cardiac myocytes was much higher in CHF-DS (Figure 4A) than in 17-week-old DR (Figure 4C). The interstitial cells and the endothelial and smooth muscle cells of the intramyocardial coronary arteries showed modest staining for ET-1, and the intensity did not differ between DS and DR.

View larger version (78K): [in this window] [in a new window]   Figure 4. Immunohistochemical detection of ET-1 in LV of CHF-DS (A and B) and 17-week-old DR rats (C). Preincubating an excess of synthetic ET-1 with a primary antibody resulted in no staining (B). Magnification x200.

Measurement of Cardiac Prepro-ET-1 mRNA
The levels of prepro-ET-1 mRNA in the LV myocardium are illustrated in Figure 5. In LVH-DS, the levels did not differ from those in the age-matched DR rats. However, at the CHF stage, the levels were 4.1-fold higher in DS than in DR rats. Thus, ET-1 mRNA and peptide levels changed in parallel, indicating that the remarkably upregulated expression of the myocardial ET-1 during the transition from LVH to CHF is mediated, at least in part, at the pretranslational level.

View larger version (39K): [in this window] [in a new window]   Figure 5. Results of quantitative RT-PCR analysis of prepro-ET-1 mRNA in LV of DS and DR rats. Vertical axis denotes amount of prepro-ET-1 (ppET-1) mRNA normalized to that of GAPDH mRNA. Values represent arbitrary units (11-wk DR values were set at 1.0, and remaining values were adjusted accordingly) and mean±SEM. n=6 per group. *P<0.01 vs 17-week DR rats.

Experiment 2
SBP and Echocardiographic Changes
There was a slight but significant difference in SBP between the Cont and Bos groups, as illustrated in Figure 6A. Bos showed 9 and 10 mm Hg lower SBP at 13 and 15 weeks, respectively (P<0.05). Doxazosin of 1 mg · kg^-1 · d^-1 decreased SBP to the same extent as bosentan throughout the course of the experiment. Figure 6B, 6C, and 6D shows changes of LVFS, LV systolic wall stress, and LV/BW, respectively (Table 3). Until 15 weeks, these 3 parameters did not differ among the 3 groups. After that period, however, bosentan attenuated both the increase of LV systolic wall stress and the decrease of LV FS (LV FS change: Cont, -10.9%/wk; Dox, -11.3%/wk; Bos, -3.5%/wk). At 17 weeks, the systolic stress was much lower in Bos (106±8.2 g/cm²; P<0.01) than in Cont (179±16 g/cm²) or Dox (160±15 g/cm²). The LV FS at 17 weeks was higher (P<0.01) in Bos (41.6±2.3%) than in Cont (27.4±1.9%) or in Dox (26.7±2.0%). In contrast, the LV/BW did not differ among Cont (4.15±0.17 mg/g), Dox (4.20±0.18 mg/g), and Bos (4.14±0.23 mg/g). Thus, chronic ET receptor blockade attenuated the progression of LV dysfunction and remodeling without reducing the LV mass. These effects were independent of its afterload reduction.

View larger version (16K): [in this window] [in a new window]   Figure 6. Effects of doxazosin and bosentan on SBP and echocardiographic functional data. A shows serial tail-cuff SBP measurements, and B, C, and D show serial transthoracic echocardiographic measurements for control group (Cont, n=14; ), doxazosin-treated group (Dox, n=12; ), and bosentan-treated group (Bos, n=14; ). Vertical axes denote LV FS in B, LV systolic wall stress in C, and estimated LV/BW in D. Values are expressed as mean±SEM. *P<0.01 vs age-matched control rats. #P<0.01 vs age-matched doxazosin-treated rats.

View this table: [in this window] [in a new window]   Table 3. Serial Measurements of Echocardiography in Vehicle-, Doxazosin-, and Bosentan-Treated DS Rats

Survival Rate
All rats of the Cont and Dox groups died of pulmonary congestion with LV dysfunction between 15 and 19 weeks (mean±SEM, 16.7±0.3 and 16.5±0.3 weeks, respectively) (Figure 7). By contrast, the longest survival in Bos was 22.7 weeks (mean±SEM, 19.3±0.4 weeks). The survival rate at 17 weeks was 93% in Bos, 42% in Dox, and 36% in Cont. The Kaplan-Meier survival analysis demonstrated a significant improvement of survival in the Bos compared with the Cont or Dox group (P<0.0001). There was no difference in survival between Cont and Dox groups (P=0.555).

View larger version (13K): [in this window] [in a new window]   Figure 7. Effect of doxazosin and bosentan on survival. Kaplan-Meier survival curves for control group (Cont, n=14; ), doxazosin-treated group (Dox, n=12; ), and bosentan-treated group (Bos, n=14; ). Doxazosin, bosentan, or placebo was administered from 11 weeks. *P<0.0001 vs Cont or Dox (log-rank test).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
To investigate the role of endogenous ET-1 in the transition from compensated LVH to CHF, we used a DS rat model. In this model with a high-salt diet, systemic hypertension resulted in established LVH at the age of 11 weeks, which was followed by marked LV dilatation and global hypokinesis (CHF) at the age of 17 weeks. We found that at the established LVH stage, the cardiac ET-1 system was not activated, but along with the transition to failure, it showed a remarkable activation. The activation was not parallel to the progression of LV hypertrophy but rather to the deterioration of LV systolic function. The immunohistochemical study revealed that myocytes were involved in the activated ET-1 system in the ventricular myocardium. Furthermore, chronic ET receptor blockade with bosentan directly attenuated the progression of LV dysfunction and improved the survival from CHF in a manner independent of the progression of LV hypertrophy. The present results for the first time clarified the critical role of the local ET system in the progression of contractile dysfunction and ventricular remodeling during the transition from LVH to CHF.

Cardiac ET-1 System Is Not Activated in Compensated Hypertrophy
ET-1 is a growth-promoting peptide able to induce hypertrophy in cultured neonatal cardiac myocytes.⁴ Stimulation with angiotensin II or stretch induces the expression of ET-1 in cultured neonatal cardiac myocytes, and ET_A receptor antagonist can block the hypertrophy evoked by these stimuli.^{27 28} These findings suggest a possible role of ET-1 as an autocrine factor for hypertrophy in these culture systems. In hypertrophy evoked by an acute aortic banding procedure or by chronic norepinephrine administration, ET-1 peptide and prepro-ET-1 mRNA levels in LV were increased.^{6 7} In those studies, however, precise and serial evaluation of LV function was not performed. Therefore, it is unclear at present whether the cardiac ET system is activated even in hypertrophied hearts with normal systolic function. We demonstrated that Dahl salt-sensitive rats at 11 weeks had a marked increase of LV mass with normal LV systolic wall stress, indicating an established compensated hypertrophy against increased afterload. The serum and myocardial ET-1 peptide and mRNA levels in DS rats did not differ from those in the age-matched DR rats. Our findings are compatible with the report by Thibault et al²⁹ that in 17-week-old spontaneously hypertensive rats (SHRs), serum and myocardial ET-1 levels were not increased compared with those in the age-matched Wistar-Kyoto rats. Although the possibility still exists that even basal levels of ET-1 in myocardium contribute to the development of compensated LVH, Li and Schiffrin³⁰ reported that chronic treatment of 12-week-old SHRs with bosentan for 4 weeks did not block cardiac hypertrophy. In the present study, bosentan did not affect the progress of LV hypertrophy. These results suggest that myocardial growth at the compensated stage is mediated through alternative pathways.

ET-1 Activation During Transition to CHF
In human CHF, it has been demonstrated that the increase of circulating ET-1 levels correlates with functional class and alterations in hemodynamics.^{31 32} The present study demonstrated that plasma and myocardial ET-1 levels increased de novo during the transition from LVH to CHF. In the LV myocardium of the CHF-DS rats, ET-1 immunoreactivity resided mainly in cardiomyocytes. The increase in ET-1 peptide levels was associated with a concomitant increase in the prepro-ET-1 mRNA levels. These findings demonstrate that cardiac synthesis of ET-1 is accelerated during the transition from LVH to CHF. In addition, although these data do not rule out a possible contribution of other organs to the increased levels of ET-1 in plasma, they suggest that the heart is one of the main sources for plasma ET-1 in CHF.

Bosentan Preserved LV Systolic Function During the Transition to CHF
LV ET-1 levels had close relationships with such LV functional measures as systolic wall stress and FS. To clarify whether the increased levels of LV ET-1 represent merely a marker of LV dysfunction or a direct contributor to LV systolic dysfunction during the transition from LVH to CHF, we examined chronic effects of endothelin receptor blockade. We showed that chronic treatment with bosentan attenuated the progression of LV dysfunction and decreased the mortality rate. At 17 weeks, the systolic wall stress of bosentan-treated animals was 41% lower than that of vehicle-treated rats. Although chronic treatment with doxazosin reduced the blood pressure to the same level as that with bosentan, it failed to improve the wall stress, indicating the minor contribution of the afterload reduction to the preserved LV function by bosentan. Thus, the chronic bosentan treatment beneficially altered the process of LV remodeling. These findings suggest that the accelerated expression of cardiac ET-1 contributes to LV systolic dysfunction and remodeling during the transition from LVH to CHF.

Bosentan Did Not Block Myocardial Growth
ACE inhibitors are now considered one of the basic pharmacological tools in the treatment of CHF. Litwin et al³³ showed that chronic ACE inhibition attenuates the transition from LVH to CHF in an aortic-banding model. In their study, an ACE inhibitor blunted the increase of LV mass and maintained smaller a LV cavity, which resulted in an improvement of both systolic and diastolic function. Hence, in ACE inhibition, the suppression of LV growth appears to play a central and key role. By contrast, our data demonstrated that the ET receptor blockade by bosentan preserved the LV systolic function but did not suppress the further increase in LV mass from LVH to CHF. LV ET-1 levels and the degree of cardiac hypertrophy did not correlate. These findings suggest that myocardial growth was mediated primarily through alternative pathways and that local ET-1 systems regulated LV function by distinct mechanisms during the transition from LVH to CHF. Two independent reports showed that acute administration of ET receptor blocker in addition to ACE inhibitors resulted in synergistic hemodynamic improvement in heart failure of rats and humans.^{34 35} Therefore, endothelin blockers can be additive therapeutic agents in patients already receiving medical treatment with ACE inhibitors.

Possible Mechanisms That Mediate Beneficial Effects of Bosentan
The mechanisms by which chronic endothelin receptor blockade ameliorated LV function during the transition to CHF without reducing LV mass should be discussed. At the CHF stage, plasma ET-1 levels were significantly elevated in DS compared with DR rats, which may have contributed to the increased afterload. Chronic treatment with bosentan decreased the SBP by 5% at 17 weeks. However, from the results of the doxazosin study, ie, that doxazosin at the same SBP level as the bosentan group did not affect the progression of LV dysfunction or improve survival, we conclude that bosentan directly modulated cardiac function in a manner independent of the level of afterload. Furthermore, LV ET-1 contents rather than plasma ET-1 levels had a closer relationship with LV systolic dysfunction, supporting the idea that local ET-1 in the heart directly regulates the LV function.

This raises the question of how local ET-1 deteriorates cardiac function. We have previously reported that substantial activation of the ß-adrenergic system with a substantial increase in the inhibitory G-coupled protein (G_i) occurs during the transition to heart failure in this animal model.^{13 36} In contrast to the finding that ET-1 has a positive inotropic action on heart muscle cells in a normal condition,³⁷ Ono et al³⁸ demonstrated that under the ß-adrenergic stimulatory condition, ET-1 exerted a negative inotropic effect on isolated myocytes through a G_i-mediated pathway. These findings suggest that long-term activation of endothelin and the sympathetic nervous system in CHF adversely affects the myocardial function synergistically. In addition, it has been reported that ET-1 represses forskolin-induced increase of cAMP in cardiac myocytes in vitro.³⁹ Thus, ET-1 might be involved in the depletion of cAMP in the failing myocardium. Hartong et al⁴⁰ recently reported that treatment of neonatal myocytes with ET-1 led to a decrease in sarcoplasmic reticulum Ca²⁺-ATPase mRNA levels. Other studies have reported that ET-1 activation may directly exert cytotoxic or cell damage effects.⁴¹ In this model, the progression of LV dysfunction is in part related to alterations in excitation-contraction coupling, as we showed previously.⁴² It is possible that chronic ET-1 blockade might improve the impaired excitation-contraction coupling directly, resulting in an attenuation of the progression in LV dysfunction. In any event, unraveling how chronic accumulation of ET-1 in cardiac myocytes deteriorates function may provide further insight into the mechanisms mediating the development of heart failure. Finally, our findings suggest that ET antagonism provides a promising strategy for chronic heart failure therapy in humans.

	Acknowledgments

 
This work was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture, Japan (A0-1, 06454291, and 07557343) and research grants from the Ministry of Health and Welfare, Japan (5A-2, 7A-2, and 7A-4). We thank W. Hayashida, MD, PhD, for his valuable suggestions on the manuscript. Bosentan was graciously supplied by F. Hoffmann-La Roche Ltd, Basel, Switzerland.

	Footnotes

 
Guest editor for this article was Joseph Loscalzo, MD, PhD, Boston University Medical Center, Boston, Mass.

Received April 21, 1998; revision received June 1, 1998; accepted June 16, 1998.

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