Role of Endothelin in Deterioration of Heart Failure Due to Cardiomyopathy in Hamsters

We and other groups have reported an increase in plasma levels of endothelin (ET)-1 in patients¹²³ and experimental animals⁴⁵ with chronic heart failure (CHF). It has been reported that plasma ET-1 level is a major predictor of mortality in patients with CHF.⁶ ET-1 is produced not only by endothelial cells⁷ but also by cardiac tissues, such as cardiac myocytes⁸ and fibroblasts.⁹ In cardiac myocytes, mechanical stretching¹⁰ and some neurohumoral factors (eg, angiotensin II and norepinephrine)¹¹¹² stimulate the production of ET-1. Moreover, ET-1 has direct effects on contractile function,¹³ protein synthesis,¹⁴¹⁵ and electrophysiological events¹⁶¹⁷ in cardiac myocytes, and these effects are mediated primarily by ET_A receptors. These observations suggest that increased ET-1 production and subsequent activation of ET_A receptors contribute to the pathophysiological changes in CHF.

We previously reported that the plasma ET-1 level and production of ET-1 in the heart were markedly increased in CHF rats with myocardial infarction induced by coronary ligation.⁵ We also showed that long-term treatment with BQ-123, an ET_A antagonist, greatly improved the survival rate of rats with CHF due to myocardial infarction.¹⁸ This beneficial effect of BQ-123 was accompanied by significant amelioration of left ventricular (LV) dysfunction and prevention of unfavorable ventricular remodeling.¹⁸

However, the pathogenesis of congestive heart failure is diverse. Not only ischemic heart disease, for which a coronary-ligated rat serves as a model, but also cardiomyopathy constitutes a primary part of the causes of CHF.¹⁹ Therefore, investigations of the roles of ET-1 in a cardiomyopathic CHF model are valuable to understand the efficacy and mechanism of ET antagonists for the treatment of CHF due to cardiomyopathy. The Bio 14.6 hamster, a model of idiopathic cardiomyopathy, is one of the representative models of CHF due to cardiomyopathy.²⁰ The pathogenesis of the disease state is not fully understood, but this model spontaneously develops cardiac necrosis, hypertrophy, and finally progression to dilation and CHF in the later stages.²⁰ Therefore, this model is appropriate for examining the effect of long-term treatment with drugs on the prognosis of CHF due to cardiomyopathy. However, either production of ET-1 in the heart or a role of ET-1 in the pathophysiology of this cardiomyopathic CHF model has not been investigated yet. We designed the present study to resolve these questions using an ET_A receptor antagonist, TA-0201.²¹

Methods

Animals and Study Protocols

We used male Bio 14.6 cardiomyopathic hamsters and F1b normal hamsters (Charles River, Yokohama, Japan). Hamsters were individually housed with a 12-hour light/dark cycle and received a powdered diet (CE-7, Nihon Clea) and tap water ad libitum.

In the present study, 2 series of experiments were performed. The aim of the first series of experiments was to investigate the levels of both preproET-1 mRNA and mature ET-1 peptide in the failing hearts of cardiomyopathic hamsters. The second series of experiments were performed to examine the effects of chronic treatment with an ET_A antagonist on survival and cardiac function of cardiomyopathic hamsters. All experimental protocols for animal studies were approved by the Animal Care and Use Committee of Tanabe Seiyaku Co Ltd.

First Series of Experiments

Measurement of Cardiac Functions and Tissue Sampling

Measurements of cardiac functions were performed by a minor modification of the method described previously.⁵¹⁸²² Bio 14.6 and F1b hamsters (52 weeks old) were anesthetized with thiobutabarbital 100 mg/kg IP. A catheter (SP-10) was inserted into the right carotid artery to measure arterial blood pressure and was advanced into the LV for evaluating LV +dP/dt/P_max. Subsequently, another catheter was inserted into the right jugular vein for the recording of central venous pressure (CVP) and was advanced into the right ventricle (RV) to measure RV +dP/dt/P_max. After measurements of cardiac functions, blood samples were collected and stored at −80°C. The heart was excised and divided into the RV and the LV including the septum. After each ventricle was weighed, all samples were frozen in liquid nitrogen and stored at −80°C.

Sandwich Enzyme Immunoassay for Determination of Heart and Plasma ET-1 Levels

The LV ET-1 level was determined as previously described in our publications.⁵¹⁸²² Sandwich enzyme immunoassay (EIA) for ET-1 was carried out with immobilized mouse monoclonal antibody AwETN40, which recognizes the N-terminal portion of ET-1, and peroxidase-labeled rabbit anti–ET-1 C-terminal peptide 15-21 Fab′.⁵¹⁸²³ The assay for ET-1 did not cross-react with ET-3 or big ET-1 (cross-reactivity <0.1%). Plasma ET-1 concentration was also measured by a sandwich EIA.

Reverse Transcription and Polymerase Chain Reaction to Evaluate the Level of PreproET-1 mRNA in the LV

Extraction of total RNA from the LV and analysis of preproET-1 mRNA expression by reverse transcription and polymerase chain reaction (RT-PCR) were performed as described previously.²⁴ Total RNA was reverse-transcribed by avian myeloblastosis virus reverse transcriptase. The gene-specific primer pair of preproET-1 was designed on the basis of the partial sequence of hamster preproET-1.²⁴ The expression of GAPDH mRNA was also determined as an internal control. The gene-specific primer pair of GAPDH was designed on the basis of the cDNA sequence for hamster GAPDH.²⁵ The sequences of the oligonucleotides were as follows: preproET-1 (sense): 5′-CGAGCTGAGAATGAAGGGGAGAG-3′; preproET-1 (antisense): 5′-GCCTTTCTGCATGGTGTTTTGGGC-3′; GAPDH (sense): 5′-CCTCAACTACATGGTCTACA-3′; and GAPDH (antisense): 5′-CCTTCCACAATGCCAAAGTT-3′.

The reaction cycles of PCR were performed in a range that shows a linear correlation between the amount of cDNA and the yield of PCR products. The amplified PCR product was electrophoresed on a 1.2% agarose gel, stained with ethidium bromide, visualized by a UV transilluminator, and photographed. The photograph was scanned by a scanner (CanoScan 600; Canon) and quantified by computer with MacBAS software (Fuji Film).

Second Series of Experiments

Effect of TA-0201, an ET_A Antagonist, on Survival Rate of Cardiomyopathic Hamsters

Bio 14.6 hamsters (52 weeks old) were used in the study of survival rate. Hamsters were randomized to receive either placebo (n=25) or TA-0201 (n=24). TA-0201 was mixed into their diet at a concentration of 30 ppm. The daily dose of TA-0201 in the treated group was ≈1.3 mg · kg⁻¹ · d⁻¹, and the plasma concentration of TA-0201 was 63.9±30.4 ng/mL (n=7). We reported that TA-0201 is an orally active ET_A antagonist.²¹ In the binding study using human cloned ET receptors, TA-0201 showed higher affinity for ET_A receptors (K_i=0.015 nmol/L) than for ET_B receptors (K_i=41 nmol/L).

Throughout the experiment, each hamster was inspected for signs of “wet tail,” a fatal diarrhea in rodents that occurs frequently in hamsters. Hamsters that died of wet tail were excluded from the study. Based on these criteria, 1 of 56 cardiomyopathic hamsters was excluded during the course of the study.

Effect of TA-0201 on Cardiac Functions of Hamsters

We also evaluated the effect of chronic treatment with TA-0201 on cardiac functions of hamsters. Hamsters were randomized to receive either placebo or TA-0201. Noncardiac death was observed in 1 animal during the course of the study. After 6 weeks of treatment, all of the surviving hamsters were anesthetized with thiobutabarbital 100 mg/kg IP, and cardiac functions were measured as described above.

Statistical Analysis

All data except the survival rate are expressed as mean±SEM. Comparison of survival in untreated and TA-0201–treated cardiomyopathic hamsters was analyzed by a log-rank test with the Kaplan-Meier method. Plasma ET-1 levels in cardiomyopathic and normal hamsters with or without TA-0201 were examined by use of 2-way ANOVA with contrasts. The analytical method used in other studies was the unpaired t test. Differences were considered significant at the level of P<0.05.

Results

First Series of Experiments

Cardiac Function and Tissue Weights

Table 1 shows mean blood pressure (MBP), heart rate (HR), and cardiac functions measured in anesthetized hamsters. In the 52-week-old cardiomyopathic hamsters, MBP, HR, LV +dP/dt/P_max, and RV +dP/dt/P_max were significantly lower than in age-matched normal hamsters. In addition, LV end-diastolic pressure (LVEDP), RVEDP, and CVP were markedly elevated in the cardiomyopathic hamsters. These data indicated that the cardiomyopathic hamsters in 52 weeks had developed severe cardiac dysfunction and CHF. LV and RV mass indices for body weight (LV/BW and RV/BW) were much higher in the cardiomyopathic hamsters than in the normal hamsters (Table 1).

PreproET-1 mRNA Expression and Tissue ET-1 Level in the LV and Plasma ET-1 Level

The expression of preproET-1 mRNA was markedly higher in cardiomyopathic hamsters than in normal hamsters (Figure 1). The values of band density of PCR products for GAPDH mRNA did not differ between the 2 groups. As shown in Figure 2A, the peptide ET-1 level in the LV of cardiomyopathic hamsters was ≈3.2-fold higher than that of normal hamsters. The plasma ET-1 concentration of the cardiomyopathic hamsters was also ≈1.7-fold higher than that of normal hamsters (Figure 2B). Plasma ET-1 level was significantly correlated with LV +dP/dt/P_max (r=−0.81, P<0.001) in the hamsters. The peptide level of ET-1 in the LV was also significantly correlated with LV +dP/dt/P_max (r=−0.67, P<0.01).

Second Series of Experiments

Effect of Chronic Administration of TA-0201 on Survival Rate of Cardiomyopathic Hamsters

Chronic treatment with TA-0201 markedly increased the survival rate of the cardiomyopathic hamsters (Figure 3). At the end of the experiment (after 9 weeks of treatment), the survival rates of cardiomyopathic hamsters were 16% in the untreated group and 65.2% in the TA-0201–treated group (Figure 3). The survival rate of the treated group was significantly higher than that of the untreated group (P<0.001).

Effect of Chronic Administration of TA-0201 on Cardiac Function and Plasma ET-1 Levels in Cardiomyopathic Hamsters

Cardiac function was measured in anesthetized hamsters after 6 weeks of treatment with or without TA-0201. At this point, the survival rate of the TA-0201–treated cardiomyopathic hamsters was significantly higher than that of the untreated hamsters (82.6% versus 50%, P<0.05).

Chronic administration of TA-0201 did not affect MBP or HR in cardiomyopathic hamsters (Figure 4). On the contrary, TA-0201–treated hamsters showed significantly higher LV +dP/dt/P_max (Figure 4) and RV +dP/dt/P_max (Figure 5) compared with the untreated hamsters. In addition, LVEDP (Figure 4), RVEDP (Figure 5), and CVP (Figure 5) were significantly lower in the TA-0201–treated compared with the untreated hamsters. With regard to the tissue weights, RV/BW and lung/BW were also significantly lower in the TA-0201–treated than in the untreated hamsters (Table 2).

In contrast to cardiomyopathic hamsters, TA-0201 showed no significant effects on hemodynamics and tissue weights in F1b hamsters (data not shown).

As shown in Figure 6, plasma ET-1 levels were higher in the cardiomyopathic than in the normal hamsters. TA-0201 did not affect plasma ET-1 levels in cardiomyopathic and normal hamsters (Figure 6).

Discussion

The present study clearly demonstrated that the production of ET-1 is markedly increased in the LV of Bio 14.6 cardiomyopathic hamsters at a stage of CHF. The chronic administration of an ET_A antagonist, TA-0201, markedly improved the survival rate of cardiomyopathic hamsters. In addition, after 6 weeks of treatment, TA-0201 effectively prevented (1) the increase in LVEDP, RVEDP, and CVP; (2) the decrease in LV +dP/dt/P_max and RV +dP/dt/P_max; and (3) RV hypertrophy. These results suggest that chronic ET blockade improves the survival rate and ameliorates both LV and RV dysfunction in cardiomyopathic hamsters. Therefore, it is strongly suggested that endogenous ET-1 plays an important role in the deterioration of CHF due to cardiomyopathy in hamsters.

Cardiomyopathy, as well as myocardial infarction, is one of the major causes of CHF.¹⁹ It has been reported that plasma ET-1 level is increased in patients with cardiomyopathy.² To date, however, the contribution of ET-1 in CHF caused by cardiomyopathy has not been investigated. Therefore, in the present study, we used Bio 14.6 cardiomyopathic hamsters (52 weeks old) to evaluate the role of ET-1 in CHF due to cardiomyopathy. At this age, not only plasma ET-1 concentration but also ET-1 production in the LV was significantly higher in the cardiomyopathic hamsters than in the age-matched normal hamsters, suggesting that ET pathway is activated in the cardiomyopathic hamsters. We also observed that LV +dP/dt/P_max was markedly lower in the cardiomyopathic than in the normal hamsters. The LV +dP/dt/P_max was negatively correlated with both plasma and LV ET-1 level in the hamsters. Plasma ET-1 concentration is well correlated with the clinical class of heart failure, New York Heart Association functional class in patients with chronic CHF.³ Taken together, the increased plasma ET-1 concentration in the cardiomyopathic hamsters may reflect the severity of cardiac dysfunction.

The long-term administration of an ET_A antagonist, TA-0201, greatly improved survival of the cardiomyopathic hamsters. Indeed, the survival rate was 16% in the untreated cardiomyopathic hamsters and 65.2% in the TA-0201–treated hamsters. To the best of our knowledge, this is the first study to describe the beneficial effect of ET blockade on survival of cardiomyopathic hamsters at the CHF stage. We have already reported the beneficial effect of BQ-123, another ET_A antagonist, in rats with CHF induced by coronary ligation.¹⁸ The administration of BQ-123 almost doubled the number of surviving rats with CHF; the survival rate of CHF rats treated with BQ-123 was 85%, compared with 43% for the untreated CHF rats.¹⁸ Therefore, ET antagonists may be beneficial for the treatment of CHF caused not only by ischemic heart disease but also by cardiomyopathy.

We observed that LV +dP/dt/P_max and RV +dP/dt/P_max were markedly lower in the 52-week-old cardiomyopathic hamsters than in the age-matched normal hamsters. Moreover, LVEDP, RVEDP, and CVP were markedly elevated in the cardiomyopathic hamsters compared with the normal hamsters. These results indicate that the cardiomyopathic hamsters at 52 weeks old already have severe cardiac dysfunction and are in an advanced stage of CHF. Therefore, the beneficial effect of TA-0201 on the survival rate of cardiomyopathic hamsters suggests that ET_A antagonists have a therapeutic potential for CHF even when treatment is initiated at an advanced stage of CHF.

With regard to cardiac function, LV and RV performances in the untreated 58-week-old hamsters had progressively deteriorated compared with those in the 52-week-olds. The present study demonstrated that long-term treatment with TA-0201 significantly prevented the progression of cardiac dysfunction. Thus, these results suggest that ET-1 contributes to the progression of CHF due to cardiomyopathy and that these effects of an ET_A antagonist on cardiac hemodynamics might favorably contribute to the prolongation of life expectancy in cardiomyopathic hamsters.

In this study, long-term treatment with TA-0201, an ET_A antagonist, did not affect the arterial pressure in the cardiomyopathic hamsters. This result is in accordance with our previous data that the administration of BQ-123 did not alter the arterial pressure in rats with CHF due to myocardial infarction.¹⁸ However, Mulder et al²⁶ and Fraccarollo et al²⁷ reported that long-term treatment with bosentan, an ET_A/ET_B-nonselective antagonist, lowers arterial pressure in rats with CHF due to myocardial infarction. One possible explanation for the discrepancy in effects on arterial pressure among ET antagonists is that the blockade of both ET_A and ET_B receptors by bosentan contributes to hypotension, because both ET_A- and ET_B-mediated mechanisms for vascular contraction exist in various vessels. Indeed, in spontaneously hypertensive rats, bosentan lowers arterial pressure,²⁸ whereas an ET_A antagonist shows no significant effect.²⁹

Furthermore, TA-0201 showed no significant effect on plasma ET-1 levels after 6 weeks of treatment in hamsters. Other investigators have demonstrated that ET_A/B-nonselective antagonists, such as bosentan, increased plasma ET-1 and have suggested that this increase was partly attributed to the blockade of ET_B receptor, which is considered to be involved in ET-1 clearance.³⁰³¹ Therefore, it is likely that long-term treatment with an ET_A antagonist does not affect ET-1 clearance in hamsters.

We clearly indicated the beneficial effects of an ET_A antagonist on survival rate and cardiac function, and the mechanisms for this improvement should be elucidated. First, because ET-1 has direct toxic effects on cardiac myocytes,³² an increase in ET-1 production in the heart may lead to myocardial injury and may contribute to the progression of CHF. Second, because ET-1 exerts a hypertrophic effect on cardiac myocytes,¹⁴¹⁵ it is likely that the upregulated ET-1 system in the heart contributes to the excessive hypertrophy of myocardium in cardiomyopathic hamsters. Third, ET-1 is known to induce ventricular arrhythmia,³³ and high arrhythmogenicity of the ventricles was reported in cardiomyopathic hamsters.³⁴ Therefore, it is likely that the inhibition of ET-1–induced arrhythmia by ET antagonists is beneficial to increasing the survival rate. Fourth, because it was reported that the acute administration of some ET antagonists caused a moderate peripheral vasodilation in humans,³⁵ we cannot exclude the possibility that the inhibition of ET-1–induced vasoconstriction partly contributes to the beneficial effects of the ET_A antagonist.

In conclusion, our study demonstrates for the first time that (1) the production of ET-1 in the LV was greatly increased in the cardiomyopathic CHF hamsters, and (2) the chronic treatment with an ET_A antagonist markedly improved the survival, cardiac function, and hypertrophy of cardiomyopathic hamsters with advanced CHF. These observations suggest that ET-1 contributes to the progression of CHF due to cardiomyopathy and that an ET_A antagonist has therapeutic potential for CHF due to cardiomyopathy.

Guest Editor for this article was Michael R. Bristow, MD, University of Colorado Health Sciences Center, Denver.

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Footnotes