Angiotensin-converting enzyme inhibitors (ACEI) have been shown to reduce morbidity and mortality in patients and animal models with heart failure (1-5). This favorable effect could be explained by the regression of hypertrophy and improved systolic and diastolic function resulting from inhibition of the intracardiac renin-angiotensin system as well as myocardial metabolism as a result of elevated kinin levels (6,7). Enalapril is an ACEI widely employed for the treatment of hypertension and heart failure. Perindopril is an ACEI with a well- tested efficacy and comparatively longer half-life than enalapril (8,9). It also exhibits higher selectivity and potency in terms of angiotensin-converting enzyme (ACE) blockade and has a better tolerance profile; in addition, perindopril exhibits lower first-dose hypotension effects and combats cardiovascular remodeling in heart failure. Although inhibition of the intracardiac renin-angiotensin system by perindopril is stronger than that by enalapril, their inhibitory actions on angiotensin type-1 (AT1)-induced increases in blood pressure and effects in heart failure are not clear.
The transition from compensated to failing cardiac hypertrophy has been attributed to a reversal to a fetal pattern of cardiomyocyte gene expression and adverse remodeling of the ventricular connective tissue matrix (10,11). Many kinds of growth factors and cytokines, such as basic fibroblast growth factor, angiotensin type-2, transforming growth factor (TGF)-β1 and collagen- III, have been suggested as playing important roles in structural remodeling of the non-myocyte compartment of the myocardium following heart failure (12,13). Therefore, it is important to determine whether ACEI have any effect on fetal gene expression or extracellular matrix remodeling in models not only with heart failure after myocardial infarction but also with dilated cardiomyopathy. We reported that quinapril treatment decreased mortality, heart weight, myocardial fibrosis and mRNA expression of TGF-β1, and improved the survival rate and cardiac function in rats with dilated cardiomyopathy after myocarditis in a dose-dependent manner (14).
In the present study, the inhibitory action against AT1 and cardioprotective properties of the ACEIs perindopril and enalapril were studied in a rat model of dilated cardiomyopathy. Subsequently the effects of long-term treatment with ACEIs on the development of myocardial damage were examined. We found that although both ACEIs can block increases in blood pressure caused by circulating AT1, perindopril confers greater protection than enalapril against injury from the renin-angiotensin system in heart failure.
Nine-week-old male Lewis rats were obtained from Charles River Japan Inc. (Kanagawa, Japan). The morbidity of experimental autoimmune myocarditis was 100% in the immunized rats using our protocol (14-16). The age-matched normal control group (Group N) comprised ten normal Lewis rats.
Assessment of the inhibitory actions of perindopril and enalapril on AT1-induced increases in blood pressure in rats with heart failure
Six weeks after immunization, the surviving Lewis rats were used for this experiment. The rats were anesthetized with sodium pentobarbital (50 mg/kg i.p., each group n = 5). Under aseptic conditions, a catheter prepared by connecting polyethylene tubing (PE10 and PE50; Becton, Dickinson and Company, Parsippany, NJ, U.S.A.) filled with sterile heparin sodium (200 U/ml) was inserted into the abdominal aorta via the femoral artery, guided under the skin and exteriorized at the back of the neck. A catheter filled with heparin was inserted into the external jugular vein, guided under the skin of the neck and exteriorized at the back of the neck. Each rat was injected subcutaneously with penicillin G (2000 units) and streptomycin (20 mg) to prevent infection. After 4-5 days, the rats were placed in cages and acclimatized for the measurement of blood pressure and injection of AT1 while conscious. Before administration of the drugs, AT1 was infused in a stepwise manner (each dose for 2 min) through the venous catheter at 0.1-3.0 μg/kg per min to obtain a dose-response curve as a control. Then, the rats were treated orally by gastric lavage once with perindopril, enalapril or vehicle (0.5% methylcellulose). Four hours after the drug administration, when the drugs exhibited the maximal hypotensive effects, AT1 was infused in the same manner at 1 μg/kg per min to 30 μg/kg per min. The animals were used repeatedly after a recovery period of 2 days or more. The blood pressure was measured using a pressure transducer (P10EX-1; NEC Medical Systems, Tokyo, Japan) and bridge amplifier (AD Instruments, Castle Hill, Australia), and was recorded on a Power Lab system (Power Lab/8sp; AD Instruments).
Twenty-eight days after immunization, the surviving rats (90/120 = 75%) were divided into six groups for oral administration of perindopril at 0.02, 0.2 and 2 mg/kg per day (Group P0.02, P0.2, P2), enalapril at 2 and 20 mg/kg per day (Group E2, E20) or vehicle alone (0.5% methylcellulose, Group V, all groups n = 15) for 1 month.
Throughout the studies, all the animals were treated in accordance with our institute's guidelines for animal experimentation.
The rats were anesthetized with 2% halothane in oxygen during the surgical procedures in order to measure the hemodynamic parameters, and then this concentration was reduced to 0.5% to minimize hemodynamic effects. Peak left ventricular pressure, left ventricular end-diastolic pressure (LVEDP), and the rates of intraventricular pressure rise and decline (± dP/dt) were recorded as described previously (14,16).
Using specimens stained with Azan-Mallory at the middle level of the left ventricle, the area of myocardial fibrosis was quantified using a color image analyzer (CIA-102; Olympus, Tokyo, Japan), making use of the differences in color (blue fibrotic area as opposed to red myocardium). The results are presented as the ratio of the fibrotic area to the area of the myocardium (16).
Ribonuclease protection assay
Apical left ventricles from Groups N, V, P and E were rapidly excised, frozen in ice-cold acetone, and stored at -80°C. Antisense complementary RNA probes for TGF-β1, collagen-III and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were generated as described in previous studies (17-20). The RNase protection assays for quantification of TGF-β1 and collagen-III mRNA levels were performed as described in earlier works (17-20). The results for each mRNA were normalized to those for GAPDH mRNA in each sample.
Data are presented as the mean ± the standard error of the mean. Statistical assessment of the groups was performed by one-way analysis of variance, followed by Tukey's method. Differences were considered significant at p < 0.05.
The inhibitory actions of perindopril and enalapril on AT1-induced increases in blood pressure in rats with heart failure
Injection of AT1 evoked hypertension in all groups. The maximum increase in mean blood pressure from the baseline value during AT1 injection was about 40 mmHg in all groups (Fig. 1). Treatment with perindopril inhibited these responses in a dose-dependent manner. Enalapril at 20 mg/kg had a slightly stronger or the same AT1 blocking effect as perindopril at 2 mg/kg.
Four of 15 (27%) rats in Group V and two of 15 (13%) in Groups P0.02 and E2 died between Day 30 and Day 56. All hearts from these rats showed extensive myocardial fibrosis and massive pericardial effusion. None of the animals in Groups P0.2, P2, E20 or N died (Table 1).
Myocardial mRNA expression of TGF-β1 and collagen-III
As shown in Fig. 2, the levels of left ventricular mRNA expression of TGF-β1 and collagen-III were markedly up-regulated in Group V compared to those in Group N. Although perindopril or enalapril treatment significantly suppressed the increased expression of TGF-β1 and collagen-III mRNA in a dose-dependent manner, perindopril treatment showed a greater effect than enalapril (Fig. 2 and Table 1).
Among the six groups with heart failure, the incidence of fibrosis was lowest in Group P2. The area of myocardial fibrosis in Group P0.2 was smaller than that in Group E2, and that in Group P2 was smaller than that in Group E20 (Fig. 3 and Table 1).
Left ventricular end-diastolic pressure was higher in Group V than in Group N (Table 1). ± dP/dt was significantly lower in Group V than in Group N. Although LVEDP was decreased in Group P and E dose-dependently, these changes were stronger in Group P than in Group E (Table 1).
We reported previously that long-term treatment with an ACEI caused a decrease in heart weight and LVEDP, and an increase ± dP/dt in a rat model with dilated cardiomyopathy (14,21). Remarkably, the area of fibrosis was greatly reduced by perindopril in a dose-dependent manner, from 29% of the myocardium in the non-treatment group to 22, 13 or 6%, respectively, in groups receiving 0.02, 0.2, 2.0 mg/kg per day, respectively. In the present study, using the same rat model of dilated cardiomyopathy, we examined the effects of the ACEIs perindopril and enalapril on increases in blood pressure caused by AT1, survival rate, progression of heart failure, TGF-β1 mRNA expression and myocardial fibrosis. Our results indicated that enalapril at 20 mg/kg inhibited AT1-induced blood pressure increases to the same extent as perindopril at 2 mg/kg, but enalapril did not decrease mortality, myocardial fibrosis, or TGF-β1 mRNA expression to the same degree as perindopril in rats with heart failure.
The main reasons perindopril is favored over enalapril, are its higher target organ specificity, and more potent and prolonged ACE blockade in tissues as well as in plasma (6-9). The pharmacological differences between perindopril and enalapril lead to different levels of improvement of objective parameters when these drugs are prescribed in patients or rats with heart failure. Musuelli et al. (8) reported that after switching from enalapril at 30 mg daily to perindopril at 4 mg daily, a further 50% improvement in functional New York Heart Association (NYHA) class was obtained in patients with heart failure.
Angiotensin-converting enzyme inhibitors increase tissue accumulation of bradykinin, which has anti-growth effects and reduces vasomotor tone. There are two kinds of renin-angiotensin system, circulating and tissue, and our interest lies in evaluating the tissue renin-angiotensin system under conditions of heart failure. Perindopril has good tissue affinity, and this affinity was more effective than enalapril in this study. Although enalapril is used for patients with heart failure, perindopril is not used in Japan. Further studies are required to elucidate which ACEIs can decrease mortality, myocardial fibrosis, tissue bradykinin accumulation and TGF-β1 mRNA expression in patients with dilated cardiomyopathy.
Acknowledgements: This research was supported by grants from Yujin Memorial Grant; the Ministry of Education, Culture, Sports, Science and Technology of Japan; and the Promotion and Mutual Aid Corporation for Private Schools of Japan.
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