Efficient Inhibition of the Development of Cardiac Remodeling by a Long-Acting Calcium Antagonist Amlodipine
Abstract—The purpose of the present study was to examine the effects of a long-acting calcium antagonist, amlodipine, on the development of cardiac remodeling. Dihydropyridine calcium antagonists have been used widely for many years in the treatment of hypertension and angina pectoris. It has been reported, however, that a prototype of dihydropyridines, nifedipine, does not reduce mortality of patients with ischemic heart disease, possibly because of reflex stimulation of the sympathetic nervous system. A calcium antagonist, amlodipine, has been reported to have potential benefits by virtue of a gradual onset of action and a long duration of effects. Amlodipine (8 mg/kg per day, once a day) or nifedipine (24 mg/kg per day, three times a day) was administered to spontaneously hypertensive 12-week-old rats for 12 weeks. Left ventricular wall thickness was measured by echocardiography, and relative amounts of myosin heavy chain isoforms were assessed by pyrophosphate gels. Expressions of “fetal type” genes and type 1 collagen gene were examined by Northern blot analysis. Amlodipine and nifedipine both markedly reduced systolic blood pressure. However, the decrease in systolic blood pressure caused by nifedipine continued for no more than 8 hours, whereas the blood pressure-lowering effect of amlodipine continued for more than 16 hours post dose. Amlodipine markedly reduced left ventricular wall thickness, whereas nifedipine only weakly attenuated an increase in the wall thickness. Amlodipine, but not nifedipine, prevented an increase in the relative amount of V3 myosin heavy chain isoform and suppressed an increase in mRNA levels of β-myosin heavy chain, skeletal α-actin, and type 1 collagen. Unlike nifedipine, amlodipine effectively preveted cardiac remodeling secondary to high blood pressure at biochemical levels and morphological levels. These results suggest that a long-acting calcium antagonist is more effective than a short-acting one in preventing organ injury in hypertensive subjects.
It is quite important to prevent end-organ damage secondary to hypertension. The heart is one of the target organs of high BP, and cardiac remodeling evoked by high BP is an important and independent risk factor of cardiac morbidity and mortality.1 Nowadays, many kinds of antihypertensive drugs have been developed. Among them, dihydropyridine calcium antagonists continue to maintain a major position, and newer dihydropyridines have been developed with the aim of improving their therapeutic efficacy as well as decreasing the frequency of adverse effects. Although clinical studies have shown that calcium antagonists have shown inhibitory effects on cardiac hypertrophy induced by hypertension,2 the therapeutic effects of calcium antagonists on cardiac morbidity and mortality caused by hypertension remain unproved. It has been reported recently that a prototype of the dihydropyridines, nifedipine, does not reduce mortality of patients with ischemic heart disease.3
Amlodipine [3-ethyl-5-methyl-2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridinedicarboxylate benzenesulphonate], a third-generation dihydropyridine-based calcium antagonist, has potential clinical benefits by virtue of a gradual onset of action and a long duration of effects.4 Amlodipine has been shown to have a favorable effect on the survival of patients with heart failure due to nonischemic dilated cardiomyopathy in the prospective randomized amlodipine survival evaluation (PRAISE) trial.5 Therefore, amlodipine may be able to reduce cardiac injury secondary to hypertension. Recently, Nayler6 has reported that orally administered amlodipine slows the progression of hypertension-induced cardiac hypertrophy in SHR. However, it remains unknown whether amlodipine ameliorates cardiac remodeling as well as cardiac hypertrophy.
We have recently reported the cardioprotective effects of an antihypertensive agent.7 In SHR, an Ang II type 1 receptor antagonist, TCV-116, not only reduced the left ventricular wall thickness determined by echocardiography but also prevented cardiac remodeling, which was evaluated by inhibition of MHC isoform switching from V1 to V3 and of cardiac interstitial fibrosis.7 The purpose of the present study was to examine the efficient effects of amlodipine over nifedipine on hypertensive cardiac remodeling.
This study was carried out in compliance with the guidelines for animal usage of the University of Tokyo.
Chemicals and Reagents
Amlodipine and nifedipine were gifts from Pfizer Pharmaceuticals, Inc (Tokyo, Japan) and Bayer Yakuhin Ltd (Tokyo, Japan), respectively. [α-32P]dCTP and [γ-32P]dATP were purchased from DuPont-New England Nuclear, and other reagents were purchased from Sigma.
Animals and Experimental Protocols
Male SHR and normotensive WKY were obtained from Charles River Japan (Tokyo, Japan). SHR were randomly divided into three groups, and amlodipine (8 mg/kg per day, once a day), nifedipine (24 mg/kg per day, three times a day), or vehicle (distilled water, three times a day) was administered daily through a stomach tube for 12 weeks from the age of 12 weeks, when cardiac hypertrophy had already developed.7 These doses were chosen from the previous report8 and our preliminary experiment (data not shown). Vehicle (distilled water, three times a day) was also administered to WKY daily through a stomach tube for 12 weeks from the age of 12 weeks. Each group consisted of 18 rats. At the age of 12, 16, and 24 weeks, 6 rats of each group were killed, and the determination of MHC isoform profiles and the Northern blot analysis were performed (Fig 1⇓). The rats killed at 24 weeks old were also used for the measurement of systolic BP and the determination of left ventricular wall thickness by echocardiography.
Measurement of Systolic BP and Echocardiographic Evaluation of Left Ventricular Wall Thickness
Systolic BP was measured every week by the tail-cuff plethysmography method in conscious rats,7 and the left ventricular wall thickness was serially measured by M-mode echocardiography every 4 weeks (Fig 1⇑). At days 0, 7, and 13 after the start of the oral administration, the changes in systolic BP were measured. Echocardiographic studies were performed with a Hewlett-Packard HP Sonos 100 mechanical sector scanner using a single element transducer with a frequency of 10 MHz as previously described.7 Conscious rats were held in the supine position, and M-mode tracings of the left ventricular wall were obtained using a two-dimensional reference sector. The thickness of the IVS was measured from M-mode tracings by using the leading edge method every 4 weeks (Fig 1⇑).
Determination of MHC Isoform
For MHC isoform analysis, left ventricles of 8-, 12-, 16-, or 24-week-old rats were used (Fig 1⇑). Left ventricular MHC isoform profiles were determined by pyrophosphate gel electrophoresis as previously described by Hoh et al.9 Briefly, a small piece of heart was homogenized in 10% glycerol and 20 mmol/L Na4P2O4, pH 8.8. After centrifugation, electrophoresis of the supernatant was performed in nondenaturing pyrophosphate gel, and relative amounts of V1, V2, and V3 MHC isoforms were analyzed by a laser densitometer after Coomassie blue staining.
Northern Blot Analysis
Left ventricles of 12-, 16-, or 24-week-old rats were used (Fig 1⇑). Total cellular RNA was extracted from the heart using 201B (Cinna Biotecx Laboratories, Inc). Twenty micrograms of total RNA was size-fractionated by 1.2% agarose gels and transferred to nylon membranes according to the manufacturer’s instructions. The 3′ untranslated region of rat skeletal α-actin (184 bp) was used as a probe as described previously.10 Type 1 collagen cDNA (≈1.2 kbp) was isolated by polymerase chain reaction using the primers specific to the triple helical region and the carboxyl-terminal region of rat α1 chain of type 1 collagen.11 A 20-mer oligonucleotide specific to the 3′ untranslated region of rat βMHC was synthesized as described before.12 cDNA was labeled by a random priming method using [α-32P]dCTP and oligo DNA was labeled at the 5′ end by T4 polynucleotide kinase using [γ-32P]dATP. Northern blot analysis was performed as described previously.13 14 In brief, the blots were hybridized with a 32P-labeled cDNA probe at 42°C for 10 hours in 50% formamide, 5× SSPE, 1% SDS, 5× Denhardt’s solution, and 100 mg/L salmon sperm DNA and washed to a stringency of 0.1% SDS at 42°C. In the case of oligo DNA, the blots were hybridized in the same solution at room temperature and washed in 6× SSC and 2× SSC at room temperature. The filter was dried and exposed to x-ray film at −80°C. The density of each band was quantified using a laser densitometer.
All values are expressed as mean±SEM of six experiments in each instance. Comparisons among three or more groups were made by one-way ANOVA followed by Dunnetts’ modified t test. Values of P<.05 were considered statistically significant.
Treatment of SHR with amlodipine (8 mg/kg per day, once a day) or nifedipine (24 mg/kg per day, three times a day) was started from 12 weeks after birth. Systolic BP at 2 hours after oral administration of these agents was measured by the tail-cuff method. At the start of the experiments, 12-week-old SHR had much higher systolic BPs (approximately 210 mm Hg, n=6) compared with the age-matched WKY (approximately 140 mm Hg, n=6). Systolic BP was gradually decreased in both amlodipine- and nifedipine-treated SHR. During the treatment (12 weeks), amlodipine and nifedipine both efficiently lowered BP in SHR compared with vehicle-treated control animals (n=6) (Fig 2A⇓). Changes in systolic BP were also recorded every 4 hours on days 0, 7, and 13 after treatment. Nifedipine exerted a maximal lowering effect on systolic BP at 0.5 hours postdose, and the effect disappeared at 8 hours postdose (Fig 2B⇓). In contrast, treatment with amlodipine induced a gradual decrease in systolic BP, and the BP-lowering effect of amlodipine was still recognized at 20 hours after the administration at days 7 and 13 (Fig 2B⇓). No significant differences were observed in heart rates and body weights among vehicle-, amlodipine-, and nifedipine-treated SHR (data not shown).
Left Ventricular Wall Thickness on Echocardiography
Two-dimensional long-axis images were clear enough to obtain adequate M-mode tracings, and the thickness of the IVS was measured from M-mode images during six successive beats as previously reported7 (Fig 3A⇓). The thickness of the IVS was increased progressively along with the elevation of systolic BP in vehicle-treated SHR (1.71±0.04 mm for 12-week-old SHR, 1.75±0.07 mm for 16-week-old SHR, 2.12±0.10 mm for 20-week-old SHR, and 2.20±0.12 mm for 24-week-old SHR). Amlodipine strongly attenuated the increase in the thickness of the IVS (1.49±0.12 mm for amlodipine-treated 20-week-old SHR versus 2.12±0.10 mm for vehicle-treated 20-week-old SHR, P<.05; 1.50±0.04 mm for amlodipine-treated 24-week-old SHR versus 2.20±0.12 mm for vehicle-treated 24-week-old SHR, P<.01). On the other hand, when SHR were treated with nifedipine, the IVS thickness was not reduced to the same level as with amlodipine treatment (1.71±0.04 mm for 12-week-old SHR, 1.84±0.11 mm for 20-week-old SHR, 1.91±0.07 mm for 24-week-old SHR) (Fig 3B⇓). There were significant differences in IVS thickness between amlodipine- and nifedipine-treated SHR at 20 (P<.05) and 24 (P<.01) weeks of age.
To elucidate whether amlodipine prevents not only morphological changes but also biochemical changes from hypertension, we first examined MHC isoforms. At the age of 8, 12, 16, and 24 weeks, SHR were killed, and the hearts were excised. The relative amounts of V3 MHC isoform increased progressively from the age of 8 weeks to 24 weeks in vehicle-treated SHR (Fig 4A⇓ and 4B⇓). Treatment with amlodipine markedly inhibited an increase in the ratio of V3 MHC isoform. The relative amount of V3 MHC in amlodipine-treated 24-week-old SHR was almost the same as that of 8-week-old SHR (Fig 4B⇓). The attenuation of the increase in V3 MHC was much less in nifedipine-treated SHR than in amlodipine-treated SHR (Fig 4A⇓ and 4B⇓).
βMHC and Skeletal α-Actin mRNA
To elucidate whether the isoformic change of MHC is regulated at pretranslational levels, we analyzed mRNA levels of βMHC by Northern blot analysis. In vehicle-treated SHR, the levels of βMHC mRNA were increased progressively along with the age (Fig 5A⇓ and 5B⇓). Nifedipine did not significantly reduce the increase in βMHC levels. On the other hand, amlodipine significantly inhibited the increase in the levels of βMHC transcripts, by approximately 70%.
It is well known that “fetal type” genes such as βMHC and skeletal α-actin genes are induced during cardiac hypertrophy (for a review see reference 15). To elucidate whether amlodipine generally attenuates the expression of “fetal type” genes, we next analyzed the expression of skeletal α-actin gene. The levels of skeletal α-actin mRNA increased progressively along with age in SHR hearts (Fig 6⇓). In SHR treated with amlodipine, the mRNA levels of skeletal α-actin were much lower than those of vehicle-treated SHR, by approximately 80%. In contrast, nifedipine had no significant effects on the mRNA levels of skeletal α-actin.
Type 1 Collagen mRNA
Cardiac collagen fibers are composed predominantly of type 1 and type 3 collagen, about 80% and 15%, respectively.16 In hypertrophic hearts, extracellular matrix proteins such as collagen and fibronectin are increased, and perivascular and interstitial fibrosis occurs as one of the features of cardiac remodeling. The increase in extracellular matrix proteins is detrimental because fibrosis is one of the main causes of diastolic dysfunction. To examine the effects of calcium antagonists on fibrosis, we finally examined the expression levels of the type 1 collagen gene by Northern blot analysis. In vehicle-treated SHR, mRNA levels of type 1 collagen were increased progressively along with age (Fig 7A⇓ and 7B⇓). Treatment with amlodipine inhibited the age-dependent increase in type 1 collagen mRNA levels in SHR. However, treatment with nifedipine did not significantly inhibit the increase (Fig 7A⇓ and 7B⇓).
Previous studies using animal models have demonstrated that angiotensin-converting enzyme inhibitors17 18 and the Ang II type 1 receptor antagonist TCV-1167 prevented hypertrophy- associated ventricular remodeling, suggesting that these agents are cardioprotective. In the present study the long-acting third-generation dihydropyridine calcium antagonist amlodipine markedly reduced left ventricular wall thickness as evaluated by echocardiography, whereas nifedipine had a minimal effect. Amlodipine prevented not only cardiac hypertrophy but also cardiac remodeling by inhibiting increases in MHC V3 isoform and in gene expressions of βMHC, skeletal α-actin, and type 1 collagen. These results suggest that amlodipine as well as angiotensin-converting enzyme inhibitors and Ang II receptor antagonists work to protect the heart from high BP.
The BP-lowering effect of amlodipine in SHR reached a maximum at 4 hours and continued up to 24 hours postdose, in contrast to nifedipine, which produced a maximal effect at 0.5 hours with a prompt loss of efficiency at 8 hours postdose (Fig 2B⇑). Therefore, a possible reason for the better effect of amlodipine than nifedipine in preventing cardiac remodeling is that amlodipine has a longer duration of BP-lowering effect.
Recently, we have reported that an Ang II type 1 receptor antagonist induces regression of cardiac hypertrophy and prevents left ventricular remodeling in SHR.7 Accumulating evidence has suggested that Ang II plays an important role in the development of left ventricular remodeling.7 19 We and others have reported that the calcium ion partly mediates Ang II-induced hypertrophic events.19 20 Actually, nifedipine partly inhibited Ang II-induced activation of mitogen-activated protein kinases, which is critical for the induction of cardiac hypertrophy.19 Therefore, the better prevention of cardiac remodeling by amlodipine over nifedipine may be due to the more efficient inhibition of calcium channels by amlodipine than nifedipine. We are now examining this hypothesis by in vitro study.
Although short-acting calcium antagonists such as nifedipine have shown favorable acute hemodynamic effects by effectively reducing afterload, long-term studies with such agents have demonstrated the potential unfavorable effects, including negative inotropic effects and neurohormonal activation.3 The fall in BP caused by nifedipine is associated with reflex activation of the sympathetic nervous system, with a consequent increase in renin release.21 On the other hand, Aber- nethy et al22 have observed that treatment with amlodipine for 14 weeks induces no increase in plasma levels of norepinephrine, epinephrine, renin, or aldosterone. In addition, Lund-Johansen et al23 have reported on hormonal responses at rest and during exercise in patients with essential hypertension treated with amlodipine or nifedipine. In amlodipine-treated patients, plasma norepinephrine levels were not significantly increased after exercise; whereas in nifedipine-treated ones, exercise strongly elevated plasma norepinephrine levels. Moreover, it has been shown that the pressure response to norepinephrine is blunted after amlodipine treatment24 but not after nifedipine treatment25 in patients with essential hypertension. These results suggest that treatment with amlodipine avoids reflex stimulation of the sympathetic nervous system. Catecholamines have been reported to also be potent inducers of cardiac hypertrophy (for a review, see reference 26), and aldosterone has been shown to play an important role in inducing fibrosis in the heart (for a review, see reference 27). The lack of stimulation of neurohormonal factors by amlodipine may partly explain why amlodipine, but not nifedipine, effectively prevents cardiac remodeling. With respect to the mechanisms, Terland et al28 have reported that in the bovine adrenal medulla, nifedipine-induced inhibition of the proton pump activity induces an increased adrenergic activity by inhibiting the catecholamine uptake into storage vesicles and that only amlodipine increases the proton pump activity in a concentration-related manner compared with several calcium antagonists such as diltiazem, felodipine, and nicardipine.
Cardiac fibrosis, which is increased in accordance with the duration of hypertension in SHR,7 is a major determinant of diastolic stiffness in hypertensive heart disease.29 In the present study, amlodipine strongly reduced type 1 collagen gene expression and interstitial fibrosis (Fig 7⇑, data not shown). Recently, Ang II (as well as aldosterone) has been demonstrated to induce cardiac fibrosis.7 27 Although the precise mechanisms of how these molecules are involved in interstitial fibrosis remain unresolved, amlodipine may prevent an increase in fibrotic tissues by inhibiting the effects or production of these humoral factors.
Amlodipine, but not nifedipine, inhibited the shift of MHC isoforms from V1 to V3 in SHR in the present study. Although in the ventricle of larger animals including humans, little or no isoform transition occurs in response to hemodynamic overload because of the predominance of V3 in the normal ventricle,30 in chronically pressure-overloaded human atria, the shift from V1 to V3 has been shown to be accompanied by atrial enlargement.31 Thus, we think at present that amlodipine, which exerts prevention of switching from V1 to V3 on the SHR heart, may also have a potential cardioprotector role in human hearts.
In conclusion, in SHR, which is a good model of human essential hypertension, cardiac remodeling was prevented by chronic treatment with a long-acting third-generation calcium antagonist, amlodipine, but not with nifedipine. The present results support the clinical usefulness of amlodipine in hypertension. However, further investigation is necessary to clarify the precise molecular mechanisms by which amlodipine reduces cardiac remodeling that is produced by pressure overload.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|MHC||=||myosin heavy chain|
|SHR||=||spontaneously hypertensive rat(s)|
This work was supported by a grant-in-aid from the Ministry of Education, Science, Sports and Culture, Japan. We wish to thank Fumiko Harima, Makiko Iwata, and Mika Ono for technical assistance.
- Received July 16, 1997.
- Revision received August 14, 1997.
- Accepted August 14, 1997.
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