Improvement of Left Ventricular Remodeling and Function by Hydroxymethylglutaryl Coenzyme A Reductase Inhibition With Cerivastatin in Rats With Heart Failure After Myocardial Infarction
Background Hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) attenuate angiotensin II-induced cellular signaling. Because angiotensin II is involved in left ventricular (LV) remodeling after myocardial infarction (MI), we examined the effects of statin treatment in an experimental model of chronic heart failure after MI.
Methods and Results Rats with extensive MI were treated with placebo or cerivastatin (0.3 mg/kg per day) as a dietary supplement or via gavage for 11 weeks starting on the 7th postoperative day. Infarct size and cholesterol levels were similar among all groups. LV cavity area, an index of LV dilatation, was reduced in MI rats on cerivastatin compared with placebo. LV end-diastolic pressure was increased in MI rats on placebo (24.1±4.1 mm Hg versus sham: 5.1±0.3 mm Hg; P<0.01), and it was significantly reduced by cerivastatin treatment (13.7±2.7 mm Hg; P<0.05 versus placebo). Cerivastatin partially normalized LV dP/dtmax and dP/dtmin, indices of LV systolic and diastolic function, which were significantly reduced in MI rats on placebo. Improvement of LV function by cerivastatin was accompanied by a reduced expression of collagen type I and β-myosin heavy chain. LV endothelial nitric oxide synthase was increased, whereas the nitrotyrosine protein level was decreased in MI rats by cerivastatin treatment.
Conclusions Cerivastatin improved LV remodeling and function in rats with heart failure. This effect was associated with an attenuated LV expression of fetal myosin heavy chain isoenzymes and collagen I. Statin treatment may retard the progression of chronic heart failure.
Received June 1, 2001; revision received July 9, 2001; accepted July 10, 2001.
A cute neurohumoral activation after myocardial infarction (MI) helps maintain cardiac output and peripheral perfusion in the acute phase of MI. However, chronic stimulation of neurohumoral systems, such as angiotensin II and endothelin-1, results in increased cardiac load and left ventricular (LV) hypertrophy, which eventually leads to ventricular enlargement and progression of heart failure.1,2 The detrimental effects of neurohumoral activation on ventricular remodeling seem to depend on the increased generation of reactive oxygen species such as superoxide anions (O2−).3–5 Hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) attenuate O2− formation and simultaneously increase the expression of endothelial nitric oxide synthase (eNOS),6–8 resulting in a beneficial shift in the balance between NO and O2− that may improve LV remodeling after MI.9 Interestingly, statins attenuate angiotensin II–induced myocyte hypertrophy10 in cultured neonatal rat cardiomyocytes in a manner that is probably mediated by the attenuation of radical generation. Therefore, we examined whether long-term treatment with cerivastatin improves LV remodeling and function in rats with extensive MI.
Study Protocol, MI, and Hemodynamic Measurements
Left coronary artery ligations were performed in adult male Wistar rats (250 to 300 g).11 On the seventh postoperative day, rats were randomly allocated to treatment with placebo or cerivastatin (0.3 mg/kg per day), which was given as either a dietary supplement or via gavage. Hemodynamic studies were performed 12 weeks after MI under barbiturate anesthesia and controlled respiration.11
Sample Collection, Determination of Infarct Size, and Ventricular Remodeling
The heart was excised and dissected into the right and left ventricles, including the septum. The LV was cut into 3 transverse sections: apex, middle ring (≈3 mm), and base. From the middle ring, 5-μm sections were cut at 100-μm intervals and stained with picrosirius red. The boundary lengths of the infarcted and noninfarcted endocardial and epicardial surfaces were traced with a planimeter digital image analyser. Infarct size (fraction of the infarcted LV) was calculated as the average of all slices and expressed as the percentage of length of circumference, and only rats with extensive infarcts (>40%) were included in the study. LV cavity area (area enclosed by LV endocardial circumference) was taken as an index of LV dilatation.
Myosin Heavy Chain Isoenzyme and Collagen I Expression
Total RNA was isolated from surviving LV myocardium (septum) using TRIzol reagent. α- and β-myosin heavy chain (MHC) mRNA was amplified by polymerase chain reaction after reverse transcription (SuperScript, Life Technologies, Germany; Table 1). Fragments of the amplification product were separated on 8% polyacrylamide gels after enzymatic digestion with Tru9I (lengths: 310 bp for α- and 257+53 bp for β-MHC), and the ratio of β- to α-MHC mRNA was quantified. mRNA expression of collagen α1(I) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was determined by competitive polymerase chain reaction using internal standards. After separation on a 2% agarose gel, amplification products were densitometrically quantified. A given mRNA level was expressed as a ratio with respect to the level of mRNA for GAPDH.
Western Blot Analysis
Crude protein extracts (20 μg) were subjected to a 7.5% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. eNOS and nitrotyrosine protein levels were detected using specific antibodies (Transduction Laboratories and Upstate Biotechnology) and were visualized by enhanced chemiluminescence.
All biochemicals were obtained from Sigma. Cerivastatin was provided by Bayer AG (Wuppertal, Germany).
Statistical analysis was performed by 2-factor ANOVA followed by a Newman-Keuls test or by the 2-tailed Student’s t test, where appropriate. Values are expressed as mean±SEM, and P<0.05 was considered statistically significant.
Global parameters are shown in Table 2. Infarct size, body weight, and plasma cholesterol levels did not differ among MI rats on placebo or cerivastatin. LV cavity area, which was increased in MI rats, was reduced by cerivastatin. Mean arterial pressure and LV systolic pressure were significantly lower in placebo-treated MI rats (Table 2), and LV end-diastolic pressure and right atrial pressure were substantially elevated compared with sham-operated animals (Figure 1). In MI rats on cerivastatin treatment, LV systolic pressure and mean arterial pressure were significantly increased, whereas LV end-diastolic pressure and right atrial pressure were reduced (P<0.05 versus placebo). Cerivastatin partially normalized LV dP/dtmax, an index of myocardial contractility, and dP/dtmin, an index of diastolic relaxation, which were both significantly reduced in MI rats on placebo (Figure 1). Reduction of systolic and diastolic function was associated with an increased ratio of LV β-MHC to α-MHC mRNA and a marked increase in LV collagen I mRNA expression in MI rats compared with sham-operated animals. Both parameters were beneficially modulated by cerivastatin treatment (Figure 2). The reduction in collagen expression significantly correlated with the improvement in dP/dtmin (r=0.72, P<0.01) and dP/dtmax (r=0.75, P<0.01).
LV eNOS protein was significantly increased in rats with chronic MI (3.6±0.1 versus 2.7±0.2 arbitrary units [aU] in sham), and it was further enhanced in both cerivastatin-treated groups. LV nitrotyrosine protein level as a marker for peroxynitrite formation was enhanced in MI rats (2.9±0.3 versus 1.8±0.4 aU in sham) and attenuated by cerivastatin (2.0±0.4 versus 1.8±0.5 aU in sham, n=3; P<0.05; Figure 2C).
The major novel result of this study was that cerivastatin improves LV systolic and diastolic function in rats with chronic heart failure after experimental MI. This effect was associated with an attenuated LV expression of fetal genes such as β-MHC, a marked reduction of collagen I gene expression, an increase in LV eNOS expression, and a decrease in tyrosine nitration.
Improvement of LV function by cerivastatin may be explained by the beneficial effects of statins on afterload, such as a direct reduction of systemic vascular resistance. However, a cardioprotective effect of cerivastatin secondary to afterload lowering is unlikely because mean arterial pressure increased during treatment. Thus, cerivastatin seems to modulate cardiac function directly, as indicated by the improvement of dP/dtmax and the attenuation of fetal β-MHC expression. In addition, we showed that statin treatment leads to a substantial reduction of LV collagen I expression, which correlated with the improvement in LV systolic and diastolic function. Myocardial fibrosis is a major feature of LV remodeling after MI, which is mainly driven by angiotensin II.1,2 Statins have been shown to prevent angiotensin II–induced hypertrophy in cultured neonatal rat cardiac myocytes, probably by attenuating angiotensin II–stimulated p21 ras activity.10 This effect could be reversed by mevalonic acid, an immediate precursor of isoprenoids, suggesting that statins reduce angiotensin II–dependent hypertrophy by blocking the isoprenylation of small (21 kDa) guanine nucleotide-binding proteins (G-proteins). The critical steps for angiotensin II–induced effects on myocardial remodeling seem to depend on the generation of reactive oxygen species.4,5 Indeed, statins have been shown to reduce the formation of O2− by preventing the isoprenylation of p21 Rac, which is critical for the assembly of NADPH oxidase.7 Furthermore, statins upregulate eNOS expression by inhibiting geranylgeranylation of Rho GTPase, another small G-protein.6,8
LV eNOS expression was markedly increased and protein tyrosine nitration was reduced by cerivastatin in our study, suggesting an interaction of cerivastatin treatment with the above-discussed intracellular pathways. Thus, the beneficial modulation of LV remodeling by cerivastatin may be mediated by an improved NO/O2− balance. This hypothesis is supported by the observed decrease of protein tyrosine nitration, indicating reduced peroxynitrite formation. Reduced NO bioavailability contributes to the deterioration of LV function after MI,9 whereas an increase of NO bioavailability combined with reduced O2− formation may synergistically improve LV remodeling and function.5,9 We interpret the slight increase in eNOS expression in MI rats on placebo as a failed counter-regulatory mechanism in response to the increase in O2− formation, which has a decisive role for LV remodeling.5 This response was presumably not sufficient to counteract the marked increase in radical generation.
Furthermore, the beneficial effects of cerivastatin on LV remodeling after MI may be mediated by an attenuation of endothelin-1 synthesis, which has been demonstrated previously in vascular endothelial cells.8 Thus, cerivastatin may alleviate the deleterious consequences of an increased expression of endothelin-1 after coronary ligation.1,2,11
In conclusion, we show for the first time that cerivastatin improves LV remodeling and function in rats with heart failure, which was associated with a marked reduction of collagen expression and an attenuated expression of fetal MHC isoenzymes. The reduction of overall mortality by statins in the secondary prevention of coronary heart disease presumably depends on plaque stabilization; however, our data indicate that statins may also retard the progression of heart failure, presumably by a beneficial modulation of cellular responses to neurohormonal activation after large MI.
This work was supported in part by the Deutsche Forschungsgemeinschaft (SFB355, B9, 10) and by Bayer AG, Wuppertal, Germany. The authors thank Claudia Liebetrau and Anna Dembny for expert technical assistance.
The first 2 authors contributed equally to this work.
Sutton MG, Sharpe N. Left ventricular remodeling after myocardial infarction: pathophysiology and therapy. Circulation. 2000; 101: 2981–2988.
Weber KT. Extracellular matrix remodeling in heart failure: a role for de novo angiotensin II generation. Circulation. 1997; 96: 4065–4082.
Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res. 2000; 86: 494–501.
Kinugawa S, Tsutsui H, Hayashidani S, et al. Treatment with dimethylthiourea prevents left ventricular remodeling and failure after experimental myocardial infarction in mice: role of oxidative stress. Circ Res. 2000; 87: 392–398.
Laufs U, Liao JK. Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. J Biol Chem. 1998; 273: 24266–24271.
Wagner AH, Köhler T, Rückschloss U, et al. Improvement of nitric oxide-dependent vasodilatation by HMG-CoA reductase inhibitors through attenuation of endothelial superoxide anion formation. Arterioscler Thromb Vasc Biol. 2000; 20: 61–69.
Hernandez-Perera O, Perez-Sala D, Navarro-Antolin J, et al. Effects of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, atorvastatin and simvastatin, on the expression of endothelin-1 and endothelial nitric oxide synthase in vascular endothelial cells. J Clin Invest. 1998; 101: 2711–2719.
Qi XL, Stewart DJ, Gosselin H, et al. Improvement of endocardial and vascular endothelial function on myocardial performance by captopril treatment in postinfarct rat hearts. Circulation. 1999; 100: 1338–1345.
Fraccarollo D, Hu K, Galuppo P, et al. Chronic endothelin receptor blockade attenuates progressive ventricular dilatation and improves cardiac function in rats with myocardial infarction. Possible involvement of myocardial endothelin system in ventricular remodeling. Circulation. 1997; 96: 3963–3973.