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Statins initiated after hypertrophy inhibit oxidative stress and prevent heart failure in rats with aortic stenosis
Journal of Molecular and Cellular Cardiology 37 (2004) 889–896 www.elsevier.com/locate/yjmcc
Original Article
Statins initiated after hypertrophy inhibit oxidative stress and prevent heart failure in rats with aortic stenosis Min-Sheng Chen a,b,1, Fang-Ping Xu a,1, Yan-Zhen Wang a,c, Gui-Ping Zhang a, Quan Yi a, Hui-Qiu Zhang d, Jian-Dong Luo a,* a Department of Pharmacology, Guangzhou Medical College, Guangzhou 510182, China Department of Internal Medicine, Guangzhou Medical College, Guangzhou 510182, China c Department of Research Laboratory of Pathophysiology, PLA General Hospital, Beijing 100853, China d Department of Pathology, Guangzhou Medical College, Guangzhou 510182, China b
Received 4 March 2004; received in revised form 21 June 2004; accepted 24 June 2004 Available online 11 August 2004
Abstract Objective. – Heart failure is a major and escalating public health problem. Recent studies have demonstrated that statins prevented chronic heart failure (CHF) in animal studies. However, it is unknown whether statins therapy initiated after left ventricular (LV) hypertrophy is evident can still effectively prevent CHF. This study tested the hypothesis that statins can prevent the transition of hypertrophy to heart failure. Methods and results. – The rats were studied at 6, 12, and 20 weeks after aortic stenosis (AS) operation. Some rats were given simvastatin (2.0 mg kg–1 per day) from 13 weeks after AS operation for 8 weeks. Coarctation of aorta in rats resulted in compensatory LV hypertrophy (LVH), concomitant with an increase of superoxide levels and cardiomyocyte apoptosis in LV tissues at 12 weeks after AS operation. This was followed by CHF with a progressive increase in superoxide levels and cardiomyocyte apoptosis in LV tissues at 20 weeks after AS operation. Simvastatin treatment initiated from 13 weeks after AS operation significantly improved LV function and reduced superoxide levels and cardiomyocyte apoptosis in LV tissues. Pretreatment of simvastatin suppressed the hydrogen peroxide-induced apoptosis of cultured cardiomyocytes from neonatal rats. Conclusions. – These data indicate that long-term administration of simvastatin improved LV function and prevented the transition of hypertrophy to CHF. Inhibition of oxidative stress and cardiomyocyte apoptosis may contribute to the benefits of simvastatin treatment on heart of rats with AS. © 2004 Elsevier Ltd. All rights reserved. Keywords: Apoptosis; Cardiac hypertrophy; Heart failure; Superoxide; Statins
1. Introduction CHF is a clinical syndrome characterized by progressive left ventricular (LV) systolic and/or diastolic dysfunction. The incidence doubles with each decade of life, and CHF is the most common hospital discharge diagnosis in individuals over age 65. Unlike most other cardiovascular diseases, the incidence and morbidity/mortality from CHF have not decreased over the last two decades and the prevalence is expected to double in the next 40 years [1,2]. Thus CHF is a * Corresponding author. Tel.: +86-20-8134-0203; fax: +86-20-8134-1922. E-mail address: [email protected] (J.-D. Luo). 1 These authors contributed equally to this work. 0022-2828/$ - see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2004.06.019
major medical problem which is expected to become more significant with time [3,4]. Therefore, it is very important to develop the new therapeutic strategies for CHF. Hydroxymethylglutaryl-coenzyme A reductase inhibitors, known as “statins”, are proven to be clinical benefit in coronary heart disease, at least in those patients who do not have overt CHF [5–8]. The beneficial potential of statins clearly relates to their inhibition of oxidative stress and proinflammatory cytokine activity, improvements in endothelial function, properties of plaque stabilization, and favorable modulation of the autonomic nervous system besides lipid-lowing effect [9–15]. Recent results from our laboratory and others have demonstrated that cardiac hypertrophy and heart failure could be prevented by statins initiated at an
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early stage when LV hypertrophy (LVH) and dysfunction were not evident in animal study [9,16,17]. However, there is very little data pertaining to the effects of pharmacological interventions with statins initiated after LVH and LV dysfunction are promoted. It is very important to elucidate cardioprotective effects of statins treatment initiates after the on-set of abnormalities as to recreate a situation that is to most clinical situations. Experimental evidence in animal models of myocardial remodeling and failure demonstrate that the failing heart is subjected to increased oxidative stress. The reactive oxygen species (ROS) may be derived from mitochondrial electron transport chain, NADH/NADPH oxidase, xanthine oxidase, nitric oxide synthase, and/or cyclooxygenase [18–22]. At a cellular level, oxidative stress can induce most of the changes that are thought to contribute to myocardial remodelingmyocyte hypertrophy, apoptosis, fetal gene expression and increased matrix metalloproteinase activity [23]. The results from both animal and human studies suggest that cardiomyocyte apoptosis plays a crucial role as the underlying mechanism being at least in part responsible for ventricular dysfunction [18,19,23]. Oxidative stress has been shown to be involved in cardiomyocyte apoptosis [24,25]. Antioxidant therapy has been demonstrated to inhibit cardiomyocyte apoptosis and preserve LV function during development of CHF [18,24]. Therefore, oxidative stress may play an important role in the pathogenesis and progression of CHF. However, direct evidence regarding kinetics of oxidative stress, apoptosis, and ventricular dysfunction during the transition of hypertrophy to heart failure has not been obtained. In the present study, we determined the time course of the oxidative stress and LV dysfunction after aortic coarctation and the overall effectiveness of simvastatin treatment initiated after LVH and dysfunction in preventing the transition of hypertrophy to CHF in rats with chronic pressure overload-induced CHF. Our results demonstrated that in rats with AS: (1) aortic coarctation induced a progressive increase of oxidative stress and cardiomyocyte apoptosis in LV tissues, ultimately resulting in heart failure, and (2) longterm administration of HMG-CoA reductase inhibitor simvastatin, initiated from the compensatory hypertrophic stage, reduced oxidative stress and cardiomyocyte apoptosis, improved LV function, and prevented transition of hypertrophy to heart failure.
2. Methods 2.1. Animals and experimental protocol This study was approved by our institutional animal research committee and conformed to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH publication No. 85-23, revised 1996). The male Sprague–Dawley rats, 3–4 weeks and 60–70 g in body weight (BW), were obtained
from Medical Laboratory Animal Center (Guangdong, China). The cardiac hypertrophy-failure of rats was induced by partial aortic coarctation according to our previous study [16]. The rats with AS were studied in the time points of 6, 12, and 20 weeks after aortic coarctation. Sham-operated rats served as age-matched controls. Simvastatin (2 mg kg–1 per day) was administered by gavage once a day for 8 weeks, beginning 13 weeks after aortic coarctation. The dose of simvastatin was determined according to our previous study [16]. 2.2. Hemodynamic measurements Cardiac hemodynamics were assessed at the end of 6, 12, and 20 weeks after aortic coarctation. In brief, the rats were anesthetized (pentobarbital; 50 mg kg–1, i.p.) and the right carotid artery was cannulated with a micromanometer-tipped catheter (SPR 407, Millar Instruments Inc., Houston, TX, USA) for recording of arterial pressure [26]. The catheter was then advanced into the left ventricle (LV) for recording of LV pressure and its maximal rate of rise (+dP/dtmax) and decrease (–dP/dtmax) [26]. 2.3. Reverse transcription-PCR (RT-PCR) Total RNA was isolated from LV tissues using TRIzol reagent (Invitrogen). The atrial natriuretic peptide (ANP) mRNA was analyzed by RT-PCR using primers specific for ANP (sense: 5′-GGGCTCCTTCTCCATCACC-3′, and antisense: 5′-CTCCAATCCTGTCAATCCTACC-3′) [27]. ANP PCR amplification was performed for 27 cycles at 94 °C for 20 s, 55 °C for 15 s, and 72 °C for 30 s. The amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, a constitutively and ubiquitously expressed gene, served as an internal standard for RT-PCR analysis. A sense primer 5′-AAGGTCGGTGTCAACCCATTTGGCCGT-3′ and antisense primer 5′-CAGTGATGGCATCCACTGTG GTC-3′ were used. Amplification was performed over 23 cycles, each involving 1 min at 94 °C, 1.5 min at 59 °C, and 2 min at 72 °C. 2.4. Superoxide measurement Myocardial superoxide levels were determined by fluorescence spectrometry according to the previous study [28]. Briefly, the fluorogenic oxidation of dihydroethidine (DHE) to ethidium (Eth) was used as a measure of O2–. Tissue homogenates were incubated with DHE (10 µmol/l) in a microtiter plate at 37 °C for 30 min, and Eth-DNA fluorescence was measured at an excitation of 475 nm and an emission of 610 nm with a fluorescence microplate reader (Bio-Tec, American). The fluorescent signal intensity reflects O2– levels. 2.5. In situ apoptosis detection The apoptosis in heart tissue sections and cultured cells was analyzed using TUNEL staining assay [29]. The TUNEL
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reaction was performed using a TdT in situ apoptosis detection kit—TACS™ blue label (R&D System Inc., MN, USA) according to the manufacturer’s directions. The apoptotic (TUNEL-positive) cardiomyocyte nucleus was stained as blue color and the number of TUNEL-positive cardiomyocyte nuclei was counted. Five sections were analyzed for each animal and five fields were observed for each section.
Keuls Multiple Comparison Test. A P value of less than 0.05 was considered statistically significant.
2.6. Cell culture
During the first 2 weeks after AS, nine of 51 rats with AS died. Thereafter, no rat died in any of the groups until the end of the experimental period. There was no significant change of morphological indexes in rats with AS compared with sham rats at 6 weeks after operation. However, AS induced a significant increase in heart weight (HW), LV weight, LV depth, interventricular depth and the ratios of HW/BW, LVW/BW, and LVW/right ventricular weight (RVW) in rats with AS compared with sham rats at 12 or 20 weeks after operation (Table 1, Fig. 1A).
Primary cultures of cardiomyocytes from the ventricles of neonatal Sprague–Dawley rats were prepared according to the previous studies [30]. Cardiomyocytes were plated at a density of 1 × 103 cells per mm2 onto six-well culture plates (Corning) or glass slips with eagle minimum essential medium containing 10% heat-inactivated fetal calf serum. In order to induce cardiomyocyte apoptosis, hydrogen peroxide (H2O2, 100 µM) was added after 24 h, and incubated for 20 h with or without simvastatin pretreatment for 1 h [31]. To detect TUNEL-positive cells, an in situ apoptosis detection kit (R&D System Inc.) was used. 2.7. Caspase-3 activity assay Caspase-3 activity was determined by using caspase-3 colorimetric assay kit (R&D System Inc.) according to the manufacturer’s directions. Briefly, the cell lysate was tested for protease activity by addition of a caspase-specific peptide that is conjugated to the color reporter molecule p-nitroanline (pNA). The cleavage of the peptide by the caspase releases the chromophore pNA, which can be quantitated spectrophyotometrically at a wavelength of 405 nm. The level of caspase enzymatic activity in the cell lysate is directly proportional to the color reaction.
3. Results 3.1. Ventricular remodeling and morphology
3.2. Hemodynamics There was no significant change of hemodynamic indexes between 6, 12, and 20 weeks after operation in the sham rats. In rats with AS, AS induced a significant increase of LVSP, and maximal rate of pressure rise and decline (LV +/–dP/dt) without a significant change of LVEDP and lung wet weight compared with sham rats at 6 or 12 weeks (Fig. 1A–E), which indicates that the heart is in a compensatory stage. At 20 weeks after operation, AS resulted in a markedly higher level of LVEDP and lung wet weight, with a concomitant enhanced ANP mRNA expression and decrease of LVSP and LV +/–dP/dt in rats with AS, which reflects the existence of congestive heart failure (Fig. 1B–F).
2.8. Statistical analysis
3.3. Effects of simvastatin treatment after compensatory hypertrophic stage on ventricular function in failing heart
Data were presented as mean ± S.E.M. Experimental means were subjected to one-way ANOVA with Newman–
In order to investigate the positive effects of simvastatin on the transition of cardiac hypertrophy to failure, simvasta-
Table 1 Morphological changes during the development of cardiac hypertrophy and heart failure in rats with AS n
BW (g)
HW/BW (g kg–1)
LVW (g)
TLVW (cm)
IVD (cm)
LVW/RVW (g g–1)
1.06 ± 0.09 1.32 ± 0.13 1.20 ± 0.11
3.25 ± 0.13 2.97 ± 0.35 2.80 ± 0.59
0.63 ± 0.08 0.83 ± 0.11 0.79 ± 0.09
0.29 ± 0.02 0.30 ± 0.01 0.30 ± 0.04
0.30 ± 0.01 0.32 ± 0.01 0.27 ± 0.04
1.92 ± 0.05 1.87 ± 0.29 1.84 ± 0.13
1.18 ± 0.10
2.85 ± 0.61
0.81 ± 0.10
0.29 ± 0.04
0.28 ± 0.04
1.86 ± 0.12
1.11 ± 0.07 1.68 ± 0.15 † 1.71 ± 0.25 ‡
3.22 ± 0.13 3.70 ± 0.20 † 3.72 ± 0.32 ‡
0.64 ± 0.06 1.17 ± 0.14 † 1.25 ± 0.23 ‡
0.30 ± 0.01 0.35 ± 0.02 † 0.38 ± 0.05 ‡
0.29 ± 0.01 0.37 ± 0.02 † 0.36 ± 0.03 ‡
1.86 ± 0.14 2.57 ± 0.22 † 2.69 ± 0.37 ‡
1.49 ± 0.25 §
3.63 ± 0.48
1.08 ± 0.21 §
0.35 ± 0.06 §
0.31 ± 0.04 §
2.64 ± 0.53 §
aBP (mmHg) HW (g)
Sham group 6 weeks 8 326.38 ± 39.92 105 ± 7.8 12 weeks 10 444.00 ± 31.37 125 ± 5.8 20 weeks 13 431.08 ± 44.64 133 ± 6.2 Sham + simvastatin (2.0 mg kg–1 per day) for 8 weeks 20 weeks 13 426.16 ± 42.53 131 ± 5.2 AS group 6 weeks 9 344.22 ± 30.27 118 ± 1.4 * 12 weeks 10 453.10 ± 26.77 139 ± 4.3 † ‡ 20 weeks 11 458.91 ± 34.39 122 ± 4.6 ‡ AS + simvastatin (2.0 mg kg–1 per day) for 8 weeks 20 weeks 12 412.92 ± 71.08 127 ± 5.9 §
BW = body weight; aBP = average blood pressure; HW = heart weight; LVW = left ventricular weight; TLVW = thickness of left ventricular wall; TIVW = thickness of interventricular wall; RVW = right ventricular weight. Values were presented as mean ± S.E.M. * P< 0.05 vs. Sham 6 weeks.† P < 0.05 vs. Sham 12 weeks.‡ P < 0.05 vs. Sham 20 weeks.§ P < 0.05 vs. AS 20 weeks.
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Fig. 1. Effects of simvastatin on LV histological change (A), ANP mRNA expression (B), LV systolic pressure (C), LV end-diastolic pressure (D), LV +/–dP/dtmax (E and F), and lung wet weight (G) in rats with AS. Cardiac hemodynamics was assessed at the end of 6, 12, and 20 weeks after aortic coarctation and simvastatin (2 mg kg–1 per day) was administered by gavage once a day for 8 weeks, beginning 13 weeks after aortic coarctation. ANP mRNA was analyzed by RT-PCR at the end of 20 weeks after aortic coarctation. Values were presented as mean ± S.E.M. Sham = sham-operation rats; Sham + Sim = sham-operation + simvastatin treatment rats; AS = aortic stenosis rats; AS + Sim = aortic stenosis + simvastatin treatment rats. n = 8–13, * P < 0.05 vs. sham, # P < 0.05 vs. AS.
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Fig. 1 (continued)
tin (2.0 mg kg–1 per day) was administrated in rats with AS at 13 weeks after operation, at which point the rats have developed significant cardiac hypertrophy. Treatment with simvastatin for 8 weeks in rats with AS significantly improved the LV function, concomitant with inhibition of enhanced ANP mRNA expression induced by AS compared to those without simvastatin treatment (Fig. 1A–E, Table 1), indicating that statins treatment after compensatory hypertrophic stage is still effective for preventing transition of hypertrophy to heart failure.
compared with sham rats at 12 and 20 weeks after operation (Fig. 3) although there was no significant difference of cardiomyocyte apoptosis in LV tissues between the rats with AS and sham rats at 6 week after operation. After a compensatory hypertrophic stage in rats with AS, treatment with simvastatin in vivo markedly inhibited cardiomyocyte apoptosis
3.4. Effects of simvastatin on oxidative stress in failing heart There was no significant difference of superoxide levels in LV tissues between the rats with AS and sham rats at 6 weeks after operation. However, superoxide levels in LV tissues of rats with AS were significant increase at 12 and 20 weeks after operation compared with sham rats at 12 and 20 weeks after operation (Fig. 2). In vivo simvastatin treatment initiated after compensatory hypertrophic stage in rats with AS markedly decreased the superoxide levels in LV tissues compared to those without simvastatin treatment (Fig. 2). 3.5. Effects of simvastatin on apoptosis in failing heart In rats with AS, cardiomyocyte apoptosis in LV tissues at 12 and 20 weeks after operation was significantly increased
Fig. 2. Effects of simvastatin on superoxide levels in LV tissues of rats with AS. Superoxide levels in LV tissues were assessed by lucigenin-enhanced chemiluminescence at the end of 6, 12, and 20 weeks after aortic coarctation and simvastatin (2 mg kg–1 per day) was administered by gavage once a day for 8 weeks, beginning 13 weeks after aortic coarctation. Values were presented as mean ± S.E.M. Sham = sham-operation rats; AS = aortic stenosis rats; AS + Sim = aortic stenosis + simvastatin treatment rats. n = 8–13, * P < 0.05 vs. sham, # P < 0.05 vs. AS.
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Fig. 3. Effects of simvastatin on cardiomyocytes apoptosis in LV tissues of rats with AS. Cardiomyocyte apoptosis in LV tissues were assessed by TUNEL staining assay at the end of 6, 12, and 20 weeks after aortic coarctation. Simvastatin (2 mg kg–1 per day) was administered by gavage once a day for 8 weeks, beginning 13 weeks after aortic coarctation. Values were presented as mean ± S.E.M. Sham = sham-operation rats; AS = aortic stenosis rats; AS + Sim = aortic stenosis + simvastatin treatment rats. n = 8–13, * P < 0.05 vs. sham, # P < 0.05 vs. AS.
in LV tissues compared to those without simvastatin treatment (Fig. 3). 3.6. Inhibitory effects of simvastatin on cardiomyocyte apoptosis induced by hydrogen peroxide in vitro Since it is difficult to distinguish a causal relationship of simvastatin treatment in vivo on the inhibition of oxidative stress and cardiomyocyte apoptosis, we examined the effect of simvastatin on hydrogen peroxide (H2O2, 100 µM)induced cardiomyocyte apoptosis using cultured cardiomyocytes. There was no significant difference in the number of apoptotic cardiomyocytes between cells treated with or without simvastatin (1–100 nM) (data not shown). Treatment with H2O2 for 20 h markedly increased the number of TUNEL staining positive cardiomyocytes and caspase-3 activity of cultured cardiomyocytes (Fig. 4A, B). Pretreatment with simvastatin for 1 h dose-dependently inhibited H2O2induced cardiomyocyte apoptosis and the augment of caspase-3 activity (Fig. 4A, B).
4. Discussion The present study demonstrates, for the first time, that in rats with AS: (1) aortic coarctation induced a progressive increase of oxidative stress and cardiomyocyte apoptosis in LV tissues, ultimately resulting in heart failure, and (2) longterm administration of HMG-CoA reductase inhibitor simvastatin, initiated from the compensatory hypertrophic stage, reduced oxidative stress and cardiomyocyte apoptosis, improved LV function, and prevented transition of hypertrophy to heart failure. CHF is a complex syndrome that consist not only of hemodynamic abnormalities but also of metabolic and neu-
Fig. 4. Effects of simvastatin on hydrogen peroxide (H2O2) induced cardiomyocyte apoptosis in cultured cardiomyocytes from neonatal rats. After 20 h H2O2 (100 µM) incubation with or without simvastatin (1–100 nM) pretreatment for 1 h, cardiomyocytes apoptosis (A) and caspase-3 activity (B) in cultured cardiomyocytes were assessed by TUNEL staining assay or by caspase-3 colorimetric assay. Values were presented as mean ± S.E.M. n = 5, * P < 0.05 vs. control, # P < 0.05 vs. H2O2.
rohormal alterations. Although hemodynamic stress in heart failure may play a role in the disease process, it is now believed that many other mechanisms, such as oxidative stress, inflammation, and endothelial dysfunction due to the activation of neurohormones and proinflammatory cytokines such as tumor necrosis factor-a, interleukin-1, and C-reactive protein (CRP) may be more important in mediating the progression of heart failure [32]. Cardiomyocyte loss by apoptosis has also been recognized as a potential cause of heart failure [33]. One of the novel findings of the present study was that myocardial oxidative stress was progressive increase, concomitant with cardiomyocyte apoptosis and LV ventricular dysfunction in the development process of cardiac hypertrophy and heart failure in rats with overload pressure. These results are consistent with data from both clinical and animal studies that the failing heart is subjected to increased oxidative stress and cardiomyocyte apoptosis [18–22,34]. These results might suggest that low levels ROS induces early cardiac hypertrophic response and higher levels of ROS results in later myocyte apoptotic response, which is an important factor for the transition of cardiac hypertrophy to heart failure. This assumption is supported by data from
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previous studies indicating that antioxidants can inhibit early hypertrophic responses and improve ventricular function in heart failure [18,21,24]. The intracellular mechanism that distinguishes the hypertrophic response elicited by low levels of ROS from the apoptotic response elicited by higher levels remains to be defined, but probably involves differences in the type, quantity and/or duration of ROS production [23]. ROS may be derived from mitochondrial electron transport chain, NADH/NADPH oxidase, xanthine oxidase, nitric oxide synthase, and/or cyclooxygenase [18–22]. Maladaptive changes in antioxidant enzyme activity also contribute to the oxidative stress of the failing heart. Oxidative stress damages the cardiomyocytes and extracellular matrix through lipid peroxidation and oxidative modification of cellular proteins and DNA. Earlier studies have shown that the increase of ROS is capable of inducing apoptosis in cultured cardiomyocytes and cardiac dysfunction in an intact heart [35]. The importance of ROS formation in cardiomyocyte apoptosis is further supported by the findings that a number of antioxidants can reduce or block cardiomyocyte apoptosis in heart failure [18,21,24]. Very recent data has demonstrated that very low levels of cardiomyocyte apoptosis (23 myocytes per 105 nuclei, compared with 1.5 myocytes per 105 nuclei in controls) are sufficient to cause a lethal, dilated cardiomyopathy and inhibition of cardiac cardiomyocyte death largely prevents the development of cardiac dilation and contractile dysfunction, the hallmarks of heart failure in FK-binding-protein–caspase-8 transgenic mice [29]. These data provide the evidence that oxidative stress and cardiomyocyte apoptosis may be important mechanisms of heart failure. Recently, several animal studies have shown that statins therapy can prevent the development of heart failure through preservation of NO production, normalization of sympathetic nerve function, and inhibition of oxidative stress [9–11,15–17]. However, it is unknown whether statins therapy initiated after compensatory hypertrophic stage is still effective in preventing the transition of hypertrophy to CHF since the statins treatment in previous studies were initiated before the abnormalities were manifest. So it is very important to elucidate the cardioprotective effects of statins treatment initiated after the onset of abnormalities to imitate a situation close to clinical practice. In the present study, our results demonstrated that long-term administration of simvastatin initiated from 13 weeks after aortic coarctation improved LV function and prevented the transition of hypertrophy to heart failure. This is the first study to evaluate the effects of statins initiated after compensatory hypertrophic stage on the transition of hypertrophy to CHF and thus the first to show beneficial effects in this setting. The mechanisms by which statins exerted beneficial effects in this setting are not completely clear. Treatment with simvastatin initiated after the compensatory hypertrophic stage reduced the superoxide levels in LV tissues and myocytes apoptosis. Several recent studies have shown that simvastatin is able to lessen oxidative stress by inhibiting
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NADPH oxidase activity and improving catalase activity of myocardium [36,37]. Other study indicated that simvastatin protected the cardiomyocytes against the mitochondrial dysfunction induced by H2O2 in vitro [38]. Taken together, these data suggest that oxidative stress may be a causal mechanism of heart failure and simvastatin may have exerted its beneficial effects via its antioxidant property. However, we can not exclude the possibility that decrease of ROS and apoptosis in myocardium treated with simvastatin is due to improved LV function secondary to other mechanisms such as preservation of nitric oxide function [11], inhibition of inflammation [39,40], and normalization of augmented sympathetic outflow [15] and this issue need to be further investigated. In summary, the present study demonstrates, for the first time, long-term administration of simvastatin improved LV function and prevented the transition of hypertrophy to CHF. Inhibition of oxidative stress and cardiomyocyte apoptosis may contribute to the benefits of simvastatin treatment on heart of rats with AS. Statins may be an effective means in improving heart function in CHF.
Acknowledgments This study was supported by grants from the Guangzhou Education Committee and the Education Ministry of the People’s Republic of China. The authors would like to thank Alicia DeMarco (Michigan State University, MI, USA) for her editorial assistance with manuscript.
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[26] Mulder P, Boujedaini H, Richard V, Henry JP, Renet S, Munter K, et al. Long-term survival and hemodynamics after endothelin-a receptor antagonism and angiotensin-converting enzyme inhibition in rats with chronic heart failure: monotherapy versus combination therapy. Circulation 2002;106:1159–64. [27] Xu FP, Chen MS, Wang YZ, Yi Q, Lin SB, Chen AF, et al. Leptin induces hypertrophy via endothelin-1 reactive oxygen species pathway in cultured neonatal rat cardiomyocytes. Circulation 2004; [in press]. [28] Zou AP, Li N, Cowley Jr AW. Production and actions of superoxide in the renal medulla. Hypertension 2001;37:547–53. [29] Wencker D, Chandra M, Nguyen K, Miao W, Garantziotis S, Factor SM, et al. A mechanistic role for cardiac myocyte apoptosis in heart failure. J Clin Invest 2003;111:1497–504. [30] Luo JD, Xie F, Zhang WW, Ma XD, Guan JX, Chen X. Simvastatin inhibits noradrenaline-induced hypertrophy of cultured neonatal rat cardiomyocytes. Br J Pharmacol 2001;132:159–64. [31] Chen HW, Chien CT, Yu SL, Lee YT, Chen WJ. Cyclosporine A regulate oxidative stress-induced apoptosis in cardiomyocytes: mechanisms via ROS generation, iNOS and Hsp70. Br J Pharmacol 2002;137:771–81. [32] Vasan RS, Sullivan LM, Roubenoff R, Dinarello CA, Harris T, Benjamin EJ, et al. Framingham Heart Study. Inflammatory markers and risk of heart failure in elderly subjects without prior myocardial infarction: the Framingham Heart Study. Circulation 2003;107:1486– 91. [33] Kang PM, Izumo S. Apoptosis and heart failure: a critical review of the literature. Circ Res 2000;86:1107–13. [34] Kang PM, Izumo S. Apoptosis and heart failure: a critical review of the literature. Circ Res 2000;86:1107–13. [35] Ide T, Tsutsui H, Hayashidani S, Kang D, Suematsu N, Nakamura Ki, et al. Mitochondrial DNA damage and dysfunction associated with oxidative stress in the failing hearts after myocardial infarction. Circ Res 2001;88:529–35. [36] Takemoto M, Node K, Nakagami H, Liao Y, Grimm M, Takemoto Y, et al. Statins as antioxidant therapy for preventing cardiac myocyte hypertrophy. J Clin Invest 2001;108:1429–37. [37] Luo JD, Zhang WW, Zhang GP, Zhong BH, Ou HJ. Effects of simvastatin on activities of endogenous antioxidant enzymes and angiotensin-converting enzyme in rat myocardium with pressureoverload cardiac hypertrophy. Acta Pharmacol Sin 2002;23:124–8. [38] Jones SP, Teshima Y, Akao M, Marban E. Simvastatin attenuates oxidant-induced mitochondrial dysfunction in cardiac myocytes. Circ Res 2003;93:697–9. [39] Plenge JK, Hernandez TL, Weil KM, Poirier P, Grunwald GK, Marcovina SM, et al. Simvastatin lowers C-reactive protein within 14 days: an effect independent of low-density lipoprotein cholesterol reduction. Circulation 2002;106:1447–52. [40] Albert MA, Danielson E, Rifai N, Ridker PM, PRINCE Investigators. Effect of statin therapy on C-reactive protein levels: the pravastatin inflammation/CRP evaluation (PRINCE): a randomized trial and cohort study. J Am Med Assoc 2001;286:64–70.
Original Article
Statins initiated after hypertrophy inhibit oxidative stress and prevent heart failure in rats with aortic stenosis Min-Sheng Chen a,b,1, Fang-Ping Xu a,1, Yan-Zhen Wang a,c, Gui-Ping Zhang a, Quan Yi a, Hui-Qiu Zhang d, Jian-Dong Luo a,* a Department of Pharmacology, Guangzhou Medical College, Guangzhou 510182, China Department of Internal Medicine, Guangzhou Medical College, Guangzhou 510182, China c Department of Research Laboratory of Pathophysiology, PLA General Hospital, Beijing 100853, China d Department of Pathology, Guangzhou Medical College, Guangzhou 510182, China b
Received 4 March 2004; received in revised form 21 June 2004; accepted 24 June 2004 Available online 11 August 2004
Abstract Objective. – Heart failure is a major and escalating public health problem. Recent studies have demonstrated that statins prevented chronic heart failure (CHF) in animal studies. However, it is unknown whether statins therapy initiated after left ventricular (LV) hypertrophy is evident can still effectively prevent CHF. This study tested the hypothesis that statins can prevent the transition of hypertrophy to heart failure. Methods and results. – The rats were studied at 6, 12, and 20 weeks after aortic stenosis (AS) operation. Some rats were given simvastatin (2.0 mg kg–1 per day) from 13 weeks after AS operation for 8 weeks. Coarctation of aorta in rats resulted in compensatory LV hypertrophy (LVH), concomitant with an increase of superoxide levels and cardiomyocyte apoptosis in LV tissues at 12 weeks after AS operation. This was followed by CHF with a progressive increase in superoxide levels and cardiomyocyte apoptosis in LV tissues at 20 weeks after AS operation. Simvastatin treatment initiated from 13 weeks after AS operation significantly improved LV function and reduced superoxide levels and cardiomyocyte apoptosis in LV tissues. Pretreatment of simvastatin suppressed the hydrogen peroxide-induced apoptosis of cultured cardiomyocytes from neonatal rats. Conclusions. – These data indicate that long-term administration of simvastatin improved LV function and prevented the transition of hypertrophy to CHF. Inhibition of oxidative stress and cardiomyocyte apoptosis may contribute to the benefits of simvastatin treatment on heart of rats with AS. © 2004 Elsevier Ltd. All rights reserved. Keywords: Apoptosis; Cardiac hypertrophy; Heart failure; Superoxide; Statins
1. Introduction CHF is a clinical syndrome characterized by progressive left ventricular (LV) systolic and/or diastolic dysfunction. The incidence doubles with each decade of life, and CHF is the most common hospital discharge diagnosis in individuals over age 65. Unlike most other cardiovascular diseases, the incidence and morbidity/mortality from CHF have not decreased over the last two decades and the prevalence is expected to double in the next 40 years [1,2]. Thus CHF is a * Corresponding author. Tel.: +86-20-8134-0203; fax: +86-20-8134-1922. E-mail address: [email protected] (J.-D. Luo). 1 These authors contributed equally to this work. 0022-2828/$ - see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2004.06.019
major medical problem which is expected to become more significant with time [3,4]. Therefore, it is very important to develop the new therapeutic strategies for CHF. Hydroxymethylglutaryl-coenzyme A reductase inhibitors, known as “statins”, are proven to be clinical benefit in coronary heart disease, at least in those patients who do not have overt CHF [5–8]. The beneficial potential of statins clearly relates to their inhibition of oxidative stress and proinflammatory cytokine activity, improvements in endothelial function, properties of plaque stabilization, and favorable modulation of the autonomic nervous system besides lipid-lowing effect [9–15]. Recent results from our laboratory and others have demonstrated that cardiac hypertrophy and heart failure could be prevented by statins initiated at an
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early stage when LV hypertrophy (LVH) and dysfunction were not evident in animal study [9,16,17]. However, there is very little data pertaining to the effects of pharmacological interventions with statins initiated after LVH and LV dysfunction are promoted. It is very important to elucidate cardioprotective effects of statins treatment initiates after the on-set of abnormalities as to recreate a situation that is to most clinical situations. Experimental evidence in animal models of myocardial remodeling and failure demonstrate that the failing heart is subjected to increased oxidative stress. The reactive oxygen species (ROS) may be derived from mitochondrial electron transport chain, NADH/NADPH oxidase, xanthine oxidase, nitric oxide synthase, and/or cyclooxygenase [18–22]. At a cellular level, oxidative stress can induce most of the changes that are thought to contribute to myocardial remodelingmyocyte hypertrophy, apoptosis, fetal gene expression and increased matrix metalloproteinase activity [23]. The results from both animal and human studies suggest that cardiomyocyte apoptosis plays a crucial role as the underlying mechanism being at least in part responsible for ventricular dysfunction [18,19,23]. Oxidative stress has been shown to be involved in cardiomyocyte apoptosis [24,25]. Antioxidant therapy has been demonstrated to inhibit cardiomyocyte apoptosis and preserve LV function during development of CHF [18,24]. Therefore, oxidative stress may play an important role in the pathogenesis and progression of CHF. However, direct evidence regarding kinetics of oxidative stress, apoptosis, and ventricular dysfunction during the transition of hypertrophy to heart failure has not been obtained. In the present study, we determined the time course of the oxidative stress and LV dysfunction after aortic coarctation and the overall effectiveness of simvastatin treatment initiated after LVH and dysfunction in preventing the transition of hypertrophy to CHF in rats with chronic pressure overload-induced CHF. Our results demonstrated that in rats with AS: (1) aortic coarctation induced a progressive increase of oxidative stress and cardiomyocyte apoptosis in LV tissues, ultimately resulting in heart failure, and (2) longterm administration of HMG-CoA reductase inhibitor simvastatin, initiated from the compensatory hypertrophic stage, reduced oxidative stress and cardiomyocyte apoptosis, improved LV function, and prevented transition of hypertrophy to heart failure.
2. Methods 2.1. Animals and experimental protocol This study was approved by our institutional animal research committee and conformed to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH publication No. 85-23, revised 1996). The male Sprague–Dawley rats, 3–4 weeks and 60–70 g in body weight (BW), were obtained
from Medical Laboratory Animal Center (Guangdong, China). The cardiac hypertrophy-failure of rats was induced by partial aortic coarctation according to our previous study [16]. The rats with AS were studied in the time points of 6, 12, and 20 weeks after aortic coarctation. Sham-operated rats served as age-matched controls. Simvastatin (2 mg kg–1 per day) was administered by gavage once a day for 8 weeks, beginning 13 weeks after aortic coarctation. The dose of simvastatin was determined according to our previous study [16]. 2.2. Hemodynamic measurements Cardiac hemodynamics were assessed at the end of 6, 12, and 20 weeks after aortic coarctation. In brief, the rats were anesthetized (pentobarbital; 50 mg kg–1, i.p.) and the right carotid artery was cannulated with a micromanometer-tipped catheter (SPR 407, Millar Instruments Inc., Houston, TX, USA) for recording of arterial pressure [26]. The catheter was then advanced into the left ventricle (LV) for recording of LV pressure and its maximal rate of rise (+dP/dtmax) and decrease (–dP/dtmax) [26]. 2.3. Reverse transcription-PCR (RT-PCR) Total RNA was isolated from LV tissues using TRIzol reagent (Invitrogen). The atrial natriuretic peptide (ANP) mRNA was analyzed by RT-PCR using primers specific for ANP (sense: 5′-GGGCTCCTTCTCCATCACC-3′, and antisense: 5′-CTCCAATCCTGTCAATCCTACC-3′) [27]. ANP PCR amplification was performed for 27 cycles at 94 °C for 20 s, 55 °C for 15 s, and 72 °C for 30 s. The amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, a constitutively and ubiquitously expressed gene, served as an internal standard for RT-PCR analysis. A sense primer 5′-AAGGTCGGTGTCAACCCATTTGGCCGT-3′ and antisense primer 5′-CAGTGATGGCATCCACTGTG GTC-3′ were used. Amplification was performed over 23 cycles, each involving 1 min at 94 °C, 1.5 min at 59 °C, and 2 min at 72 °C. 2.4. Superoxide measurement Myocardial superoxide levels were determined by fluorescence spectrometry according to the previous study [28]. Briefly, the fluorogenic oxidation of dihydroethidine (DHE) to ethidium (Eth) was used as a measure of O2–. Tissue homogenates were incubated with DHE (10 µmol/l) in a microtiter plate at 37 °C for 30 min, and Eth-DNA fluorescence was measured at an excitation of 475 nm and an emission of 610 nm with a fluorescence microplate reader (Bio-Tec, American). The fluorescent signal intensity reflects O2– levels. 2.5. In situ apoptosis detection The apoptosis in heart tissue sections and cultured cells was analyzed using TUNEL staining assay [29]. The TUNEL
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reaction was performed using a TdT in situ apoptosis detection kit—TACS™ blue label (R&D System Inc., MN, USA) according to the manufacturer’s directions. The apoptotic (TUNEL-positive) cardiomyocyte nucleus was stained as blue color and the number of TUNEL-positive cardiomyocyte nuclei was counted. Five sections were analyzed for each animal and five fields were observed for each section.
Keuls Multiple Comparison Test. A P value of less than 0.05 was considered statistically significant.
2.6. Cell culture
During the first 2 weeks after AS, nine of 51 rats with AS died. Thereafter, no rat died in any of the groups until the end of the experimental period. There was no significant change of morphological indexes in rats with AS compared with sham rats at 6 weeks after operation. However, AS induced a significant increase in heart weight (HW), LV weight, LV depth, interventricular depth and the ratios of HW/BW, LVW/BW, and LVW/right ventricular weight (RVW) in rats with AS compared with sham rats at 12 or 20 weeks after operation (Table 1, Fig. 1A).
Primary cultures of cardiomyocytes from the ventricles of neonatal Sprague–Dawley rats were prepared according to the previous studies [30]. Cardiomyocytes were plated at a density of 1 × 103 cells per mm2 onto six-well culture plates (Corning) or glass slips with eagle minimum essential medium containing 10% heat-inactivated fetal calf serum. In order to induce cardiomyocyte apoptosis, hydrogen peroxide (H2O2, 100 µM) was added after 24 h, and incubated for 20 h with or without simvastatin pretreatment for 1 h [31]. To detect TUNEL-positive cells, an in situ apoptosis detection kit (R&D System Inc.) was used. 2.7. Caspase-3 activity assay Caspase-3 activity was determined by using caspase-3 colorimetric assay kit (R&D System Inc.) according to the manufacturer’s directions. Briefly, the cell lysate was tested for protease activity by addition of a caspase-specific peptide that is conjugated to the color reporter molecule p-nitroanline (pNA). The cleavage of the peptide by the caspase releases the chromophore pNA, which can be quantitated spectrophyotometrically at a wavelength of 405 nm. The level of caspase enzymatic activity in the cell lysate is directly proportional to the color reaction.
3. Results 3.1. Ventricular remodeling and morphology
3.2. Hemodynamics There was no significant change of hemodynamic indexes between 6, 12, and 20 weeks after operation in the sham rats. In rats with AS, AS induced a significant increase of LVSP, and maximal rate of pressure rise and decline (LV +/–dP/dt) without a significant change of LVEDP and lung wet weight compared with sham rats at 6 or 12 weeks (Fig. 1A–E), which indicates that the heart is in a compensatory stage. At 20 weeks after operation, AS resulted in a markedly higher level of LVEDP and lung wet weight, with a concomitant enhanced ANP mRNA expression and decrease of LVSP and LV +/–dP/dt in rats with AS, which reflects the existence of congestive heart failure (Fig. 1B–F).
2.8. Statistical analysis
3.3. Effects of simvastatin treatment after compensatory hypertrophic stage on ventricular function in failing heart
Data were presented as mean ± S.E.M. Experimental means were subjected to one-way ANOVA with Newman–
In order to investigate the positive effects of simvastatin on the transition of cardiac hypertrophy to failure, simvasta-
Table 1 Morphological changes during the development of cardiac hypertrophy and heart failure in rats with AS n
BW (g)
HW/BW (g kg–1)
LVW (g)
TLVW (cm)
IVD (cm)
LVW/RVW (g g–1)
1.06 ± 0.09 1.32 ± 0.13 1.20 ± 0.11
3.25 ± 0.13 2.97 ± 0.35 2.80 ± 0.59
0.63 ± 0.08 0.83 ± 0.11 0.79 ± 0.09
0.29 ± 0.02 0.30 ± 0.01 0.30 ± 0.04
0.30 ± 0.01 0.32 ± 0.01 0.27 ± 0.04
1.92 ± 0.05 1.87 ± 0.29 1.84 ± 0.13
1.18 ± 0.10
2.85 ± 0.61
0.81 ± 0.10
0.29 ± 0.04
0.28 ± 0.04
1.86 ± 0.12
1.11 ± 0.07 1.68 ± 0.15 † 1.71 ± 0.25 ‡
3.22 ± 0.13 3.70 ± 0.20 † 3.72 ± 0.32 ‡
0.64 ± 0.06 1.17 ± 0.14 † 1.25 ± 0.23 ‡
0.30 ± 0.01 0.35 ± 0.02 † 0.38 ± 0.05 ‡
0.29 ± 0.01 0.37 ± 0.02 † 0.36 ± 0.03 ‡
1.86 ± 0.14 2.57 ± 0.22 † 2.69 ± 0.37 ‡
1.49 ± 0.25 §
3.63 ± 0.48
1.08 ± 0.21 §
0.35 ± 0.06 §
0.31 ± 0.04 §
2.64 ± 0.53 §
aBP (mmHg) HW (g)
Sham group 6 weeks 8 326.38 ± 39.92 105 ± 7.8 12 weeks 10 444.00 ± 31.37 125 ± 5.8 20 weeks 13 431.08 ± 44.64 133 ± 6.2 Sham + simvastatin (2.0 mg kg–1 per day) for 8 weeks 20 weeks 13 426.16 ± 42.53 131 ± 5.2 AS group 6 weeks 9 344.22 ± 30.27 118 ± 1.4 * 12 weeks 10 453.10 ± 26.77 139 ± 4.3 † ‡ 20 weeks 11 458.91 ± 34.39 122 ± 4.6 ‡ AS + simvastatin (2.0 mg kg–1 per day) for 8 weeks 20 weeks 12 412.92 ± 71.08 127 ± 5.9 §
BW = body weight; aBP = average blood pressure; HW = heart weight; LVW = left ventricular weight; TLVW = thickness of left ventricular wall; TIVW = thickness of interventricular wall; RVW = right ventricular weight. Values were presented as mean ± S.E.M. * P< 0.05 vs. Sham 6 weeks.† P < 0.05 vs. Sham 12 weeks.‡ P < 0.05 vs. Sham 20 weeks.§ P < 0.05 vs. AS 20 weeks.
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Fig. 1. Effects of simvastatin on LV histological change (A), ANP mRNA expression (B), LV systolic pressure (C), LV end-diastolic pressure (D), LV +/–dP/dtmax (E and F), and lung wet weight (G) in rats with AS. Cardiac hemodynamics was assessed at the end of 6, 12, and 20 weeks after aortic coarctation and simvastatin (2 mg kg–1 per day) was administered by gavage once a day for 8 weeks, beginning 13 weeks after aortic coarctation. ANP mRNA was analyzed by RT-PCR at the end of 20 weeks after aortic coarctation. Values were presented as mean ± S.E.M. Sham = sham-operation rats; Sham + Sim = sham-operation + simvastatin treatment rats; AS = aortic stenosis rats; AS + Sim = aortic stenosis + simvastatin treatment rats. n = 8–13, * P < 0.05 vs. sham, # P < 0.05 vs. AS.
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Fig. 1 (continued)
tin (2.0 mg kg–1 per day) was administrated in rats with AS at 13 weeks after operation, at which point the rats have developed significant cardiac hypertrophy. Treatment with simvastatin for 8 weeks in rats with AS significantly improved the LV function, concomitant with inhibition of enhanced ANP mRNA expression induced by AS compared to those without simvastatin treatment (Fig. 1A–E, Table 1), indicating that statins treatment after compensatory hypertrophic stage is still effective for preventing transition of hypertrophy to heart failure.
compared with sham rats at 12 and 20 weeks after operation (Fig. 3) although there was no significant difference of cardiomyocyte apoptosis in LV tissues between the rats with AS and sham rats at 6 week after operation. After a compensatory hypertrophic stage in rats with AS, treatment with simvastatin in vivo markedly inhibited cardiomyocyte apoptosis
3.4. Effects of simvastatin on oxidative stress in failing heart There was no significant difference of superoxide levels in LV tissues between the rats with AS and sham rats at 6 weeks after operation. However, superoxide levels in LV tissues of rats with AS were significant increase at 12 and 20 weeks after operation compared with sham rats at 12 and 20 weeks after operation (Fig. 2). In vivo simvastatin treatment initiated after compensatory hypertrophic stage in rats with AS markedly decreased the superoxide levels in LV tissues compared to those without simvastatin treatment (Fig. 2). 3.5. Effects of simvastatin on apoptosis in failing heart In rats with AS, cardiomyocyte apoptosis in LV tissues at 12 and 20 weeks after operation was significantly increased
Fig. 2. Effects of simvastatin on superoxide levels in LV tissues of rats with AS. Superoxide levels in LV tissues were assessed by lucigenin-enhanced chemiluminescence at the end of 6, 12, and 20 weeks after aortic coarctation and simvastatin (2 mg kg–1 per day) was administered by gavage once a day for 8 weeks, beginning 13 weeks after aortic coarctation. Values were presented as mean ± S.E.M. Sham = sham-operation rats; AS = aortic stenosis rats; AS + Sim = aortic stenosis + simvastatin treatment rats. n = 8–13, * P < 0.05 vs. sham, # P < 0.05 vs. AS.
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Fig. 3. Effects of simvastatin on cardiomyocytes apoptosis in LV tissues of rats with AS. Cardiomyocyte apoptosis in LV tissues were assessed by TUNEL staining assay at the end of 6, 12, and 20 weeks after aortic coarctation. Simvastatin (2 mg kg–1 per day) was administered by gavage once a day for 8 weeks, beginning 13 weeks after aortic coarctation. Values were presented as mean ± S.E.M. Sham = sham-operation rats; AS = aortic stenosis rats; AS + Sim = aortic stenosis + simvastatin treatment rats. n = 8–13, * P < 0.05 vs. sham, # P < 0.05 vs. AS.
in LV tissues compared to those without simvastatin treatment (Fig. 3). 3.6. Inhibitory effects of simvastatin on cardiomyocyte apoptosis induced by hydrogen peroxide in vitro Since it is difficult to distinguish a causal relationship of simvastatin treatment in vivo on the inhibition of oxidative stress and cardiomyocyte apoptosis, we examined the effect of simvastatin on hydrogen peroxide (H2O2, 100 µM)induced cardiomyocyte apoptosis using cultured cardiomyocytes. There was no significant difference in the number of apoptotic cardiomyocytes between cells treated with or without simvastatin (1–100 nM) (data not shown). Treatment with H2O2 for 20 h markedly increased the number of TUNEL staining positive cardiomyocytes and caspase-3 activity of cultured cardiomyocytes (Fig. 4A, B). Pretreatment with simvastatin for 1 h dose-dependently inhibited H2O2induced cardiomyocyte apoptosis and the augment of caspase-3 activity (Fig. 4A, B).
4. Discussion The present study demonstrates, for the first time, that in rats with AS: (1) aortic coarctation induced a progressive increase of oxidative stress and cardiomyocyte apoptosis in LV tissues, ultimately resulting in heart failure, and (2) longterm administration of HMG-CoA reductase inhibitor simvastatin, initiated from the compensatory hypertrophic stage, reduced oxidative stress and cardiomyocyte apoptosis, improved LV function, and prevented transition of hypertrophy to heart failure. CHF is a complex syndrome that consist not only of hemodynamic abnormalities but also of metabolic and neu-
Fig. 4. Effects of simvastatin on hydrogen peroxide (H2O2) induced cardiomyocyte apoptosis in cultured cardiomyocytes from neonatal rats. After 20 h H2O2 (100 µM) incubation with or without simvastatin (1–100 nM) pretreatment for 1 h, cardiomyocytes apoptosis (A) and caspase-3 activity (B) in cultured cardiomyocytes were assessed by TUNEL staining assay or by caspase-3 colorimetric assay. Values were presented as mean ± S.E.M. n = 5, * P < 0.05 vs. control, # P < 0.05 vs. H2O2.
rohormal alterations. Although hemodynamic stress in heart failure may play a role in the disease process, it is now believed that many other mechanisms, such as oxidative stress, inflammation, and endothelial dysfunction due to the activation of neurohormones and proinflammatory cytokines such as tumor necrosis factor-a, interleukin-1, and C-reactive protein (CRP) may be more important in mediating the progression of heart failure [32]. Cardiomyocyte loss by apoptosis has also been recognized as a potential cause of heart failure [33]. One of the novel findings of the present study was that myocardial oxidative stress was progressive increase, concomitant with cardiomyocyte apoptosis and LV ventricular dysfunction in the development process of cardiac hypertrophy and heart failure in rats with overload pressure. These results are consistent with data from both clinical and animal studies that the failing heart is subjected to increased oxidative stress and cardiomyocyte apoptosis [18–22,34]. These results might suggest that low levels ROS induces early cardiac hypertrophic response and higher levels of ROS results in later myocyte apoptotic response, which is an important factor for the transition of cardiac hypertrophy to heart failure. This assumption is supported by data from
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previous studies indicating that antioxidants can inhibit early hypertrophic responses and improve ventricular function in heart failure [18,21,24]. The intracellular mechanism that distinguishes the hypertrophic response elicited by low levels of ROS from the apoptotic response elicited by higher levels remains to be defined, but probably involves differences in the type, quantity and/or duration of ROS production [23]. ROS may be derived from mitochondrial electron transport chain, NADH/NADPH oxidase, xanthine oxidase, nitric oxide synthase, and/or cyclooxygenase [18–22]. Maladaptive changes in antioxidant enzyme activity also contribute to the oxidative stress of the failing heart. Oxidative stress damages the cardiomyocytes and extracellular matrix through lipid peroxidation and oxidative modification of cellular proteins and DNA. Earlier studies have shown that the increase of ROS is capable of inducing apoptosis in cultured cardiomyocytes and cardiac dysfunction in an intact heart [35]. The importance of ROS formation in cardiomyocyte apoptosis is further supported by the findings that a number of antioxidants can reduce or block cardiomyocyte apoptosis in heart failure [18,21,24]. Very recent data has demonstrated that very low levels of cardiomyocyte apoptosis (23 myocytes per 105 nuclei, compared with 1.5 myocytes per 105 nuclei in controls) are sufficient to cause a lethal, dilated cardiomyopathy and inhibition of cardiac cardiomyocyte death largely prevents the development of cardiac dilation and contractile dysfunction, the hallmarks of heart failure in FK-binding-protein–caspase-8 transgenic mice [29]. These data provide the evidence that oxidative stress and cardiomyocyte apoptosis may be important mechanisms of heart failure. Recently, several animal studies have shown that statins therapy can prevent the development of heart failure through preservation of NO production, normalization of sympathetic nerve function, and inhibition of oxidative stress [9–11,15–17]. However, it is unknown whether statins therapy initiated after compensatory hypertrophic stage is still effective in preventing the transition of hypertrophy to CHF since the statins treatment in previous studies were initiated before the abnormalities were manifest. So it is very important to elucidate the cardioprotective effects of statins treatment initiated after the onset of abnormalities to imitate a situation close to clinical practice. In the present study, our results demonstrated that long-term administration of simvastatin initiated from 13 weeks after aortic coarctation improved LV function and prevented the transition of hypertrophy to heart failure. This is the first study to evaluate the effects of statins initiated after compensatory hypertrophic stage on the transition of hypertrophy to CHF and thus the first to show beneficial effects in this setting. The mechanisms by which statins exerted beneficial effects in this setting are not completely clear. Treatment with simvastatin initiated after the compensatory hypertrophic stage reduced the superoxide levels in LV tissues and myocytes apoptosis. Several recent studies have shown that simvastatin is able to lessen oxidative stress by inhibiting
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NADPH oxidase activity and improving catalase activity of myocardium [36,37]. Other study indicated that simvastatin protected the cardiomyocytes against the mitochondrial dysfunction induced by H2O2 in vitro [38]. Taken together, these data suggest that oxidative stress may be a causal mechanism of heart failure and simvastatin may have exerted its beneficial effects via its antioxidant property. However, we can not exclude the possibility that decrease of ROS and apoptosis in myocardium treated with simvastatin is due to improved LV function secondary to other mechanisms such as preservation of nitric oxide function [11], inhibition of inflammation [39,40], and normalization of augmented sympathetic outflow [15] and this issue need to be further investigated. In summary, the present study demonstrates, for the first time, long-term administration of simvastatin improved LV function and prevented the transition of hypertrophy to CHF. Inhibition of oxidative stress and cardiomyocyte apoptosis may contribute to the benefits of simvastatin treatment on heart of rats with AS. Statins may be an effective means in improving heart function in CHF.
Acknowledgments This study was supported by grants from the Guangzhou Education Committee and the Education Ministry of the People’s Republic of China. The authors would like to thank Alicia DeMarco (Michigan State University, MI, USA) for her editorial assistance with manuscript.
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