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Effect of simvastatin on remodeling of the left ventricle and aorta in L-NAME-induced hypertension
Life Sciences 74 (2004) 1211 – 1224 www.elsevier.com/locate/lifescie
Effect of simvastatin on remodeling of the left ventricle and aorta in L-NAME-induced hypertension Fedor Simko a,*, Jana Matuskova a, Ivan Luptak a, Kristina Krajcirovicova a, Jarmila Kucharska b, Anna Gvozdjakova b, Pavel Babal c, Olga Pechanova d a
Department of Pathophysiology, School of Medicine, Comenius University, Sasinkova 4, 813 72 Bratislava, Slovak Republic b Pharmacobiochemical Laboratory, School of Medicine, Comenius University, Bratislava, Slovak Republic c Department of Pathology, School of Medicine, Comenius University, Bratislava, Slovak Republic d Institute of Normal and Pathologic Physiology, Slovak Academy of Sciences, Bratislava, Slovak Republic Received 26 March 2003; accepted 17 July 2003
Abstract 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have been shown to prevent or reverse hypertrophy of the LV in several models of left ventricular hypertrophy. The aim of the present study was to determine whether treatment with simvastatin can prevent hypertension, reduction of tissue nitric oxide synthase activity and left ventricular (LV) remodeling in NG-nitro – L-arginine methyl ester(L-NAME)-induced hypertension. Four groups of rats were investigated: control, simvastatin (10 mg/kg), L-NAME (40 mg/kg) and LNAME + simvastatin (in corresponding doses). Animals were sacrificed and studied after 6 weeks of treatment. The decrease of NO-synthase activity in the LV, kidney and brain was associated with hypertension, LV hypertrophy and fibrosis development and remodeling of the aorta in the L-NAME group. Simvastatin attenuated the inhibition of NO-synthase activity in kidney and brain, partly prevented hypertension development and reduced the concentration of coenzyme Q in the LV. Nevertheless, myocardial hypertrophy, fibrosis and enhancement of DNA concentration in the LV, and remodeling of the aorta were not prevented by simultaneous simvastatin treatment in the L-NAME treated animals. We conclude that the HMG-CoA reductase inhibitor simvastatin improved nitric oxide production and partially prevented hypertension development, without preventing remodeling of the left ventricle and aorta in NO-deficient hypertension. D 2003 Elsevier Inc. All rights reserved. Keywords: L-NAME; NO-deficient hypertension; Simvastatin; Left ventricular hypertrophy; Fibrosis; Nitric oxide; Aorta remodeling
* Corresponding author. Tel.: +42-1-2-59357-276; fax: +42-1-2-59357-601. E-mail address: [email protected] (F. Simko). 0024-3205/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2003.07.032
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Introduction Although left ventricular hypertrophy (LVH) is an adaptive reaction to increased haemodynamic load, it represents an independent risk factor of increased cardiovascular morbidity and mortality (Simko, 2002). Thus, a number of drugs are being tested with the aim to disclose their potential to prevent the development of or reverse left ventricular hypertrophy (LVH) (Brown et al., 2001; Pereira and Mandarim-de-Lacerda, 2002; Simko, 1994; Simko, 1996; Simko et al., 2002). According to recent data, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) seem to protect against pathological myocardial growth. Statins reduce cardiovascular mortality in primary and secondary prevention of coronary heart disease in patients with hypercholesterolemia. Yet, the reduction of cholesterol level can only partly explain the statin protective effect (Lee et al., 2002). It was shown recently in several laboratories that pleiotropic effects of statins may involve the reduction of pathological myocardial remodeling. Statins inhibit hydroxy-3methylglutharyl CoA reductase, an enzyme controlling the synthesis of mevalonate. This aminoacid, beside its role in cholesterol synthesis, is a regulator of cellular gowth (Goldstein and Brown, 1990). Indeed, simvastatin prevented angiotensin II- (Takemoto et al., 2001) and by norepinephrine-induced (Luo et al., 2001) growth of fetal cardiomyocytes in culture and angiotensin II infusion- or by aortic constrictionstimulated myocardial hypertrophy in vivo (Takemoto et al., 2001). Simvastatin also reversed myocardial hypertrophy and fibrosis in models of aortic stenosis (Luo et al., 1999) and of transgenic hypertrophic cardiomyopathy (Patel et al., 2001). Cerivastatin reversed cardiac hypertrophy, fibrosis and remodeling in rats transgenic for human renin and angiotensinogen independently of cholesterol reduction (Dechend et al., 2001a). Furthermore, in a murine model of postinfarcted heart failure, amelioration of LV structural remodeling by fluvastatin was even associated with improved survival (Hayashidani et al., 2002). Another important property of statins seems to be their endothelium protective effect. Treatment of SHR with atorvastatin attenuated the endothelial dysfunction, as assed by an improvement of carbachol induced vasorelaxation of aortic segments and upregulation of vascular ecNOS expression and enhancement of ecNOS activity (Wassmann et al., 2001). Both atorvastatin and simvastatin increased NOS activity in vascular endothelial cells (Hernandez-Perera et al., 1998). Moreover, cerivastatin reduced blood pressure in angiotensin II-induced hypertension in a transgenic rat model harbouring the human renin gene (Dechend et al., 2001a; Dechend et al., 2001b). Since the model of L-NAME-induced hypertension and concomitant myocardial hypertrophy and fibrosis (Pereira and Mandarim-de-Lacerda, 2001) is characterized by inhibition of the NOS activity in various organs (Bernatova et al., 1996) and deterioration of vasodilating ability including the renal artery (Holecyova et al., 1996), we supposed that simvastatin could act beneficially within the model of NO-deficient hypertension. The hypothesis was tested, whether or not simvastatin is able to prevent hypertension and to modify the remodeling of the heart and aorta in the NO-deficient hypertension model.
Materials and methods Animals and treatment Male Wistar rats, 15 weeks old, were randomly divided into four groups (n = 8 in each group). The first group served as a control. In the second group, L-NAME (Sigma Chemical Co, Germany) was
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given 40 mg/kg/day. The third group received simvastatin (Merck Sharp & Dohme B.V., Netherland) 10 mg/kg/day. The fourth group received simultaneously L-NAME (40 mg/kg/day) and simvastatin 10 mg/ kg/day. The substances were given in tap water for 6 weeks. All animals were housed at a temperature of 22–24 jC in individual cages and fed a regular pellet diet ad libitum. The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No 8523, revised 1985). The systolic blood pressure was measured by non-invasive tail–cuff plethysmography (Hugo Sachs Elektronik KG, Freiburg, Germany) each week. After 6 weeks, the animals were sacrificed by decapitation, the body weight (BW), heart weight (HW), left ventricle weight (LVW) and right ventricle weight (RVW) were determined and the LVW/BW as well as RVW/BW ratio were calculated (Table 1). Samples of the left ventricle were used for the determination of NO-synthase activity, fibrosis and DNA concentration. NOS activity was determined also in the kidney and brain. Assay of NO-synthase activity Total NO-synthase activity was determined in crude homogenates of tissues by measuring the formation of [3H]-citruline from [3H]–L-arginine (Amersham International plc, UK), as described by Bredt and Snyder (1990) with some modifications. Briefly 50Al of 10% homogenates were incubated in the presence of 50 nmol/l Tris-HCl, pH 7.4, 20 Amol/l [3H]-L-arginine (specific activity 5 GBq/ mmol, about 100 000 dpm/min) 30 nmol/l calmodulin, 1 mmol/l h-NADPH, 3Amol/l BH4 and 2 mmol/l Ca2 + in a total volume of 100 Al. After 10 min incubation at 37 jC, the reaction was stopped by addition of 1 ml of 20 mmol/l HEPES buffer pH 5.5, containing 2 mmol/l EDTA, 2 mmol/l EGTA and 1 mmol/l L-citrulline. The samples were centrifuged at 10 000 g for 1 min at 4 jC and the suppernatant was applied to 1 ml Dowex 50 WX-8 columns (Na+ form). L-[3H] Cit was eluted with 1 ml of water and measured by liquid scintillation counting. NO synthase activity was expressed as picokatal per gram of protein (pkat .(g.protein) 1. Determination of coenzyme (Co) Q9 and Q10 levels Coenzyme Q 10 homologues were determined by isocratic high-performance liquid chromatography (LKB, Sweeden) according to Lang et al. (Lang et al., 1986) with some modifications (Kucharska et al., 1998). After homogenization of myocardial tissue (20–50 mg) and extraction by a mixture of hexane–ethanol with addition of butylhydroxytoluene and sodium dodecylsulphate, the organic phase was evaporated under nitrogen. The residue was dissolved in ethanol and injected into a Separon SGX C18 7 um 3 150 mm column (Tessek, Czech Republic). Elution was performed with methanol/acetonitril/ethanol (6/2/2 v/v/v, Merck). Spectrophotometric detection at 275 nm and external standards (Sigma) were used. The concentrations of compounds were calculated as nmol/g of wet weight. Determination of deoxyribonucleic acid (DNA) concentration DNA concentration was analysed according Sambrook et al. (Sambrook et al., 1989).
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Histologic and morphometric determination of myocardial fibrosis and morphometry of aorta The LV samples were oriented perpendicularly to the sectioning plane. Samples of aorta were put in an upright position on cellulosic filter membranes to maintain a round shape. The tissues were fixed for 24 hours in 10% phosphate buffered formalin. Then, they were processed routinely in paraffin and serial 5Am thick sections were stained with hematoxylin and eosin and by Van Gieson’s staining and analysed. Morphometric evaluation was performed on an Olympus light microscope equipped with a two dimensional image analyzer (Impor Pro, Slovakia) as described elsewhere (Babal et al., 1997). Van Gieson’s staining was applied to enhance the red color contrast of collagen. Myocardial fibrosis was expressed as a percentage of the whole muscle area on three step sections 50 Am one after the other of each heart ventricle specimen. The area of aortic wall cross-section was expressed in mm2. Media + intima of the aorta were measured with omission of the adventitia. Statistical analysis The results are expressed as mean F S.E.M.. Differences were considered significant if the P-value was less than 0.05. For statistical analysis, one way analysis of variance (ANOVA) and the Bonferroni test were used.
Results Cardiovascular parameters After six weeks of treatment, SBP was 123 F 1,8 mmHg in the control group. In the L-NAME group SBP increased by 48% (P < 0.05). In the L-NAME+ simvastatin group SBP was higher than in controls by 32% (P < 0.05) but lower if compared to L-NAME group by 11% (P < 0.05) (Fig. 1). After the five week of experiment, the LW/BW ratio was 1,08 F 0.02 mg/g w.w. in controls. In the L-NAME group the ratio increased by 17% (P < 0.05) v control group and in the L-NAME + simvastatin group the LVW/BW ratio did not decrease in comparison to L-NAME group and was higher by 23% (P < 0.05) when compared to the control group (Fig. 2). The RVW/BW ratio was not affected in any group.
Table 1 Effect of simvastatine and/or L-NAME treatment on the body weight (BW), left ventricle weight (LVW), right ventricle weight (RVW) and right ventricle to body weight ratio (RVW/BW) in NO-deficient rats Control Body weight [g] Right ventricle weight [mg] Left ventricle weight [mg] RVW/BW [mg/g]
433.00 158.60 465.48 0.37
F F F F
9.35 9.90 9.87 0.02
Simvastatine
L-NAME
409.00 166.10 432.72 0.41
396.67 137.78 499.01 0.35
F F F F
9.42 10.65 13.92 0.02
Values are means F SEM (*p < 0.05 compared to simvastatine).
F F F F
Simvastatine + L-NAME 11.46 11.00 16.28 0.02
401.50 139.70 529.18 0.35
F F F F
10.30 4.67 21,827* 0.01
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Fig. 1. Effect of 6-week L-NAME, simvastatin and L-NAME + simvastatin treatment on systolic blood pressure. C - control, S -10 mg/kg/day simvastatin, L – 40 mg/kg/day L-NAME, LS - L-NAME + simvastatin in corresponding doses. *P < 0.05 compared to control: + P < 0.05 compared to L-NAME group.
Fig. 2. Effect of 6-week L-NAME, simvastatin and L-NAME + simvastatin treatment on the LVW/BW ratio and cross section area of the aorta. C - control, S - 10 mg/kg/day simvastatin, L – 40 mg/kg/day L-NAME, LS - L-NAME + simvastatin in corresponding doses. *P < 0.05 compared to control.
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DNA concentration The concentration of DNA was 593 F 46 Ag/gww in the left ventricle of the control group. In the L-NAME group, DNA concentration increased by 56% (P < 0.05) v control. In the L-NAME + simvastatin group DNA concentration was not changed when compared to the L-NAME group (Fig. 3). LV fibrosis and morphometry of the aorta Fibrosis of the left ventricle was increased in the L-NAME group when compared to control (P < 0.05) and no significant change between the L-NAME + simvastatin and L-NAME groups was observed (Fig. 3, Fig. 6). The cross sectional area of the aorta was increased by 28% (P < 0.05) in the L-NAME group when compared to control. Simvastatin added to L-NAME failed to change significantly the cross section area when compared to the L-NAME group (Fig. 2).
Fig. 3. Effect of 6-week L-NAME, simvastatin and L-NAME + simvastatin treatment on DNA concentration and fibrosis in the LV. C - control, S - 10 mg/kg/day simvastatin, L – 40 mg/kg/day L-NAME, LS - L-NAME + simvastatin in corresponding doses. *P < 0.05 compared to control.
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Fig. 4. Effect of 6-week L-NAME, simvastatin and L-NAME + simvastatin treatment on NO-synthase activity in the left ventricle, kidney and brain. C - control, S - 10 mg/kg/day simvastatin, L – 40 mg/kg/day L-NAME, LS- L-NAME + simvastatin in corresponding doses. *P < 0.05 compared to control: + P < 0.05 compared to L-NAME group.
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NO-synthase activity NO-synthase activity was 5.0 F 0.31 pkat/g prot. in the left ventricle of the control group. In the LNAME group, NO-synthase activity was inhibited by 29% (P < 0.05) v control and addition of simvastatin to L-NAME did not improve the NO-synthase activity (Fig. 4). In the kidney, NO-synthase activity was 21.07 F 1.55 pkat/g prot in the control group. In the LNAME group, NO-synthase activity was inhibited by 29% (P < 0.05) v control and the addition of simvastatin to L-NAME increased NO-synthase activity by 26% (P < 0.05) v L-NAME group (Fig. 4). In the brain of the control group, NO-synthase activity was 9.4 F 10.70 pkat/g prot. In the L-NAME group, NO-synthase activity was inhibited by 37% (P < 0.05) v control and addition of simvastatin to LNAME increased NO-synthase activity by 46% (P < 0.05) v L-NAME group (Fig. 4). Co Q9 and Co Q10 concentrations Concentration of CoQ9 was 204.03 F 18.89 nmol/gww and of CoQ10 18.37 F 1.62 nmol/gww in the left ventricle of the control group. In the L-NAME group, Co Q9 and Q10 concentrations were only
Fig. 5. Effect of 6-week L-NAME, simvastatin and L-NAME + simvastatin treatment on the concentration of CoQ9 and CoQ10 in the left ventricle. C - control, S - 10 mg/kg/day simvastatin, L – 40 mg/kg/day L-NAME, LS - L-NAME + simvastatin in corresponding doses. *P < 0.05 compared to control.
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slightly decreased but the addition of simvastatin to L-NAME decreased the concentration of CoQ9 by 37% (P < 0.05) and of Q10 by 39% (P < 0.05) v the control group (Fig. 5).
Discussion The present study demonstrates the influence of the HMG-CoA reductase inhibitor simvastatin on the growth of the heart and aorta in L-NAME induced hypertension. L-NAME caused a reduction of NO synthase activity in the heart, kidney and brain, an increase in systolic blood pressure, hypertrophy of the LV and aorta, and fibrosis of the LV. Simultaneous treatment with simvastatin attenuated the systolic blood pressure increase and improved NO synthase production in the kidney and brain. Yet, the increase of left ventricular weight, thickness of the aorta, DNA concentration in the LV and left ventricular fibrosis development remained unaffected. Hypertrophy of the LV in the L-NAME model of hypertension was shown to be linked to increased fibrosis (Bernatova et al., 2000; Pereira and Mandarim-de-Lacerda, 2001; Pechanova et al., 1997) and protein remodeling of the left ventricle (Bernatova et al., 2000; Pechanova et al., 1997). In our previous experiment the ACE-inhibitor, captopril, completely prevented LVH and fibrosis development, however, NO synthase activity remained inhibited in all organs investigated (LV, brain, kidney and aorta) suggesting that prevention of remodeling was achieved by mechanisms different from the restoration of NO synthase activity (Pechanova et al., 1997). In this experiment, NO synthase activity was investigated in three organs with potentially different effects of NO-deficiency on the blood pressure. While the left ventricle is an organ with relatively autonomic blood flow control, in which NO production alteration is not supposed to have significant impact on blood pressure, NO-deficiency in the kidney may lead to vasoconstriction of renal artery and stimulate renin and angiotensin II production with general vasoconstriction and hypertension. Also inhibition of NO synthase in the rat brain was previously shown to increase blood pressure (Gerova et al., 1995). It seems reasonable to suppose that improvement of NO synthase activity in the kidney and brain might have played an important role in partial prevention of hypertension in this experiment. Interestingly, despite restoration of NOS activity in the kidney and brain and a reduction of blood pressure, simvastatin failed to prevent remodeling of the LV and aorta in our experiment. This result is partly in agreement with findings of Nahrendorf et al. (Nahrendorf et al., 2002). These authors found that left ventricular hypertrophy after myocardial infarction was attenuated and global function was improved by cerivastatin in rats. When the statin was applied simultaneously with the L-NAME, all positive effects were abolished (Nahrendorf et al., 2002). Several explanations of the inability of simvastatin to prevent remodeling of the heart and aorta in the model of L-NAME induced hypertension in our experiment are plausible. First, in the presence of L-NAME, simvastatin was not able to restore NO synthase activity in the myocardial tissue of the LV. It has been shown previously that local deficiency of NO per se, independently of blood pressure enhancement, was able to induce fibrosis of the left ventricular myocardium (Fig. 6) (Pechanova et al., 1999) and intimal remodeling of aorta (Rossi and ColombiniNetto, 2001). It seems that remodeling of the heart is relatively independent of blood pressure alterations in the L-NAME model and may be more closely associated with inhibition of local NO production. Thus, the lack of improvement of the local NOS activity in the LV by simvastatin, with potential local NO-deficiency in LV myocardium, might have participated in preserving the process of remodeling.
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Fig. 6. Left ventricle of a control rat (C), control treated with simvastatin (S), focal myocardial fibrosis in the left ventricular myocardium in the L-NAME treated (L) and L-NAME + simvastatin (LS) treated rat. Van Gieson, 200 .
Moreover, the prevention of hypertension was incomplete, although significant (162 mmHg in LNAME and simvastatin group v 182 mmHg in L-NAME group). Enhancement of blood pressure by 32% compared to control may have represented large enough afterload to provoke LV hypertrophy development. Second, the mechanism of L-NAME-induced LVH is a complex and not completely understood process (Simko and Simko, 2000). Besides the deficiency of NO production, with its antiproliferative effect on myocytes and fibrocytes in vivo and in vitro (Rossi and Colombini-Netto, 2001; Simko and Simko, 2000), the activation of RAS through depressed NO dependent relaxing ability of the renal artery (Holecyova et al., 1996) can participate in this process. Indeed, increased plasma renin activity was reported in NO-deficient hypertension and was associated with enhanced activity of local ACE in the LV and aorta (Takemoto et al., 1997). Statins seem to interfere with the renin-angiotensin system. Simvastatin have been shown to reduce angiotensin II-induced LV hypertrophy in vivo (Takemoto et al., 2001) and the same statin (simvastatin) induced reversion of LVH and fibrosis and improvement of the LV function in the model of aortic stenosis in rats. This LVH regression was associated with the attenuation of myocardial ACE activity and reduction of angiotensin II (Luo et al., 1999). However, several additional humoral systems, i.e. the sympathetic system (K-Laflamme et al., 1998), endothelin (Sventek et al., 1996) and aldosterone (Usui et al., 1998), which are supposed to participate in L-NAME induced hypertension and LVH development (Simko and Simko, 2000), are not influenced by
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simvastatin. Restoring the neurohormonal balance in the NO-deficient model, which could be necessary for hindering the pathologic hypertrophic growth, seems to be more complicated than in other models of LVH. Third, the protective effect of HMG-CoA reductase inhibitors against pathologic remodeling of the myocardium may be associated with the antioxidant nature of statins (Takemoto et al., 2001). Members of the small G proteins family, Rac 1 and RhoA have been suggested to be mediators of cardiac hypertrophic process (Van Aelst and D’Souza-Schorey, 1997). Furthermore, Rac1 was shown to mediate AngII-induced intracellular oxidation, which may significantly participate on the myocardial hypertrophic growth (Takemoto et al., 2001). Statins downregulate the activity of small G proteins in cardiomyocytes both in vivo and vitro enhancing thus eNOS expression (Laufs and Liao, 1998) and preventing the development of cardiac hypertrophy (Takemoto et al., 2001; Laufs et al., 2002), supposedly through an antioxidant mechanism involving inhibition of Rac1 (Takemoto et al., 2001). To investigate the activity of RhoGTPase family was beyond the scope of this publication. However, in accordance with the finding of Nahrendorf’s laboratory, where the protective effect of cerivastatin against pathologic LV remodeling after myocardial infarction was abolished by L-NAME, and on the basis of our experiment, where inhibition of myocardial NOS activity and development of LV hypertrophy in the model of L-NAME induced hypertension were not prevented by simvastatin, we speculate that in the presence of L-NAME the inhibition of small G-proteins by a statin is not functioning in the myocardium, omitting thus both improvement of endothelial NO production and the antioxidant effect of statins. In contrast to the antioxidant activity of statins, data have emerged in the literature that statins can undesirably decrease the level of CoQ in various organs including the heart (Bliznakov, 2002). Coenzyme Q is not only involved in the bioenergetic function of the cell, but also has strong antioxidant properties and may serve as an indicator of the antioxidant status of the myocardium (Bliznakov, 2002; Kucharska et al., 2000). In our work coenzyme Q 9 and Q10 levels were not decreased in the LV myocardium of simvastatin treated control rats. However, the simultaneous treatment with L-NAME and simvastatin significantly decreased the concentration of both CoQ9 and CoQ10 in the LV. The decreased antioxidant capacity of the myocardium, potentially resulting in increased oxidative stress, may have participated on the preservation of the remodeling process of the LV in the NO-deficient model of hypertrophic growth. Limitations of the work It is a matter of view, which parameter is optimal to characterize the altered NO production. Acetylcholine–induced vessel relaxation (Takase et al., 1996), inorganic nitrate in the urine (Forte et al., 1997), tissue NOS activity (Takase et al., 1996), cGMP concentration (Matsuoka et al., 1996), and most frequently expression of eNOS (Laufs and Liao, 1998; Laufs et al., 2002; Takemoto et al., 2001) and seldomly iNOS (Luvara et al., 1998) at mRNA and/or protein level were used to characterize nitric oxide production. Since in previous papers from our laboratory NOS activity in the heart and other organs were measured (Pechanova et al., 1997; Bernatova et al., 1999), we considered appropriate to use NOS activity as a potential indicator of NO production, in order the comparison of the protective effect of various substances could be possible. To characterize the level of fibrosis in the myocardium of the LV, the morphometric evaluation was used. However, more thorough information may be yielded by biochemical analysis of soluble and
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insoluble collagen protein fraction, as was performed in our previous works (Pechanova et al., 1997; Bernatova et al., 2000) or by investigation of collagen I protein expression.
Conclusion We conclude that simvastatin did not prevent pathological remodeling of the heart and aorta in the LNAME-induced hypertension despite improving NO synthase activity in the kidney and brain and attenuation of hypertension development. Although statins seem to be promising means of protection of the haemodynamically overloaded heart according to recent literature data, the level of their protective ability seems to depend on the model used and should be considered individually for each type of haemodynamic overload.
Acknowledgements This work was supported by the VEGA grants No 1/0532/03 and No 2/3185/23 and APVT grant No 51-013802.
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Luvara, G., Pueyo, M.E., Philippe, M., Mandet, C., Savoie, F., Henrion, D., Michel, J.B., 1998. Chronic blockade of NO synthase activity induces a proinflammatory phenotype in the arterial wall. Prevention by angiotensin II antagonism. Atherosclerosis and Thrombosis in Vascular Biology 18, 1408 – 1416. Matsuoka, H., Nakata, M., Keisuke, K., Koga, Y., Nomura, G., Toshima, M., Imaizumi, T., 1996. Chronic L-arginine administration attenuates cardiac hypertrophy in spontaneously hypertensive rats. Hypertension 27, 14 – 18. Nahrendorf, M., Hu, K., Hiller, K.H., Galuppo, P., Fraccarollo, D., Schweizer, G., Haase, A., Ertl, G., Bauer, W.R., Bauersachs, J., 2002. Impact of hydroxymethylglutaryl coenzyme a reductase inhibition on left ventricular remodeling after myocardial infarction: an experimental serial cardiac magnetic resonance imaging study. Journal of the American College of Cardiology 40, 1695 – 1700. Patel, R., Nagueh, S.F., Tsybouleva, N., Abdellatif, M., Lutucuta, S., Kopelen, H.A., Quinones, M.A., Zoghbi, W.A., Entman, M.L., Roberts, R., Marian, A.J., 2001. Simvastatin induces regression of cardiac hypertrophy and fibrosis and improves cardiac function in a transgenic rabbit model of human hypertrophic cardiomyopathy. Circulation 104, 317 – 324. Pereira, L.M., Mandarim-de-Lacerda, C.A., 2001. Stereology of cardiac hypertrophy induced by NO blockade in rats treated with enalapril and verapamil. Analytical Quantitative Cytology and Histology 23, 330 – 338. Pereira, L.M., Mandarim-de-Lacerda, C.A., 2002. Myocardial changes after spironolactone in spontaneous hypertensive rats. A laser scanning confocal microscopy study. Journal of Cellular and Molecular Medicine 6, 49 – 57. Pechanova, O., Bernatova, I., Pelouch, V., Simko, F., 1997. Protein remodeling of the heart in NO-deficient hypertension: the effect of captopril. Journal of Molecular and Cellular Cardiology 29, 3365 – 3374. Pechanova, O., Bernatova, I., Pelouch, V., Babal, P., 1999. L-NAME-induced protein remodeling and fibrosis in the rat heart. Physiological Research 48, 353 – 362. Rossi, M.A., Colombini-Netto, M., 2001. Chronic inhibition of NO synthesis per se promotes structural intimal remodeling of the rat aorta. Journal of Hypertension 19, 1567 – 1579.
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Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Isolation of DNA from mammalian cells. In: Ford, N. (Ed.), Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, New York, pp. 9.16 – 9.19. Sventek, P., Li, J.S., Grove, K., Deschepper, C.F., Schiffrin, E.L., 1996. Vascular structure and expression of endothelin-1 gene in L-NAME-treated spontaneously hypertensive rats. Hypertension 7, 49 – 55. Simko, F., 1994. Pathophysiological principles of the relation between myocardial hypertrophy of the left ventricle and its regression. Physiological Research 43, 259 – 266. Simko, F., 1996. Left ventricular hypertrophy regression as a process with variable biological implications. Canadian Journal of Cardiology 12, 507 – 551. Simko, F., Simko, J., 2000. The potential role of nitric oxide in the hypertrophic growth of the left ventricle. Physiological Reseach 49, 37 – 46. Simko, F., 2002. Physiologic and pathologic myocardial hypertrophy: physiologic and pathologic regression of hypertrophy? Medical Hypotheses 58, 11 – 14. Simko, F., Luptak, I., Matuskova, J., Babal, P., Pechanova, O., Bernatova, I., Hulin, I., 2002. Heart remodeling in the hereditary hypertriglyceridemic (hHTG) rat: effect of captopril and nitric oxide deficiency. Annals of the New York Academy of Sciences 967, 454 – 462. Takase, H., Moreau, P., Kung, C.F., Nava, E., Lu¨scher, T.F., 1996. Antihypertensive therapy prevents endothelial dysfunction in chronic nitric oxide deficiency. Effect of verapamil and trandolapril. Hypertension 27, 25 – 31. Takemoto, M., Egashira, K., Usui, M., Numaguchi, K., Tomita, H., Tsutsui, H., Shimokawa, H., Sueishi, K., Takeshita, A., 1997. Important role of tissue angiotensin-converting enzyme activity in the pathogenesis of coronary vascular and myocardial structural changes induced by long-term blockade of nitric oxide synthesis in rats. Journal of Clinical Investigation 99, 278 – 287. Takemoto, M., Node, K., Nakagami, H., Liao, Y., Grimm, M., Takemoto, Y., Kitakaze, M., Liao, J.K., 2001. Statins as antioxidant therapy for preventing cardiac myocyte hypertrophy. Journal of Clinical Investigation 108, 1429 – 1437. Usui, M., Ichiki, T., Katoh, M., Egashira, K., Takeshita, A., 1998. Regulation of Ang II receptor expression by nitric oxide in rat adrenal gland. Hypertension 32, 527 – 533. Van Aelst, L., D’Souza-Schorey, C., 1997. Rho GTPases and signalling networks. Genes and Development 11, 2295 – 2322. Wassmann, S., Laufs, U., Baumer, A.T., Muller, K., Ahlbory, K., Linz, W., Itter, G., Rosen, R., Bohm, M., Nickening, G., 2001. HMG-CoA reductase inhibitors improve endothelial dysfunction in normocholesterolemic hypertension via reduced production of reactive oxygen species. Hypertension 37, 1450 – 1457.
Effect of simvastatin on remodeling of the left ventricle and aorta in L-NAME-induced hypertension Fedor Simko a,*, Jana Matuskova a, Ivan Luptak a, Kristina Krajcirovicova a, Jarmila Kucharska b, Anna Gvozdjakova b, Pavel Babal c, Olga Pechanova d a
Department of Pathophysiology, School of Medicine, Comenius University, Sasinkova 4, 813 72 Bratislava, Slovak Republic b Pharmacobiochemical Laboratory, School of Medicine, Comenius University, Bratislava, Slovak Republic c Department of Pathology, School of Medicine, Comenius University, Bratislava, Slovak Republic d Institute of Normal and Pathologic Physiology, Slovak Academy of Sciences, Bratislava, Slovak Republic Received 26 March 2003; accepted 17 July 2003
Abstract 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have been shown to prevent or reverse hypertrophy of the LV in several models of left ventricular hypertrophy. The aim of the present study was to determine whether treatment with simvastatin can prevent hypertension, reduction of tissue nitric oxide synthase activity and left ventricular (LV) remodeling in NG-nitro – L-arginine methyl ester(L-NAME)-induced hypertension. Four groups of rats were investigated: control, simvastatin (10 mg/kg), L-NAME (40 mg/kg) and LNAME + simvastatin (in corresponding doses). Animals were sacrificed and studied after 6 weeks of treatment. The decrease of NO-synthase activity in the LV, kidney and brain was associated with hypertension, LV hypertrophy and fibrosis development and remodeling of the aorta in the L-NAME group. Simvastatin attenuated the inhibition of NO-synthase activity in kidney and brain, partly prevented hypertension development and reduced the concentration of coenzyme Q in the LV. Nevertheless, myocardial hypertrophy, fibrosis and enhancement of DNA concentration in the LV, and remodeling of the aorta were not prevented by simultaneous simvastatin treatment in the L-NAME treated animals. We conclude that the HMG-CoA reductase inhibitor simvastatin improved nitric oxide production and partially prevented hypertension development, without preventing remodeling of the left ventricle and aorta in NO-deficient hypertension. D 2003 Elsevier Inc. All rights reserved. Keywords: L-NAME; NO-deficient hypertension; Simvastatin; Left ventricular hypertrophy; Fibrosis; Nitric oxide; Aorta remodeling
* Corresponding author. Tel.: +42-1-2-59357-276; fax: +42-1-2-59357-601. E-mail address: [email protected] (F. Simko). 0024-3205/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2003.07.032
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Introduction Although left ventricular hypertrophy (LVH) is an adaptive reaction to increased haemodynamic load, it represents an independent risk factor of increased cardiovascular morbidity and mortality (Simko, 2002). Thus, a number of drugs are being tested with the aim to disclose their potential to prevent the development of or reverse left ventricular hypertrophy (LVH) (Brown et al., 2001; Pereira and Mandarim-de-Lacerda, 2002; Simko, 1994; Simko, 1996; Simko et al., 2002). According to recent data, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) seem to protect against pathological myocardial growth. Statins reduce cardiovascular mortality in primary and secondary prevention of coronary heart disease in patients with hypercholesterolemia. Yet, the reduction of cholesterol level can only partly explain the statin protective effect (Lee et al., 2002). It was shown recently in several laboratories that pleiotropic effects of statins may involve the reduction of pathological myocardial remodeling. Statins inhibit hydroxy-3methylglutharyl CoA reductase, an enzyme controlling the synthesis of mevalonate. This aminoacid, beside its role in cholesterol synthesis, is a regulator of cellular gowth (Goldstein and Brown, 1990). Indeed, simvastatin prevented angiotensin II- (Takemoto et al., 2001) and by norepinephrine-induced (Luo et al., 2001) growth of fetal cardiomyocytes in culture and angiotensin II infusion- or by aortic constrictionstimulated myocardial hypertrophy in vivo (Takemoto et al., 2001). Simvastatin also reversed myocardial hypertrophy and fibrosis in models of aortic stenosis (Luo et al., 1999) and of transgenic hypertrophic cardiomyopathy (Patel et al., 2001). Cerivastatin reversed cardiac hypertrophy, fibrosis and remodeling in rats transgenic for human renin and angiotensinogen independently of cholesterol reduction (Dechend et al., 2001a). Furthermore, in a murine model of postinfarcted heart failure, amelioration of LV structural remodeling by fluvastatin was even associated with improved survival (Hayashidani et al., 2002). Another important property of statins seems to be their endothelium protective effect. Treatment of SHR with atorvastatin attenuated the endothelial dysfunction, as assed by an improvement of carbachol induced vasorelaxation of aortic segments and upregulation of vascular ecNOS expression and enhancement of ecNOS activity (Wassmann et al., 2001). Both atorvastatin and simvastatin increased NOS activity in vascular endothelial cells (Hernandez-Perera et al., 1998). Moreover, cerivastatin reduced blood pressure in angiotensin II-induced hypertension in a transgenic rat model harbouring the human renin gene (Dechend et al., 2001a; Dechend et al., 2001b). Since the model of L-NAME-induced hypertension and concomitant myocardial hypertrophy and fibrosis (Pereira and Mandarim-de-Lacerda, 2001) is characterized by inhibition of the NOS activity in various organs (Bernatova et al., 1996) and deterioration of vasodilating ability including the renal artery (Holecyova et al., 1996), we supposed that simvastatin could act beneficially within the model of NO-deficient hypertension. The hypothesis was tested, whether or not simvastatin is able to prevent hypertension and to modify the remodeling of the heart and aorta in the NO-deficient hypertension model.
Materials and methods Animals and treatment Male Wistar rats, 15 weeks old, were randomly divided into four groups (n = 8 in each group). The first group served as a control. In the second group, L-NAME (Sigma Chemical Co, Germany) was
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given 40 mg/kg/day. The third group received simvastatin (Merck Sharp & Dohme B.V., Netherland) 10 mg/kg/day. The fourth group received simultaneously L-NAME (40 mg/kg/day) and simvastatin 10 mg/ kg/day. The substances were given in tap water for 6 weeks. All animals were housed at a temperature of 22–24 jC in individual cages and fed a regular pellet diet ad libitum. The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No 8523, revised 1985). The systolic blood pressure was measured by non-invasive tail–cuff plethysmography (Hugo Sachs Elektronik KG, Freiburg, Germany) each week. After 6 weeks, the animals were sacrificed by decapitation, the body weight (BW), heart weight (HW), left ventricle weight (LVW) and right ventricle weight (RVW) were determined and the LVW/BW as well as RVW/BW ratio were calculated (Table 1). Samples of the left ventricle were used for the determination of NO-synthase activity, fibrosis and DNA concentration. NOS activity was determined also in the kidney and brain. Assay of NO-synthase activity Total NO-synthase activity was determined in crude homogenates of tissues by measuring the formation of [3H]-citruline from [3H]–L-arginine (Amersham International plc, UK), as described by Bredt and Snyder (1990) with some modifications. Briefly 50Al of 10% homogenates were incubated in the presence of 50 nmol/l Tris-HCl, pH 7.4, 20 Amol/l [3H]-L-arginine (specific activity 5 GBq/ mmol, about 100 000 dpm/min) 30 nmol/l calmodulin, 1 mmol/l h-NADPH, 3Amol/l BH4 and 2 mmol/l Ca2 + in a total volume of 100 Al. After 10 min incubation at 37 jC, the reaction was stopped by addition of 1 ml of 20 mmol/l HEPES buffer pH 5.5, containing 2 mmol/l EDTA, 2 mmol/l EGTA and 1 mmol/l L-citrulline. The samples were centrifuged at 10 000 g for 1 min at 4 jC and the suppernatant was applied to 1 ml Dowex 50 WX-8 columns (Na+ form). L-[3H] Cit was eluted with 1 ml of water and measured by liquid scintillation counting. NO synthase activity was expressed as picokatal per gram of protein (pkat .(g.protein) 1. Determination of coenzyme (Co) Q9 and Q10 levels Coenzyme Q 10 homologues were determined by isocratic high-performance liquid chromatography (LKB, Sweeden) according to Lang et al. (Lang et al., 1986) with some modifications (Kucharska et al., 1998). After homogenization of myocardial tissue (20–50 mg) and extraction by a mixture of hexane–ethanol with addition of butylhydroxytoluene and sodium dodecylsulphate, the organic phase was evaporated under nitrogen. The residue was dissolved in ethanol and injected into a Separon SGX C18 7 um 3 150 mm column (Tessek, Czech Republic). Elution was performed with methanol/acetonitril/ethanol (6/2/2 v/v/v, Merck). Spectrophotometric detection at 275 nm and external standards (Sigma) were used. The concentrations of compounds were calculated as nmol/g of wet weight. Determination of deoxyribonucleic acid (DNA) concentration DNA concentration was analysed according Sambrook et al. (Sambrook et al., 1989).
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Histologic and morphometric determination of myocardial fibrosis and morphometry of aorta The LV samples were oriented perpendicularly to the sectioning plane. Samples of aorta were put in an upright position on cellulosic filter membranes to maintain a round shape. The tissues were fixed for 24 hours in 10% phosphate buffered formalin. Then, they were processed routinely in paraffin and serial 5Am thick sections were stained with hematoxylin and eosin and by Van Gieson’s staining and analysed. Morphometric evaluation was performed on an Olympus light microscope equipped with a two dimensional image analyzer (Impor Pro, Slovakia) as described elsewhere (Babal et al., 1997). Van Gieson’s staining was applied to enhance the red color contrast of collagen. Myocardial fibrosis was expressed as a percentage of the whole muscle area on three step sections 50 Am one after the other of each heart ventricle specimen. The area of aortic wall cross-section was expressed in mm2. Media + intima of the aorta were measured with omission of the adventitia. Statistical analysis The results are expressed as mean F S.E.M.. Differences were considered significant if the P-value was less than 0.05. For statistical analysis, one way analysis of variance (ANOVA) and the Bonferroni test were used.
Results Cardiovascular parameters After six weeks of treatment, SBP was 123 F 1,8 mmHg in the control group. In the L-NAME group SBP increased by 48% (P < 0.05). In the L-NAME+ simvastatin group SBP was higher than in controls by 32% (P < 0.05) but lower if compared to L-NAME group by 11% (P < 0.05) (Fig. 1). After the five week of experiment, the LW/BW ratio was 1,08 F 0.02 mg/g w.w. in controls. In the L-NAME group the ratio increased by 17% (P < 0.05) v control group and in the L-NAME + simvastatin group the LVW/BW ratio did not decrease in comparison to L-NAME group and was higher by 23% (P < 0.05) when compared to the control group (Fig. 2). The RVW/BW ratio was not affected in any group.
Table 1 Effect of simvastatine and/or L-NAME treatment on the body weight (BW), left ventricle weight (LVW), right ventricle weight (RVW) and right ventricle to body weight ratio (RVW/BW) in NO-deficient rats Control Body weight [g] Right ventricle weight [mg] Left ventricle weight [mg] RVW/BW [mg/g]
433.00 158.60 465.48 0.37
F F F F
9.35 9.90 9.87 0.02
Simvastatine
L-NAME
409.00 166.10 432.72 0.41
396.67 137.78 499.01 0.35
F F F F
9.42 10.65 13.92 0.02
Values are means F SEM (*p < 0.05 compared to simvastatine).
F F F F
Simvastatine + L-NAME 11.46 11.00 16.28 0.02
401.50 139.70 529.18 0.35
F F F F
10.30 4.67 21,827* 0.01
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Fig. 1. Effect of 6-week L-NAME, simvastatin and L-NAME + simvastatin treatment on systolic blood pressure. C - control, S -10 mg/kg/day simvastatin, L – 40 mg/kg/day L-NAME, LS - L-NAME + simvastatin in corresponding doses. *P < 0.05 compared to control: + P < 0.05 compared to L-NAME group.
Fig. 2. Effect of 6-week L-NAME, simvastatin and L-NAME + simvastatin treatment on the LVW/BW ratio and cross section area of the aorta. C - control, S - 10 mg/kg/day simvastatin, L – 40 mg/kg/day L-NAME, LS - L-NAME + simvastatin in corresponding doses. *P < 0.05 compared to control.
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DNA concentration The concentration of DNA was 593 F 46 Ag/gww in the left ventricle of the control group. In the L-NAME group, DNA concentration increased by 56% (P < 0.05) v control. In the L-NAME + simvastatin group DNA concentration was not changed when compared to the L-NAME group (Fig. 3). LV fibrosis and morphometry of the aorta Fibrosis of the left ventricle was increased in the L-NAME group when compared to control (P < 0.05) and no significant change between the L-NAME + simvastatin and L-NAME groups was observed (Fig. 3, Fig. 6). The cross sectional area of the aorta was increased by 28% (P < 0.05) in the L-NAME group when compared to control. Simvastatin added to L-NAME failed to change significantly the cross section area when compared to the L-NAME group (Fig. 2).
Fig. 3. Effect of 6-week L-NAME, simvastatin and L-NAME + simvastatin treatment on DNA concentration and fibrosis in the LV. C - control, S - 10 mg/kg/day simvastatin, L – 40 mg/kg/day L-NAME, LS - L-NAME + simvastatin in corresponding doses. *P < 0.05 compared to control.
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Fig. 4. Effect of 6-week L-NAME, simvastatin and L-NAME + simvastatin treatment on NO-synthase activity in the left ventricle, kidney and brain. C - control, S - 10 mg/kg/day simvastatin, L – 40 mg/kg/day L-NAME, LS- L-NAME + simvastatin in corresponding doses. *P < 0.05 compared to control: + P < 0.05 compared to L-NAME group.
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NO-synthase activity NO-synthase activity was 5.0 F 0.31 pkat/g prot. in the left ventricle of the control group. In the LNAME group, NO-synthase activity was inhibited by 29% (P < 0.05) v control and addition of simvastatin to L-NAME did not improve the NO-synthase activity (Fig. 4). In the kidney, NO-synthase activity was 21.07 F 1.55 pkat/g prot in the control group. In the LNAME group, NO-synthase activity was inhibited by 29% (P < 0.05) v control and the addition of simvastatin to L-NAME increased NO-synthase activity by 26% (P < 0.05) v L-NAME group (Fig. 4). In the brain of the control group, NO-synthase activity was 9.4 F 10.70 pkat/g prot. In the L-NAME group, NO-synthase activity was inhibited by 37% (P < 0.05) v control and addition of simvastatin to LNAME increased NO-synthase activity by 46% (P < 0.05) v L-NAME group (Fig. 4). Co Q9 and Co Q10 concentrations Concentration of CoQ9 was 204.03 F 18.89 nmol/gww and of CoQ10 18.37 F 1.62 nmol/gww in the left ventricle of the control group. In the L-NAME group, Co Q9 and Q10 concentrations were only
Fig. 5. Effect of 6-week L-NAME, simvastatin and L-NAME + simvastatin treatment on the concentration of CoQ9 and CoQ10 in the left ventricle. C - control, S - 10 mg/kg/day simvastatin, L – 40 mg/kg/day L-NAME, LS - L-NAME + simvastatin in corresponding doses. *P < 0.05 compared to control.
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slightly decreased but the addition of simvastatin to L-NAME decreased the concentration of CoQ9 by 37% (P < 0.05) and of Q10 by 39% (P < 0.05) v the control group (Fig. 5).
Discussion The present study demonstrates the influence of the HMG-CoA reductase inhibitor simvastatin on the growth of the heart and aorta in L-NAME induced hypertension. L-NAME caused a reduction of NO synthase activity in the heart, kidney and brain, an increase in systolic blood pressure, hypertrophy of the LV and aorta, and fibrosis of the LV. Simultaneous treatment with simvastatin attenuated the systolic blood pressure increase and improved NO synthase production in the kidney and brain. Yet, the increase of left ventricular weight, thickness of the aorta, DNA concentration in the LV and left ventricular fibrosis development remained unaffected. Hypertrophy of the LV in the L-NAME model of hypertension was shown to be linked to increased fibrosis (Bernatova et al., 2000; Pereira and Mandarim-de-Lacerda, 2001; Pechanova et al., 1997) and protein remodeling of the left ventricle (Bernatova et al., 2000; Pechanova et al., 1997). In our previous experiment the ACE-inhibitor, captopril, completely prevented LVH and fibrosis development, however, NO synthase activity remained inhibited in all organs investigated (LV, brain, kidney and aorta) suggesting that prevention of remodeling was achieved by mechanisms different from the restoration of NO synthase activity (Pechanova et al., 1997). In this experiment, NO synthase activity was investigated in three organs with potentially different effects of NO-deficiency on the blood pressure. While the left ventricle is an organ with relatively autonomic blood flow control, in which NO production alteration is not supposed to have significant impact on blood pressure, NO-deficiency in the kidney may lead to vasoconstriction of renal artery and stimulate renin and angiotensin II production with general vasoconstriction and hypertension. Also inhibition of NO synthase in the rat brain was previously shown to increase blood pressure (Gerova et al., 1995). It seems reasonable to suppose that improvement of NO synthase activity in the kidney and brain might have played an important role in partial prevention of hypertension in this experiment. Interestingly, despite restoration of NOS activity in the kidney and brain and a reduction of blood pressure, simvastatin failed to prevent remodeling of the LV and aorta in our experiment. This result is partly in agreement with findings of Nahrendorf et al. (Nahrendorf et al., 2002). These authors found that left ventricular hypertrophy after myocardial infarction was attenuated and global function was improved by cerivastatin in rats. When the statin was applied simultaneously with the L-NAME, all positive effects were abolished (Nahrendorf et al., 2002). Several explanations of the inability of simvastatin to prevent remodeling of the heart and aorta in the model of L-NAME induced hypertension in our experiment are plausible. First, in the presence of L-NAME, simvastatin was not able to restore NO synthase activity in the myocardial tissue of the LV. It has been shown previously that local deficiency of NO per se, independently of blood pressure enhancement, was able to induce fibrosis of the left ventricular myocardium (Fig. 6) (Pechanova et al., 1999) and intimal remodeling of aorta (Rossi and ColombiniNetto, 2001). It seems that remodeling of the heart is relatively independent of blood pressure alterations in the L-NAME model and may be more closely associated with inhibition of local NO production. Thus, the lack of improvement of the local NOS activity in the LV by simvastatin, with potential local NO-deficiency in LV myocardium, might have participated in preserving the process of remodeling.
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Fig. 6. Left ventricle of a control rat (C), control treated with simvastatin (S), focal myocardial fibrosis in the left ventricular myocardium in the L-NAME treated (L) and L-NAME + simvastatin (LS) treated rat. Van Gieson, 200 .
Moreover, the prevention of hypertension was incomplete, although significant (162 mmHg in LNAME and simvastatin group v 182 mmHg in L-NAME group). Enhancement of blood pressure by 32% compared to control may have represented large enough afterload to provoke LV hypertrophy development. Second, the mechanism of L-NAME-induced LVH is a complex and not completely understood process (Simko and Simko, 2000). Besides the deficiency of NO production, with its antiproliferative effect on myocytes and fibrocytes in vivo and in vitro (Rossi and Colombini-Netto, 2001; Simko and Simko, 2000), the activation of RAS through depressed NO dependent relaxing ability of the renal artery (Holecyova et al., 1996) can participate in this process. Indeed, increased plasma renin activity was reported in NO-deficient hypertension and was associated with enhanced activity of local ACE in the LV and aorta (Takemoto et al., 1997). Statins seem to interfere with the renin-angiotensin system. Simvastatin have been shown to reduce angiotensin II-induced LV hypertrophy in vivo (Takemoto et al., 2001) and the same statin (simvastatin) induced reversion of LVH and fibrosis and improvement of the LV function in the model of aortic stenosis in rats. This LVH regression was associated with the attenuation of myocardial ACE activity and reduction of angiotensin II (Luo et al., 1999). However, several additional humoral systems, i.e. the sympathetic system (K-Laflamme et al., 1998), endothelin (Sventek et al., 1996) and aldosterone (Usui et al., 1998), which are supposed to participate in L-NAME induced hypertension and LVH development (Simko and Simko, 2000), are not influenced by
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simvastatin. Restoring the neurohormonal balance in the NO-deficient model, which could be necessary for hindering the pathologic hypertrophic growth, seems to be more complicated than in other models of LVH. Third, the protective effect of HMG-CoA reductase inhibitors against pathologic remodeling of the myocardium may be associated with the antioxidant nature of statins (Takemoto et al., 2001). Members of the small G proteins family, Rac 1 and RhoA have been suggested to be mediators of cardiac hypertrophic process (Van Aelst and D’Souza-Schorey, 1997). Furthermore, Rac1 was shown to mediate AngII-induced intracellular oxidation, which may significantly participate on the myocardial hypertrophic growth (Takemoto et al., 2001). Statins downregulate the activity of small G proteins in cardiomyocytes both in vivo and vitro enhancing thus eNOS expression (Laufs and Liao, 1998) and preventing the development of cardiac hypertrophy (Takemoto et al., 2001; Laufs et al., 2002), supposedly through an antioxidant mechanism involving inhibition of Rac1 (Takemoto et al., 2001). To investigate the activity of RhoGTPase family was beyond the scope of this publication. However, in accordance with the finding of Nahrendorf’s laboratory, where the protective effect of cerivastatin against pathologic LV remodeling after myocardial infarction was abolished by L-NAME, and on the basis of our experiment, where inhibition of myocardial NOS activity and development of LV hypertrophy in the model of L-NAME induced hypertension were not prevented by simvastatin, we speculate that in the presence of L-NAME the inhibition of small G-proteins by a statin is not functioning in the myocardium, omitting thus both improvement of endothelial NO production and the antioxidant effect of statins. In contrast to the antioxidant activity of statins, data have emerged in the literature that statins can undesirably decrease the level of CoQ in various organs including the heart (Bliznakov, 2002). Coenzyme Q is not only involved in the bioenergetic function of the cell, but also has strong antioxidant properties and may serve as an indicator of the antioxidant status of the myocardium (Bliznakov, 2002; Kucharska et al., 2000). In our work coenzyme Q 9 and Q10 levels were not decreased in the LV myocardium of simvastatin treated control rats. However, the simultaneous treatment with L-NAME and simvastatin significantly decreased the concentration of both CoQ9 and CoQ10 in the LV. The decreased antioxidant capacity of the myocardium, potentially resulting in increased oxidative stress, may have participated on the preservation of the remodeling process of the LV in the NO-deficient model of hypertrophic growth. Limitations of the work It is a matter of view, which parameter is optimal to characterize the altered NO production. Acetylcholine–induced vessel relaxation (Takase et al., 1996), inorganic nitrate in the urine (Forte et al., 1997), tissue NOS activity (Takase et al., 1996), cGMP concentration (Matsuoka et al., 1996), and most frequently expression of eNOS (Laufs and Liao, 1998; Laufs et al., 2002; Takemoto et al., 2001) and seldomly iNOS (Luvara et al., 1998) at mRNA and/or protein level were used to characterize nitric oxide production. Since in previous papers from our laboratory NOS activity in the heart and other organs were measured (Pechanova et al., 1997; Bernatova et al., 1999), we considered appropriate to use NOS activity as a potential indicator of NO production, in order the comparison of the protective effect of various substances could be possible. To characterize the level of fibrosis in the myocardium of the LV, the morphometric evaluation was used. However, more thorough information may be yielded by biochemical analysis of soluble and
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insoluble collagen protein fraction, as was performed in our previous works (Pechanova et al., 1997; Bernatova et al., 2000) or by investigation of collagen I protein expression.
Conclusion We conclude that simvastatin did not prevent pathological remodeling of the heart and aorta in the LNAME-induced hypertension despite improving NO synthase activity in the kidney and brain and attenuation of hypertension development. Although statins seem to be promising means of protection of the haemodynamically overloaded heart according to recent literature data, the level of their protective ability seems to depend on the model used and should be considered individually for each type of haemodynamic overload.
Acknowledgements This work was supported by the VEGA grants No 1/0532/03 and No 2/3185/23 and APVT grant No 51-013802.
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