The 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase inhibitors, or statins, are used to treat hypercholesterolemia and coronary artery disease and are among the most frequently prescribed medications in clinical practice.1 However, clinical trials of statins for primary and secondary prevention of coronary artery disease excluded patients with heart failure.1,2 Despite the frequent intersection of coronary artery disease with heart failure, the relation of statin therapy to heart failure outcome is thus not well characterized. A recent preliminary study suggested that statins may be a therapeutic option to improve survival in individuals with diastolic heart failure, whereas an angiotensin-converting enzyme (ACE) inhibitor, blocker of the type 1a receptor for angiotensin II (ARB), β-blocker, or calcium antagonist had no significant effect on survival.3
Left ventricular (LV) remodeling is generally accepted as a determinant of the clinical course of heart failure.4 Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are important players in the development of myocardial fibrosis associated with heart failure.5,6 MMPs are zinc-dependent enzymes that contribute to turnover of the extracellular matrix (ECM). Expression of MMP genes and the gelatinolytic activity of MMPs are increased in the left ventricle of animals or humans with failing hearts.6,7 Previous studies have assessed the expression and activity of MMPs after the occurrence of LV dilation, with preventive pharmacological inhibition of these enzymes resulting in suppression of LV remodeling in animal models of heart failure.8
Endothelin (ET) is a potent vasoactive peptide with hypertrophic and growth-promoting activities. It is produced by endothelial cells as well as by cardiac myocytes,9 and it modulates ECM remodeling through stimulation of collagen synthesis by fibroblasts.10 Administration of ET-1 also induced MMP activation in the isolated, blood-perfused normal rat heart and LV dilation in vivo.11 The plasma concentration of ET-1 is increased in patients with heart failure and correlates with disease severity.9 In addition, the local ET system is activated in the failing heart,9,12 possibly contributing to the progression of heart failure and increasing the severity of LV dysfunction in the setting of heart failure. Statins have been shown to inhibit ET-1 production at the level of gene transcription.13 However, the possible effects of statins on cardiac remodeling and changes in the local ET system associated with hypertensive heart failure are unclear.
We have now investigated whether pitavastatin might be of therapeutic benefit for heart failure by evaluating its effects on the local ET system and MMP activity in hypertensive rats. We also examined the effects of this drug on the transition to heart failure by administering it before or after the development of LV hypertrophy (LVH).
Male Dahl-Iwai salt-sensitive (DS) rats (DIS/Eis; Eisai, Tokyo, Japan) were fed laboratory chow containing 0.3% NaCl (low-salt diet) from weaning until 7 weeks of age, after which the diet was either left unchanged (control group) or switched to laboratory chow containing 8% NaCl (high-salt diet). Animals on this latter diet serve as models of hypertensive LVH and heart failure at 12 and 19 weeks of age, respectively.14 Both food and tap water were provided ad libitum throughout the experiment. The rats on the high-salt diet were divided into the following 3 groups: those subjected to oral administration of pitavastatin (0.3 mg per kilogram of body weight per day) (Kowa, Tokyo, Japan) from 7 to 19 weeks of age (Pitava-E group); those subjected to treatment with pitavastatin at the same dose from 12 to 19 weeks of age (Pitava-L group); and those treated with vehicle (0.5% carboxymethylcellulose) from 7 to 19 weeks of age (untreated group). Animal care and treatment were in accordance with the guidelines of Nagoya University Graduate School of Medicine and with the NIH Guide for the Care and Use of Laboratory Animals (NIH publication no. 85-23, revised 1996).
Physiological Measurements and Determination of Serum Lipid Concentrations
Systolic blood pressure of conscious rats was measured weekly by the tail-cuff method. At 12 and 19 weeks of age, animals were subjected to transthoracic echocardiographic analysis with a SONOS 5500 system (Philips Medical Systems, Bothell, WA). Wall thickness and LV end-diastolic diameter were obtained from a short-axis view at the level of the papillary muscles. The thickness of the interventricular septum and the LV posterior wall were measured, and fractional shortening was calculated. The early diastolic myocardial velocity was derived from tissue Doppler imaging of the epicardium as a measure of diastolic function.15 After physiological measurements, the rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg), and arterial blood was immediately collected from the abdominal aorta. Serum was isolated and assayed for total cholesterol, high-density lipoprotein-cholesterol, and triglycerides by enzymatic methods.15
Transverse sections (thickness, 3 μm) of LV tissue were prepared and stained either with Azan Mallory solution for evaluation of fibrosis or with Sirius red F3BA (0.5% in saturated aqueous picric acid) (Aldrich, Milwaukee, WI) for evaluation of collagen deposition. To determine the extents of interstitial fibrosis and collagen deposition in the left ventricle, we selected 5 fields at random and calculated the ratio of the area of Azan Mallory-stained fibrosis or collagen-specific staining to the total area of the myocardium with the use of WinROOF software version 5.0 (Mitani, Tokyo, Japan).
Quantitation of Gene Expression
Total RNA was extracted from LV tissue, and the abundance of specific mRNAs was determined by reverse transcription and real-time polymerase chain reaction analysis with a Prism 7700 Sequence Detector (Perkin-Elmer, Foster City, CA). Sequences of primers and TaqMan probes specific for atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), β-myosin heavy chain, ACE, transforming growth factor (TGF)–β1, collagen types 1 and 3, prepro–ET-1, endothelin-converting enzyme-1, ET type A (ETA) receptor, ET type B receptor, TIMP-1 or -2, and MMP-2, -9, -13, or -14 were described previously.16–18 The sequences of forward and reverse primers and the detection probe specific for fibronectin were 5′-CTACTCAAGCCCTGAGGATGGA-3′, 5′-TGACTGTGTACTCAGAACCCGG-3′, and 5′-TTTTCCCTGCGCCTGATGGTGA-3′, respectively, and those for connective tissue growth factor (CTGF) were 5′-CAATACCTTCTGCAGGCTGGAG-3′, 5′-GGATGCACTTTTTGCCCTTCTT-3′, and 5′-CAGGCCCTGTGAAGCTGACCTAGAGGAAAA-3′, respectively.
TaqMan rodent glyceraldehyde-3-phosphate dehydrogenase control reagents (Perkin-Elmer) were used for detection of glyceraldehyde-3-phosphate dehydrogenase mRNA as an internal standard.
Measurement of Plasma and Myocardial Levels of ET-1
ET-1 was extracted from plasma and LV tissue and was quantified with an enzyme immunoassay kit (Immuno-Biological Lab, Gunma, Japan). In brief, LV tissue was disrupted with a Polytron homogenizer in 10 volumes of 1 mol/L acetic acid and 20 mmol/L HCl, and the homogenate was boiled and then centrifuged. ET-1 was extracted from the resulting supernatant with a Sep-Pak C18 cartridge and measured. Plasma was mixed with 10% acetic acid, after which ET-1 was extracted and measured as for the LV homogenate.
Immunohistochemical Staining for ET-1 in LV Tissue
Transverse sections (3 μm in thickness) of LV tissue were incubated with rabbit polyclonal antibodies to ET-1 (Immuno-Biological Lab), and immune complexes were subsequently detected by incubation with horseradish peroxidase-conjugated goat antibodies to rabbit immunoglobulin G (Pierce, Rockford, IL). Peroxidase activity was visualized with diaminobenzidine and hydrogen peroxide. The sections were counterstained with hematoxylin. The specificity of ET-1 detection was confirmed by staining of consecutive sections with primary antibodies that had been pretreated with ET-1 (Peptide Institute, Osaka, Japan).
LV tissue was homogenized in an ice-cold solution containing 50 mmol/L Tris-HCl (pH 7.5) and 150 mmol/L NaCl. The homogenate was centrifuged and the resulting supernatant was subjected to gelatin zymography as described previously.19 Bands of gelatinolytic activity were quantitated with image analysis software (NIH Image 1.62).
Data are presented as means±SEM. Paired data were analyzed by the paired Student t test. Differences in multiple parameters between 2 groups were evaluated by 2-way analysis of variance, and significant differences were compared by 1-way analysis of variance followed by Dunnett post hoc test. Survival rate was analyzed by the standard Kaplan-Meier method with a log-rank test. A P value of <0.05 was considered statistically significant.
Systolic Blood Pressure and Serum Lipid Levels
DS rats fed with high-salt diet from 7 weeks of age progressively developed hypertension. Systolic blood pressure was significantly higher in all animals on the high-salt diet than in those of the control group at 12 and 19 weeks of age (Table 1). Treatment with pitavastatin did not affect blood pressure. The serum concentrations of total cholesterol and high-density lipoprotein-cholesterol were significantly higher in rats fed with high-salt diet than in those of the control group at 12 and 19 weeks of age, they were not affected by treatment with pitavastatin (Table 2). The serum concentration of triglycerides did not differ among the 4 groups of rats (Table 2). We chose a dose of pitavastatin that would not affect serum lipid levels or blood pressure on the basis of the results of previous studies with rat models.20–22 These studies used doses ranging from 0.1 to 10 mg/kg per day, whereas we used a dose of 0.3 mg/kg per day. Statins lack a cholesterol-lowering effect in rats because the induced HMG-CoA reductase activity overcomes the inhibitory action of these drugs, resulting in a net increase in cholesterol synthesis.23 Other studies have also shown that treatment with statins had no effect on cholesterol levels in mice or rats.24,25 These rodent models thus seem to be suitable for isolating the effects of statins on cardiac phenotypes from their cholesterol-lowering effect apparent in humans.
LV Geometry and Function
The left ventricle/body weight ratio was significantly greater in the untreated group than in the control group at 19 weeks of age, and this difference was significantly reduced by early or late pitavastatin treatment (Table 1). Although there was no difference in LV wall thickness among the 3 groups of rats on the high-salt diet, the LV end-diastolic diameter was significantly greater in the untreated group than in both the control group and the pitavastatin-treated groups at 19 weeks. The early diastolic myocardial velocity and LV fractional shortening were significantly smaller in the untreated group than in the control group at 19 weeks; both of these parameters were significantly greater in the Pitava-L group than in the untreated group, whereas early diastolic myocardial velocity was also significantly greater in the Pitava-E group than in the untreated group. The ratio of lung weight to body weight was significantly greater in untreated rats than in the control group, and this difference was significantly reduced by pitavastatin treatment. Pitavastatin thus improved diastolic and systolic function in animals on the high-salt diet at 19 weeks of age.
Kaplan-Meier analysis revealed that the survival rate of rats in the untreated group was markedly reduced compared with that of animals in the control group (Fig. 1). Animals in the Pitava-E and Pitava-L groups began to die from 13 to 17 weeks of age, respectively. Although the survival rate did not differ significantly between the 2 pitavastatin-treated groups (P=0.56), that in each of the pitavastatin-treated groups was markedly greater than that in the untreated group.
LV Fibrosis and Collagen Deposition
Marked interstitial fibrosis and collagen deposition were detected in the heart of rats in the untreated group at 19 weeks of age. Indeed, the areas of fibrosis (Figs. 2A, B, E) and collagen deposition (Figs. 2F, G, J) were 4.6-fold and 2.8-fold greater in the untreated group than in the control group. These differences were significantly reduced by early or late treatment with pitavastatin (Fig. 2).
Expression of Hypertrophic and Profibrotic Genes
Hemodynamic overload resulted in up-regulation of the expression of fetal-type genes, including those for ANP, BNP, and β-myosin heavy chain, as well as of that of the ACE gene, the genes for the growth factors TGF-β1 and CTGF, and those for the ECM proteins fibronectin and collagen types 1 and 3 in the left ventricle of untreated rats at 19 weeks of age (Table 3). With the exception of the changes in the expression of the ANP gene and of the BNP gene in the Pitava-L group, these increases in gene expression were inhibited by pitavastatin treatment.
Circulating and Myocardial Levels of ET-1
The myocardial abundance of prepro–ET-1, endothelin-converting enzyme-1, and ETA receptor mRNAs was significantly higher in the untreated group than in the control group at 19 weeks of age, whereas that of ET type B receptor mRNA was increased at both 12 and 19 weeks (Fig. 3). This up-regulation of the ET-1 system induced by the high-salt diet was inhibited by pitavastatin administration. The localization of cells containing the mature ET-1 peptide in the left ventricle was examined by immunohistochemical analysis. ET-1 immunoreactivity was detected in both endothelial cells and myocardial cells, its intensity being greater in the untreated group than in either the control group or the pitavastatin-treated groups at 19 weeks of age (Figs. 4A–E). The myocardial and circulating levels of ET-1 in the untreated group were also significantly greater than those in the control group, as revealed by enzyme immunoassay (Figs. 4F, G). Early or late pitavastatin administration attenuated these increases in ET-1 concentration in both LV tissue and plasma. The abundance of prepro–ET-1 mRNA thus changed in parallel with the tissue level of the mature peptide, suggesting that the synthesis of prepro–ET-1 mRNA is an important step in local activation of the ET-1 system.
Expression of MMPs and TIMPs
The abundance of MMP-2, -9, -13, and -14 as well as TIMP-1 and -2 mRNAs in the left ventricle was significantly greater in the untreated group than in the control group at 19 weeks of age (Fig. 5A). The differences in the amounts of MMP-2, -9, and -13 as well as TIMP-1 and -2 mRNAs were significantly reduced by early pitavastatin treatment, whereas those in the amounts of MMP-2, -9, and -14 as well as TIMP-2 mRNAs were significantly reduced by late pitavastatin treatment.
Electrophoretic zymography also showed that the total gelatinase activity of MMP-2 and the ratio of mature MMP-2 to total MMP-2 were increased in LV tissue of untreated rats compared with those in control rats at 19 weeks (Fig. 5B). The gelatinolytic activity of MMP-9 was also greater in the untreated group than in the control group (Fig. 5B). Early or late treatment with pitavastatin inhibited these changes in MMP-2 and MMP-9 activity.
We have shown that pitavastatin prevented heart failure when administered either before or after the development of LVH, and that this effect was independent of changes in blood pressure or serum lipid levels. Pitavastatin is a synthetic HMG-CoA reductase inhibitor and manifests a cholesterol-lowering effect 10 times greater than that of pravastatin or simvastatin.26,27 DS rats fed with high-salt diet manifest marked diastolic and systolic dysfunction at 19 weeks of age and subsequently die of congestive heart failure. In the present study, we found that pitavastatin ameliorated cardiac dysfunction and prolonged survival even when administered after development of LVH in this rat model of heart failure. Furthermore, inhibition of the transition from LVH to heart failure by statin treatment was accompanied by inhibition of changes in the cardiac ET-1 system and MMP activity in this animal model.
The Renin-angiotensin System and Isoprenylation as Targets of Pitavastatin in Suppression of the ET-1 System in DS Rats
DS rats provide a useful model for studying the distinct transition from LVH to heart failure. Cardiac angiotensin II and ET-1 signaling are activated differentially in these animals during this transition.28 During the transition to heart failure, the angiotensin II system remains activated and the ET-1 system becomes activated in a manner that parallels the progression of myocardial dysfunction.28 In models of systolic heart failure, blockade of ET receptors attenuates systolic dysfunction and LV fibrosis, resulting in the prevention of overt heart failure.9,29 These observations support the notion that attenuation of activation of the ET system improves cardiac function and prevents the transition to heart failure. In the present study, the increases both in the plasma and cardiac levels of ET-1 and in the cardiac abundance of prepro–ET-1 mRNA associated with heart failure were greatly inhibited by pitavastatin treatment. Cross-talk between the angiotensin II and ET-1 systems has been demonstrated.30,31 Angiotensin II has thus been shown to induce ET-1 synthesis in cardiomyocytes in vitro.31 Furthermore, lovastatin has been shown to inhibit the activity of angiotensin II,31 thus accounting for the down-regulation of ET-1 expression by this drug.32 We have now shown that pitavastatin prevented the up-regulation of ACE gene expression in the failing heart of DS rats, an effect that might contribute to the down-regulation of ET-1 expression. The administration of mevalonate to pravastatin-treated rats revealed that inhibition of the mevalonate pathway is important for the reduction in ET-1 levels induced by pravastatin.33 Statins inhibit the synthesis of isoprenoid intermediates of cholesterol biosynthesis that are important in the posttranslational modification of members of the Ras and Rho families of small GTP-binding proteins.34 The inhibition of ET-1 expression by pitavastatin treatment observed in the present study may thus have resulted from suppression both of the renin-angiotensin system and of isoprenylation required for ET-1 gene transcription.
ECM Synthesis and Degradation as Targets of Pitavastatin in Inhibition of Cardiac Remodeling in DS Rats
Cardiac remodeling results from an imbalance between ECM synthesis and degradation and is thought to be central to the pathophysiology of heart failure.35 The relative levels of MMP-2 and MMP-9 are increased in the LV myocardium of humans with end-stage heart failure.6,35 MMP activity, especially that of MMP-9, has been shown to increase during the transition from compensatory hypertrophy to heart failure in DS rats fed with high-salt diet.17 In the present study, collagen accumulated in the failing heart of DS rats despite associated increases in MMP mRNA and activity levels. The up-regulation of collagen types 1 and 3 genes and of other profibrotic genes may thus overcome the activation of MMPs and contribute to cardiac remodeling. Collagen deposition in the heart was inhibited by pitavastatin treatment initiated either before or after the development of LVH, and this effect was accompanied by inhibition of both collagen and MMP gene expression. Both MMP-2 and MMP-9 degrade collagen in basement membranes, whereas MMP-13 degrades fibrillar collagen.36 MMP-14 (MT1-MMP) is a membrane-type MMP and participates in activation of MMP-2 at the cell surface.37 In the present study, the expression of the genes for all 4 of these MMPs in the heart of DS rats was increased by feeding the high-salt diet, and these effects were inhibited by pitavastatin treatment.
MMP activity is strictly regulated by endogenous inhibitors, including the MMP-specific family of TIMPs, in various tissues including the myocardium.38 An imbalance between MMPs and TIMPs has been thought to contribute to pathologic remodeling of the heart. We have now shown that the abundance of TIMP-1 and TIMP-2 mRNAs was significantly increased in the left ventricle of 19-week-old DS rats fed with high-salt diet, consistent with previous observations with humans or rats with heart failure.6,17 Such an increase in TIMP mRNA abundance might reflect a compensatory mechanism to counteract the associated increase in MMP gene expression. Treatment with pitavastatin inhibited the up-regulation of TIMP gene expression, although initiation of treatment after the development of LVH was not able to prevent the change in TIMP-1 gene expression.
Production of ET-1 as a Target of Pitavastatin in Down-regulation of MMP Activity in DS Rats
Several studies have linked ET-1 to the regulation of MMPs. Environmental stress was thus shown to increase vascular MMP-2 activity via stimulation of ET-1 synthesis and ETA receptor activation.39 In addition, selective antagonism of the ETA receptor reduced MMP-2 activity in the kidney40 and heart41 of hypertensive rats. Dual ET receptor antagonists have also been shown to induce down-regulation of MMP-2 activity.42 The beneficial effects of pitavastatin observed in the present study might therefore be attributable in part to inhibition of the production of ET-1 and consequent down-regulation of the activities of MMPs in the left ventricle of DS rats fed with high-salt diet.
Increase in the Survival Rate of DS Rats Induced by Pitavastatin Treatment
DS rats on a high-salt diet develop heart failure around 19 weeks of age,14 and this condition is a major cause of death. Treatment with pitavastatin induced a significant increase in the survival rate of these animals and improved cardiac function even when first administered after the development of LVH. Statin treatment has previously been shown to reduce mortality from heart failure in animals and humans.3,15,43 A recent clinical study thus showed that statin therapy was more effective in improving survival in individuals with diastolic heart failure than was treatment with an ACE inhibitor, ARB, β-blocker, or calcium antagonist.3 Consistent with the results of a previous study,44 we have now shown that pitavastatin inhibited the progression of LVH, fibrosis, and remodeling, all of which effects might underlie the prevention of heart failure.
The Pitava-E group in the present study showed an early decrease in the surviving fraction similar to that observed in the untreated group, although at 19 weeks the surviving fractions for the Pitava-E and Pitava-L groups were identical and were signficantly greater than that for the untreated group. The reason for this apparent difference in early mortality between the 2 treatment groups is unclear and warrants further investigation.
We have shown that treatment with pitavastatin improved LV function, inhibited remodeling of the cardiac ECM, and increased survival in a rat model of heart failure without exerting antihypertensive or lipid-lowering effects. The prevention of heart failure by pitavastatin in this model was apparent even when treatment was initiated after the development of LVH and may result from beneficial modulation of signaling by angiotensin II and ET-1. Our results thus support the notion that statins may prove effective for the treatment of heart failure in humans.
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