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University of Missouri School of Medicine (J.H., A.W.-C., M.R.H., J.T., J.R.S.), Diabetes and Cardiovascular Laboratory (J.H., A.W.-C., M.Q., M.R.H., J.T., J.R.S.), and Harry S. Truman Veterans Affairs Medical Center (J.H., A.W.-C., J.T., J.R.S.), Columbia, Missouri 65212; Wake Forest University School of Medicine (C.F.), Winston-Salem, North Carolina 27157; State University of New York, Stony Brook, School of Medicine at Winthrop-University Hospital (A.T.), Stony Brook, New York 11794; The New York Harbor Veterans Affairs Healthcare System (R.M.), Brooklyn Campus, Brooklyn, New York 11209; and University of Arizona and Arizona Veterans Affairs Medical Center (S.A.C., C.S.), Tucson, Arizona 85724-5218
Address all correspondence and requests for reprints to: James R. Sowers, M.D., F.A.C.E., F.A.C.P., F.A.H.A., Professor of Medicine and Physiology, Department of Internal Medicine, Division of Endocrinology, D109 HSC Diabetes Center, Columbia, Missouri 65212. E-mail: sowersj{at}deptofmed.arizona.edu.
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Introduction |
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3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins), independent of their cholesterol-lowering properties, exert direct beneficial actions on cardiac function. Many of the salutary actions of statins are thought to be mediated by decreasing reactive oxygen species (ROS) production in various tissues. ROS derived from a phagocytic type of nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase plays a critical role in Ang-II-mediated cardiac hypertrophy, fibrosis, and remodeling (12, 13, 14, 15). NADPH oxidase is a multicomponent enzyme complex that consists of the membrane-bound heterodimer of gp91phox (phagocytic oxidase) and p22phox, the cytosolic regulatory subunits p40phox, p47phox, and p67phox and the small GTP-binding protein, Rac1 (13, 14, 15). A critical process in the activation of NADPH oxidase is the prenylation of Rac1 at its C-terminal domain, which determines its translocation to the membrane, exchange of GDP for GTP at its regulatory domain, and eventual activation (13, 14, 15). Statins, in addition to lowering cholesterol by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase enzyme, also reduce cellular levels of isoprenoids and inhibit the subsequent isoprenylation of small G proteins. Lack of Rac1 isoprenylation leads to cytosolic sequestration and loss of biological activity. This is one proposed cellular mechanism mediating the antioxidant actions of statins.
In rodent models of cardiac hypertrophy and remodeling, such as after aortic banding, chronic Ang-II infusion, and chronic hypertension, statin treatment reduces these adverse cardiac changes (16, 17). Alterations in neurohumoral factors, enhanced cardiac sympathetic drive, activation or inhibition of systemic RAS, and changes in levels of circulating inflammatory cytokines may all play a role in the development of cardiac hypertrophy and oxidative stress in these models precluding definitive conclusions on the role of alterations in cardiac RAS and oxidative stress in mediating the cardioprotective actions of statins. Therefore, in this study, we evaluated the impact of chronic statin treatment in a rodent model, a transgenic rat harboring the mouse renin gene TG (mREN-2)27 (Ren2) rat, in which tissue-specific elevation of renin and Ang-II is the primary pathogenic mechanism responsible for the observed cardiovascular phenotype (18, 19, 20, 21). In addition, use of this insulin-resistant rodent model (21) facilitates the investigation of the impact of chronic increases in tissue Ang-II, as observed in insulin-resistant states such as obesity, hypertension, and diabetes on cardiac function and remodeling. We further hypothesized that the beneficial effects of statin therapy would be mediated in part through inhibition of NADPH oxidase generation of ROS in the myocardium. To that end, we investigated the effects of rosuvastatin, a highly potent synthetic statin, on cardiac oxidative stress and remodeling in Ren2 rats and its nontransgenic littermate Sprague Dawley (SD) rats.
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Materials and Methods |
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Rosuvastatin treatment
Ren2 (6–7 wk old) and age-matched SD rats were randomly assigned to placebo-treated control (Ren2-C and SD-C, respectively) or rosuvastatin (Crestor; AstraZeneca, Macclesfield, Cheshire, UK) treatment groups (Ren2-RSV and SD-RSV). Rosuvastatin (10 mg/kg) in saline or an equal volume of saline was administered ip to the rosuvastatin or control groups, respectively, for 21 d.
Systolic blood pressure (SBP) and total body and heart weight
Restraint conditioning was initiated on the day of initial blood pressure measurements. SBP was measured in triplicate, on separate occasions throughout the day, using the tail-cuff method (Harvard Systems, student oscillometric recorder) before initiation of treatment and on d 19 or 20 (22, 23). In addition, total body weight was obtained before initiation of treatment and at the time the animals were killed. Heart weights were obtained at the time the animals were killed.
Echocardiography
Left ventricular (LV) contractility and dimensions were measured by echocardiography after treatment with rosuvastatin or vehicle. Echocardiography was performed using a 12-MHz linear transducer probe (Sonos 5500; Philips Medical Systems, Andover, MA) after the induction of anesthesia (pentobarbital sodium 20 mg/kg ip; Abbott Laboratories, Chicago, IL). LV systolic and diastolic internal dimensions were recorded from M-mode images using averaged measurements from three to five consecutive cardiac cycles. LV ejection fraction was calculated using the modified Simpson’s rule. LV mass was calculated using the truncated ellipsoid equation. Electrocardiogram was monitored continuously during the measurement.
Light microscopy
Harvested LV was immersed and fixed in 3% paraformaldehyde (22). After fixation, tissues were placed in histological cassettes and dehydrated with ethanol, infiltrated with low-melting (50 C) paraplast, and embedded in high-melting (56 C) paraplast. Paraffin sections (4 µm) of heart tissue were stained with Verhoeff-van Gieson (VVG), which stains elastin (black), nuclei (blue black), collagen (red), and connective tissue (yellow). Slides were then analyzed with a 50i microscope (Nikon, Tokyo, Japan) and x4, x10, and x40 images were captured with a cool snapcf camera. Morphometric analysis was performed using MetaVue software (Boyce Scientific Inc., Gray Summit, MO). In each image, the outer line of adventitia, media and lumen of 15 to 20 arteries were traced, and the percentage area was then calculated by subtracting the combined areas for media and lumen from the total area and then dividing by the total area.
Myocardial oxidative stress markers
Products of lipid peroxidation, tissue malondialdehyde (MDA) levels and nitrotyrosine content by immunostaining were determined as a surrogate end marker for oxidative stress (22). To determine MDA levels, butylated hydroxytoluene (5 mM) was added to cardiac tissue (100 mg) to prevent new lipid peroxidation. Samples were then homogenized in a buffer solution (0.25 M sucrose, 0.5 mM EDTA, 50 mM HEPES, protease inhibitors) on ice and centrifuged at 4 C at 15,000 rpm for 10 min. The supernatant was collected for MDA and O-phthaldialdehyde protein assays. Supernatant (200 µl) was used to measure free MDA using a MDA-586 spectrophotometric assay kit (22). Total protein was measured by O-phthaldialdehyde fluorometric assay on a FLX-800 fluorometer (BioTek, Winooski, VT).
To assess myocardial nitrotyrosine content, LV sections were deparaffinized, rehydrated, and epitopes were retrieved in citrate buffer. Endogenous peroxidases were quenched with 3% H2O2, and nonspecific binding sites were blocked with avidin, biotin, and finally with protein block (Dako, Carpinteria, CA). The sections were then incubated with 1:200 primary antibody, rabbit polyclonal antinitrotyrosine antibody (Chemicon, Temecula, CA). Sections were then washed and incubated with the secondary antibodies, linked, and labeled with Strepavidin (Dako) for 30 min each. After several rinses with distilled water, diaminobenzidine was applied for 10 min. The sections were again rinsed with distilled water, stained with hematoxylin for 1 min, rehydrated, and mounted with a permanent media. The slides were evaluated under a bright-field (Nikon 50i) microscope and the x40 images were captured with a cool snapcf camera. Images were analyzed and the signal intensities measured with MetaVue (Boyce Scientific).
NADPH oxide-dependent superoxide production in cardiac homogenates
Superoxides reduce the tetrazolium dye XTT to a soluble formazan product that can be readily quantified in solution (23). Heart tissue was homogenized using prechilled glass-on-glass Dounce homogenizer in homogenization buffer [20 mM Tris HCl, 50 mM KH2PO4 (pH 7.6), protease inhibitors, phosphatase inhibitors]. The homogenate was centrifuged at 10,000 x g for 10 min and supernatant used for assessing superoxide production. Superoxide dismutase (SOD)-inhibitable NADPH-dependent activity was assessed in cardiac homogenates, using a modified protocol (23, 24). SOD-inhibitable reduction of XTT was determined in a total volume of 0.2 ml of 10 mM potassium phosphate buffer (pH 7.5) containing 500 µM XTT (Sigma Chemical, St. Louis, MO) and 50 µg of cardiac homogenate with or without 100 U SOD (Sigma). Samples were equilibrated at 37 C and the reaction initiated by adding NADPH (100 µM, final concentration) for 20 min. After 20 min, absorbance was measured at a dual wavelength of 490/630 nm using a BioTek Elx microplate reader. The quantity of superoxides produced was determined using the molar extinction coefficient of 2.16 x 104 M–1 sec–1 for XTT.
Myocardial immunostaining of NADPH oxidase subunit and Rac1 expression
Immunostaining of NADPH oxidase subunits was conducted as previously described (22). Heart tissues were sectioned, deparaffinized in CitriSolv, and rehydrated in ethanol and HEPES wash buffer. Nonspecific binding sites were blocked with rabbit blocker for gp91phox, p22phox, p40phox, and goat blocker for Rac1. Tissue sections were subsequently incubated with primary antibodies (1:100); polyclonal goat anti-gp91phox, goat anti-p22phox, goat anti-p40phox (Santa Cruz Biotechnology, Santa Cruz, CA) and monoclonal mouse anti-Rac1 (Upstate Cell Signaling, Lake Placid, NY) in a humidified chamber. After 24 h, the slides were washed and incubated with Alexa fluor-labeled secondary antibodies (1:300) for 4 h in the dark. Subsequently the sections were washed and mounted with Mowiol. The slides were examined using a laser confocal scanning microscope, images were captured by using the Laser-sharp software (Bio-Rad), and signal intensities were measured by MetaVue analysis (22).
Statistical analysis
All values are expressed as mean ± SE. Statistical analyses were performed in SPSS 13.0 (SPSS Inc., Chicago, IL) using paired or unpaired Student’s t tests or ANOVA with Fisher’s least significant differences as appropriate. P < 0.05 was considered statistically significant.
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). However, rosuvastatin treatment had no significant effect on total body weight. Before rosuvastatin treatment (6–7 wk of age), SBP was significantly higher in Ren2 when compared with age-matched SD rats (Table 1). Administration of rosuvastatin to Ren2 or SD rats for 3 wk failed to significantly affect SBP (Table 1).
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, A and B). Consistent with this finding, there were increases in heart weight to body weight ratio in the Ren2 rats, compared with SD rats that was reduced by rosuvastatin (Fig. 1C). Furthermore, the Ren2 rats exhibited significant systolic dysfunction compared with the SD rats as evidenced by the reduced ejection fraction. Rosuvastatin prevented the decrease in ejection fraction (Fig. 1D).
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, A and B). Rosvastatin significantly blunted this increase. Interestingly, there were no significant differences observed among the average area of media and the lumens of intramural arteries in SD-C, Ren2-C, and Ren2-RSV groups of rats (Fig. 2B). The perivascular fibrosis visualized in the Ren2 consistently appeared to emanate into the interstitium of the endomysium between cardiomyocytes. In addition, other microscopic changes in Ren2 rats included luminal narrowing of small intramural coronary arteries and increased thickness of the arteriolar wall.
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). Nitrotyrosine, a specific marker for protein modification by nitric oxide-derived oxidants, was assessed by analysis of the bright-field images from nitrotyrosine stained heart tissue sections. Myocardial nitrotyrosine content was increased in the Ren2 when compared with SD rats (Fig. 3, B and C). Rosuvastatin treatment attenuated nitrotyrosine levels in both the Ren2 and SD rats. Similarly, levels of MDA (an end product of lipid peroxidation) were significantly lowered in both SD and Ren2 rats when compared with their respective untreated cohorts (Fig. 3D). However, levels of MDA were similar in the untreated Ren2 and matched SD rats.
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, A and B). Furthermore, there were reductions in NADPH oxidase subunit and Rac1 expression in the statin-treated Ren2 and subunit expression in the SD group.
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Discussion |
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Results of this investigation also confirmed that this hypertensive (18, 19, 20, 21, 22) model, when compared with aged matched (9–10 wk old) SD rats, had LV hypertrophy, myocardial remodeling, and increased myocardial ROS. The most notable morphological alteration was that of coronary perivascular fibrosis. Treatment of Ren2 rats for 3 wk with low statin (10 mg/kg·) resulted in significant decreases of the perivascular adventitia area but did not significantly alter the lumen diameter of the intramural coronary arteries. Because there were no significant reductions in blood pressure after low-dose statin treatment, the reduction in LV mass and morphological improvement cannot be attributed simply to after load reduction and suggest that rosuvastatin had direct pleiotrophic effects on the myocardium.
These data also show that the Ren2 heart manifests increased NADPH oxidase activity, compared with age-matched SD normotensive controls. In parallel with increased NADPH oxidase activity, there was an increase in lipid peroxidation products, as measured by nitrotyrosine staining, in Ren2 myocardial tissue; these markers, including MDA, were reduced significantly by low-dose statin therapy. NADPH oxidase catalyzes the one-electron reduction of molecular oxygen to superoxide anion, which can react to form short-lived peroxynitrite. Peroxynitrite forms stable 3-nitrotyrosine conjugated molecules (13, 14). Indeed, 3-nitrotyrosine staining was particularly pronounced in the perivascular regions, areas of increased inflammatory-induced fibrosis. Oxidative stress is a known stimulus for fibrosis; ROS can activate redox-sensitive transcription factors such as nuclear factor-
ß which, in turn, promotes fibrosis. In vitro studies have shown that Ang-II increased ROS in cultured myocardial fibroblasts as well as cardiomyocytes (11, 30). The myocardium expresses a relatively limited endogenous antioxidant capacity as contributed by both enzymatic and nonenzymatic free radical scavengers and antioxidants. This property renders the heart more susceptible than other tissues to oxidative stress, with attendant structural and functional abnormalities (31). Elevated myocardial levels of oxidative stress have been linked to LV hypertrophy, abnormal metabolic signaling, and LV systolic and diastolic dysfunction (30, 31). The current observation that in vivo statin treatment significantly decreases NADPH oxidase activity and ROS levels, in concert with decreased LV mass and cardiac interstitial and perivascular fibrosis, is the first such report in a chronic in vivo model associated with increased tissue Ang-II.
Myocardial immunohistochemistry demonstrated that the increased NADPH oxidase activity and nitrotyrosine staining were associated with increases in Rac1, gp91phox, p40 phox, and p22phox subunits of NADPH oxidase. In concert with decreases in NADPH oxidase/MDA/nitrotyrosine, there was a significant reduction in Rac1 expression after low-dose statin therapy in the Ren2 rat. Given the critical role of Rac1 translocation to the membrane in the activation of the NADPH complex, the effect of statin therapy to decrease membrane translocation appears to be important underlying mechanism for the cardioprotective effects of this therapy (15). There have been prior reports that treatment of rat cardiomyocytes with Ang-II causes hypertrophy of the cells, which is partly dependent on Rac1 translocation to cell membranes and consequent activation of the NADPH oxidase complex (15, 32, 33, 34). Thus, the ability of statins to inhibit the activation of small molecular weight G proteins appears to account for part of their cardioprotective pleiotropic effects (16, 17).
That statin therapy reduced the expression of gp91phox in the Ren2 rat has not been previously reported. gp91phox is the catalytic subunit and the major membrane component to which the p40 phox, p47 phox, and p67 phox complex binds via Rac1 (35). Although increased levels of this cell membrane-associated NADPH oxidase subunit may contribute to increased membrane enzyme activity (12), there are disparate reports on the role of this subunit in Ang-II mediated cardiomyocyte hypertrophy (12, 36). In gp91phox-deficient mice, basal blood pressure is lower than wild-type counterparts, and subpressor doses of Ang-II infused over 2 wk fail to induce cardiac hypertrophy (12). However, in a transgenic mouse with elevated plasma Ang-II levels and gp91phox deficiency, development of cardiac hypertrophy occurred (41). Thus, impact of its reduction with statin therapy in LV mass regression in the Ren2 remains to be clarified.
That the p40phox subunit was elevated in this model of chronic elevation of Ang-II in the myocardium and that the levels of this subunit were reduced with statin therapy are also novel observations. There is very little known about the role of p40phox in cardiac redox function. Nonetheless, there is evidence that it acts as a bridge between p47 phox and p67 phox to assemble the NADPH oxidase complex on the membrane (37). p40phox may be derived from fibroblasts or mononuclear cells that have infiltrated the myocardium (38, 39, 40). This notion is consistent with our current observation of increased perivascular fibrosis of the Ren2 rat. That p40phox subunit expression decreased in parallel with decreased NADPH oxidase activity and products of oxidative stress, perivascular fibrosis, and monocyte infiltration after 3 wk of statin therapy suggest that this subunit may be of relevance in Ang-II-induced oxidative stress and resultant cardiac remodeling in this model of chronic cardiac overexpression of Ang-II.
In summary, this investigation demonstrates a rosuvastatin-mediated reduction in oxidative stress such as reduced myocardial remodeling including ventricular mass regression in the Ren2 rodent model. These beneficial effects of statin therapy are related partially to reduced cardiac membrane translocation/activation of the NADPH oxidase enzyme subunits, NADPH oxidase activity, and consequent reductions in oxidative stress.
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Acknowledgments |
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Footnotes |
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Current address for R.M.: Laboratory of Clinical Investigation, 10-CRC-Hatfield Clinical Research, National Institutes of Health, Bethesda, Maryland.
Disclosure Summary: All authors have nothing to declare.
First Published Online February 22, 2007
Abbreviations: Ang-II, Angiotensin-II; LV, left ventricular; MDA, malondialdehyde; NADPH, nicotinamide adenine dinucleotide phosphate reduced; phox, phagocytic oxidase; RAS, renin-angiotensin system; Ren2, mouse renin gene TG (mREN-2)27; ROS, reactive oxygen species; SD, Sprague-Dawley; SBP, systolic blood pressure; SOD, superoxide dismutase; VVG, Verhoeff-van Gieson.
Received October 4, 2006.
Accepted for publication February 1, 2007.
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with Rac1 GTPase mediates free radical production during myocardial hypertrophy. Cardiovasc Res 71:342–351 12/13- and reactive oxygen species-dependent activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase by angiotensin receptor stimulation in rat neonatal cardiomyocytes. J Biol Chem 280:18434–18441
and stimulated macrophages in cardiovascular disease. Curr Vasc Pharmacol 4:205–213[CrossRef][Medline]This article has been cited by other articles:
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