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1Department of Molecular and Cellular Physiology, 2Division of Cardiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana; 3Division of Cardiology and Department of Pathology, Albert Einstein College of Medicine, Bronx, New York; and 4Institute of Molecular Cardiology, University of Louisville, Louisville, Kentucky
Submitted 31 March 2006 ; accepted in final form 12 July 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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nitric oxide; 3-hydroxy-3-methylglutaryl coenzyme A; myocardial infarction; ventricular remodeling; echocardiography
It is now widely accepted that statins increase NO bioavailability via activation of endothelial nitric oxide synthase (eNOS). Indeed, several groups have identified multiple molecular mechanisms for statin-mediated eNOS activation (26). Specifically, statins have been shown to increase eNOS mRNA half-life by inhibition of Rho (15) and phosphorylation of eNOS protein by activation of the phosphatidylinositol 3-kinase/Akt pathway (13). Statin-mediated modulation of eNOS expression and activity may prove beneficial in deterring the progression of cardiovascular diseases, especially congestive heart failure (CHF). Endothelial dysfunction leads to compromised eNOS activity in animal models and humans with CHF (2, 4, 5). A previous study of CHF in mice demonstrated that genetic overexpression of eNOS preserved cardiac performance and improved survival (11). Such results suggest that pharmacological approaches to improve eNOS activity would exert protective effects in the setting of CHF.
We hypothesized that HMG CoA reductase inhibition with a low dose of simvastatin would attenuate the severity of CHF via induction of eNOS. We induced CHF in mice by permanent ligation of the left coronary artery (LCA) and evaluated survival, left ventricular (LV) function, cardiac hypertrophy, and pulmonary edema.
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MATERIALS AND METHODS |
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Eight- to ten-week-old male C57BL/6J mice utilized in the study were obtained from Jackson Laboratory (Bar Harbor, ME). In addition, eNOS–/– mice were originally donated by Dr. Paul Huang (Massachusetts General Hospital). The eNOS–/– mice were back crossed onto the C57BL/6J background for at least 15 generations and generated in our breeding colony. The eNOS–/– mice that were used were 8–10 wk of age.
Animals
All animals received humane care in compliance with the "Principals of Laboratory Animal Care" formulated by the National Society of Medical Research and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996). The experimental protocol for the present study was reviewed and approved by the Institutional Animal Care and Use Committee of the Albert Einstein College of Medicine.
Simvastatin Preparation
Preparation. Pure simvastatin powder was obtained from Merck (Rahway, NJ) in the inactive lactone prodrug form. Simvastatin was converted to its active dihydroxy-open acid form by dissolving the pure powder in ethanol and sodium hydroxide followed by pH neutralization with hydrochloric acid. The dihydroxy-open acid form of simvastatin was then diluted with PBS and aliquoted into single-dose vials containing 150 µl of simvastatin solution.
Administration. The experimental protocols for the present study are depicted in Fig. 1, A and B. Wild-type mice were subjected to permanent ligation of the LCA or to sham surgery and then randomized to receive intraperitoneal injections of simvastatin (0.25 mg/kg) or saline vehicle injections daily (QD) for 7 days followed by 21 days of every other day (QOD) injections during the 28-day protocol with the initial dose given 2 h after myocardial infarction or sham surgery (Fig. 1A). An additional study group of wild-type mice (Fig. 1B) were dosed with simvastatin (0.25 mg/kg) QD for 5 days starting at 48 h after LCA occlusion followed by 21 days of simvastatin therapy (0.25 mg/kg QOD).
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Myocardial Infarction Protocol
Ligation of the LCA was performed as previously described (12). Mice were anesthetized with intraperitoneal injections of pentobarbital sodium (50 mg/kg) and ketamine hydrochloride (50 mg/kg). The animals were then attached to a surgical board ventral side up, orally intubated with polyethylene-90 tubing, and connected to a rodent ventilator (Harvard Apparatus, Natick, MA). Core body temperature was maintained at 37.0°C throughout the procedure with a rectal thermometer and infrared heat lamp. Baseline two-dimensional echocardiography was performed followed by a median sternotomy with an electric cautery. The LCA was microscopically visualized and completely occluded with a 7-0 suture mounted on a tapered needle (12). The chest wound was then reapproximated, and the animals received a subcutaneous injection of butorphanol tartrate (0.1 mg/kg). Animals were extubated, provided with supplemental oxygen (100%), and allowed to recover in a temperature-controlled environment. Mice that survived 24 h after myocardial infarction were enrolled in the survival study and monitored for 28 days. After 28 days, mice were anesthetized and evaluated by two-dimensional echocardiography, and heart and lung tissue were excised to calculate heart-to-body weight ratios and pulmonary edema, respectively.
An additional group of wild-type mice were subjected to permanent LCA ligation to assess the effects of simvastatin treatment on the extent of myocardial infarction. Mice were randomized to receive simvastatin (0.25 mg/kg) (n = 8) or saline vehicle (n = 8) 2 h after LCA ligation. Mice were allowed to recover for 48 h and then anesthetized; myocardial area at risk and infarct size were then assessed by in vivo Evans blue injection and by the ex vivo 2,3,5-triphenyltetrazolium chloride staining method, as previously described (12).
Cardiac Hypertrophy
Cardiac hypertrophy was assessed by dividing the ex vivo heart weight by the body weight of the mouse. The resulting heart-to-body weight ratio provides an index of cardiac hypertrophy.
Brightfield Microscopy for Myocyte Size and Infarct Size
Midventricular myocardial specimens were obtained from mice receiving either vehicle or simvastatin subsequent to myocardial infarction. Thick sections were fixed in paraformaldehyde, embedded in paraffin, and sectioned into 10-µm sections. Slides were stained with hematoxylin and eosin to assess cellular hypertrophy. Using transmitted light, we viewed slides on a Nikon TE2000E2, which was fitted with a x60 Plan Apo oil immersion objective. Images were captured and planimetrically analyzed with MetaMorph software (version 6.3r2). For the myocyte hypertrophy data, at least 50 myocytes with centrally located nuclei were evaluated from multiple LV sites from each sample. The myocyte cross-sectional areas were expressed relative to the cross-sectional areas of the nuclei.
Additional slides were fixed, mounted, and stained with Masson's trichrome to delineate the infarcted region. The infarcted region (indicated by the aqua-colored stain) was evaluated on a Nikon TE2000U with a x2 Plan objective. Using the circumferential infarct size determination method, we measured the inner circumference of the infarct and then the entire inner LV circumference with the use of image analysis software (NIH Image J, version 1.34p; National Institutes of Health, Bethesda, MD). The infarct size is expressed as a percentage of the LV inner circumference.
Echocardiographic Assessment of Left Ventricular Function
In vivo transthoracic echocardiography of the LV by a 15-MHz linear array transducer (15L8) interfaced with a Sequoia C256 (Acuson, Mountain View, CA) was performed as previously described (12). Mice were lightly anesthetized with pentobarbital sodium (40 mg/kg). Ventricular parameters were measured by the leading-edge technique. M-mode (sweep speed = 200 mm/s) echocardiograms were captured from parasternal and short- and long-axis two-dimensional views of the LV at the midpapillary level. Left ventricular end- diastolic diameters (LVEDD), left ventricular end-systolic diameters (LVESD), aortic diameter, aortic velocity time integral, and heart rate were measured at baseline and at 28 days after permanent occlusion of the LCA. LV percent fractional shortening (FS) was calculated according to the following equation: LV%FS = [(LVEDD – LVESD)/LVEDD] x 100. Stroke volume was calculated from the product of the aortic cross-sectional area [(aortic diameter/2)2 x 
] and the aortic velocity time integral. All data were calculated from 10 cardiac cycles per experiment.
Pulmonary Edema
Lungs from mice subjected to myocardial infarction or sham operation were excised after 28 days to determine pulmonary fluid accumulation. Excised lungs were immediately weighed (wet weight) and placed in a drying oven (Econotherm Laboratory Oven, Precision Systems, Natick, MA) for 7 days at 40°C. Lungs were weighed after the drying procedure (dry weight), and the difference between wet and dry weights represented pulmonary fluid.
Cholesterol and Triglyceride Determinations
Serum was collected after 28 days from an additional group of sham-operated mice randomized to receive simvastatin (0.25 mg/kg; n = 10) or vehicle (n = 9) injections (QOD) for 28 days to determine cholesterol and triglyceride content.
Total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglyceride concentrations were assessed by Christus Schumpert Health System Pathology Laboratory, using the Dimension Clinical Chemistry System (Dade Behring, Deerfield, Illinois) and Flex reagents.
Liver Enzyme Determinations
Serum was collected at 28 days postsurgery from sham-operated mice (n = 8 per group) randomized to injections of simvastatin (0.25 mg/kg QOD) or saline vehicle.
Serum samples were analyzed for aspartate aminotransferase and alanine aminotransferase with a spectrophotometric method (Sigma, St. Louis, MO) to assess the effects of prolonged simvastatin therapy on hepatic function.
Myocardial eNOS Western Blotting
Mice (n = 8 per group) were subjected to the immediate simvastatin therapy protocol (Fig. 1A) in the absence of LCA occlusion. Cardiac lysates were centrifuged to remove any particulate, and protein concentration of the cleared lysate was measured by the Bio-Rad Dc protein assay. Equal amounts of protein were loaded into each lane and separated on a 6% polyacrilamide gel. Protein was transferred to polyvinylidene difluoride overnight at 30 V and then blocked in 5% milk-Tris-buffered saline-Tween 20 (TBST) at room temperature for 3 h. Membranes were washed three times with TBST and then incubated with mouse anti-eNOS (1:4,000) (BD Transduction Labs) in 5% BSA TBST overnight at 4°C. Membranes were then washed three times with TBST and then incubated with horseradish peroxidase-linked anti-mouse secondary antibody (Amersham) at 1:2,000 in 5% BSA-TBST at room temperature for 3 h. Membranes were then washed three times with TBST, incubated with enhanced chemiluminescence reagents (Amersham), and then exposed to film. Densitometric analysis was performed using NIH Image software.
Cardiac Tissue Nitrate and Nitrite Levels
Additional mice (n = 8 per group) were subjected to the immediate simvastatin therapy protocol (Fig. 1A) in the absence of LCA occlusion. At 28 days after baseline, cardiac tissue homogenates were centrifuged for 20 min at 12,500 g, and the supernatants were normalized to the total protein content according to the Bradford assay. Each sample was then filtered to remove proteins, and resultant cleared homogenates were spectrophotometrically analyzed for nitrate and nitrite (NOx) levels using a commercially available system (NO quantitation kit from Active Motif). Samples were analyzed in triplicate, and absorbance was measured at a wavelength of 540 ± 2 nm on a Multiskan Spectrum (Thermo Electron) plate reader controlled by SkanIt Software (version 2.1).
Statistical Analysis
Data were analyzed by Student's unpaired t-test or ANOVA with Bonferroni post hoc analysis using StatView (SAS Institute, Cary, NC) software. Data are reported as means ± SE, with differences accepted as significant at P < 0.05.
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RESULTS |
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Simvastatin treatment did not alter circulating lipid levels in sham-operated mice treated with simvastatin (n = 10) for 28 days (Table 1). No changes were observed in total cholesterol, low-density lipoprotein cholesterol, or high-density lipoprotein cholesterol levels compared with mice receiving saline vehicle (n = 9). Similarly, there were no significant changes in serum triglyceride levels between the simvastatin-treated mice and mice receiving saline vehicle (Table 1).
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Simvastatin therapy exerted no significant changes in hepatic enzyme levels in mice after the 28-day experimental protocol (Table 2). Liver enzyme levels in sham-operated mice receiving simvastatin (n = 8) were similar to levels in mice receiving vehicle (n = 8).
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Studies were performed in sham-operated control (n = 5 per group) mice to determine the circulating levels of simvastatin in mice treated with simvastatin (0.25 mg/kg QD) for 7 days followed by simvastatin (0.25 mg/kg QOD) for 21 days. Plasma simvastatin was undetectable in animals receiving saline vehicle and 13.4 ± 5.94 ng/ml in mice receiving 0.25 mg/kg simvastatin QOD for 28 days.
Myocardial Infarct Size
The extent of myocardial infarction was examined in simvastatin-treated (n = 8) and saline vehicle control (n = 8) mice at 48 h after permanent LCA occlusion (Fig. 2A). The area-at-risk per LV was 44.2 ± 2.6% in simvastatin-treated and 43.0 ± 4.6% in vehicle mice (P = not significant between groups). The infarct size per LV was also similar in both groups, with 41.7 ± 1.2% in the simvastatin group and 38.6 ± 3.6% in the saline vehicle group (P = not significant between groups). In addition, infarct size per area at risk was 92.8 ± 2.3% in the simvastatin group, which was similar (P = not significant between groups) to that observed in the saline vehicle group (90.8 ± 4.6%).
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Survival
Both saline-treated mice and simvastatin-treated mice subjected to myocardial infarction exhibited significant (P < 0.05) mortality compared with sham-operated mice receiving saline vehicle (Fig. 3). Myocardial infarcted mice receiving saline vehicle experienced an overall survival rate of 25% during the 28-day protocol compared with mice treated with simvastatin, which resulted in 45% survival. This represents a 45% improvement in survival compared with the saline vehicle control group (P < 0.05 between groups). In contrast to immediate treatment with simvastatin, delayed treatment (48 h after LCA occlusion) of simvastatin did not improve survival (26%, n = 19) compared with the saline vehicle group (P = not significant between groups).
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Permanent ligation of the LCA resulted in significant LV dilatation in mice receiving saline vehicle (Fig. 4A). However, simvastatin treatment of mice (QD for 7 days and QOD for 21 days) subjected to myocardial infarction significantly attenuated LV dilation in both diastole and systole at 28 days after myocardial infarction compared with vehicle-treated controls (P < 0.01 between groups). In fact, LV dimensions in simvastatin-treated mice were similar to those observed in sham-operated control mice (P = not significant between groups).
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LV function data in sham, myocardial infarction + vehicle, and myocardial infarction + simvastatin mice 28 days after myocardial infarction are presented in Fig. 4B. Fractional shortening was significantly (P < 0.01) attenuated at 28 days after myocardial infarction in saline vehicle mice (18.6 ± 1.8%) compared with sham-operated mice (31.8 ± 2.3%). Mice receiving simvastatin displayed fractional shortening values (26.2 ± 2.1%) that were significantly improved over vehicle-treated myocardial infarction mice (P < 0.01 between groups).
Assessment of LV ejection fraction (EF) revealed similar findings in that vehicle-treated myocardial infarcted mice demonstrated a significant (P < 0.01 vs. sham) reduction in EF at 28 days after myocardial infarction (37.8 ± 4.3%) compared with sham-operated mice (65.4 ± 2.3%) (Fig. 4B). Mice receiving simvastatin exhibited a significant (P < 0.01) improvement in EF (58.9 ± 6.0%) compared with mice receiving vehicle and demonstrated recovery of EF that approached the values observed in sham-operated controls (Fig. 4B). No significant differences in EF were observed at baseline between the study groups.
Cardiac Hypertrophy
Permanent LCA ligation resulted in significant (P < 0.01) increases in the heart-to-body weight ratio at 28 days after myocardial infarction in both vehicle (9.2 ± 1.4 mg/g) and simvastatin-treated mice (6.1 ± 0.32 mg/g) compared with sham-operated controls (4.6 ± 0.13 mg/g), indicative of cardiac hypertrophy (Fig. 5A). However, simvastatin therapy significantly attenuated the degree of hypertrophy compared with the vehicle group (P < 0.05 between study groups).
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Pulmonary Edema
Examination of pulmonary fluid (Fig. 6) in mice subjected to myocardial infarction and treated with saline vehicle revealed significant (P < 0.01) pulmonary edema (128.4 ± 14 mg) compared with sham-operated control mice (96.1 ± 2.7 mg). Interestingly, mice receiving simvastatin displayed no appreciable pulmonary edema (103.8 ± 6 mg) after myocardial infarction compared with sham-operated mice (P = not significant between groups).
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eNOS–/– mice subjected to myocardial infarction demonstrated significant (P < 0.01) decreases in survival (Fig. 7A) in the saline vehicle group (32%) compared with eNOS–/– sham-operated mice (100%). However, simvastatin treatment failed to improve survival (28%) at 28 days after myocardial infarction compared in eNOS–/– mice (P = not significant between groups).
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Data for cardiac dimensions and LV function in eNOS–/– subjected to myocardial infarction and subsequent heart failure are presented in Fig. 7, B–E. Simvastatin treatment failed to prevent ventricular dilatation or dysfunction in eNOS–/– mice in the setting of heart failure.
Cardiac Hypertrophy in eNOS–/– Mice
Analyses of heart-to-body weight ratios in eNOS–/– mice subjected to myocardial infarction revealed similar results in vehicle (7.9 ± 0.3 mg/g) and simvastatin-treated (8.2 ± 0.7 mg/g) groups (P = not significant between groups). Thus simvastatin did not reduce cardiac hypertrophy in eNOS–/– mice at 28 days after myocardial infarction.
Pulmonary Edema in eNOS–/– Mice
In eNOS–/– mice receiving saline vehicle, pulmonary fluid accumulation was 139.4 ± 14 mg compared with 126.8 ± 7 mg in eNOS–/– mice treated with simvastatin (P = not significant between study groups). Thus simvastatin therapy failed to ameliorate the severity of pulmonary edema in the absence of eNOS following myocardial infarction.
Cardiac eNOS Protein Levels
Myocardial eNOS protein levels were measured in sham-operated animals that received either saline vehicle or simvastatin therapy for 28 days (Fig. 8). There was no change in cardiac eNOS protein levels in animals receiving simvastatin (0.25 mg/kg) compared with vehicle animals.
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Myocardial tissue NOx levels were determined in sham-operated animals that received simvastatin (0.25 mg/kg) or saline vehicle for 28 days (Fig. 8B). Simvastatin therapy resulted in a significant (P < 0.05) increase in total NOx levels.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The pleiotropic effects of statins have been investigated in multiple diseases. The CARE trial (22) provided evidence of cardiovascular benefits despite cholesterol levels in the normal range before statin treatment. Furthermore, multiple experimental studies have demonstrated reductions in myocardial infarct size in the absence of cholesterol lowering (16, 26). Recently, Jones et al. (10) demonstrated that statin-mediated protection against myocardial ischemia-reperfusion injury was eNOS dependent because eNOS–/– mice treated with rosuvastatin displayed no reduction in infarct size. In addition, Endres et al. (7) demonstrated that the protection afforded by statins in stroke was dependent on the presence and activation of eNOS. The present study adds to a compelling and growing body of evidence that supports the lipid-lowering independent cardiovascular benefits of statin treatment.
Recent studies investigated the effects of prolonged statin treatment in more chronic cardiovascular diseases. Patel et al. (20) found that simvastatin induced regression of hypertrophy and fibrosis as well as improved cardiac function in a rabbit model of cardiac hypertrophy induced by overexpression of the 
-myosin heavy chain-Q403. In addition, in a rat infarct model of heart failure, Bauersachs et al. (3) reported improvements in LV developed pressure and dP/dt following cerivastatin treatment. These improvements were attributed to favorable remodeling after myocardial infarction as reduced gene levels of -myosin heavy chain and collagen I were observed (3). Another heart failure study by Hayashidani et al. (8) reported improved survival, LV systolic and diastolic dimensions, and LV fractional shortening after fluvastatin treatment at a dose of 10 mg·kg–1·day–1. Despite the use of a very high dose of fluvastatin in their study, eNOS protein expression was not increased at 28 days postinfarction. The authors suggested that fluvastatin protected without augmenting eNOS protein levels. However, the authors failed to consider changes in the serine 1177 phosphorylation status or increased activity of eNOS. Finally, Landmesser et al. (14) investigated the role of statin therapy with atorvastatin (50 mg·kg–1·day–1) in a mouse model of ischemia-induced heart failure. The authors reported improved survival and cardiac function. In addition, a loss of these protective effects in eNOS-deficient mice was also observed. The Landmesser study suggests that statins mediate improved survival by favorable remodeling and by the proangiogenic effects of atorvastatin in the setting of CHF.
Fluvastatin has been show to inhibit matrix metaloproteinase-1 expression in human vascular endothelial cells (9), which could possibly be applied to the myocardium itself because it plays a critical role in the development of heart failure. It has been shown that LV hypertrophy can be attenuated in a mouse model of myocardial infarction by matrix metaloproteinase-1 inhibition (23).
Statin-mediated angiogenesis is now widely accepted as multiple studies have shown neovascularization from statin administration (13, 25). The study by Kureishi et al. (13) demonstrates that statin-mediated angiogenesis occurs via Akt activation of eNOS and suggests that phosphorylation of eNOS, rather than upregulation of protein expression, is responsible for the proangiogenic effects of statins. The angiogenic effects of statin administration may play a critical role in the improvements of survival and LV function observed in the present study. Recently, endothelial progenitor cell mobilization has been shown to be dependent on NO (1). Increased NO bioavailability as a result of statin therapy may have increased circulating endothelial progenitor cells in our model of heart failure, resulting in the growth of new blood vessels in the myocardium.
The present study convincingly demonstrates that low-dose (i.e., 0.25 mg·kg–1·day–1) simvastatin treatment initiated after coronary occlusion significantly improves survival, preserves cardiac function, and reduces pulmonary edema. Previous studies of statin therapy in the setting of CHF have been focused on very high dosages of statins, ranging from 10 to 50 mg/kg, in rodents. The highest dosages of statins that are administered to humans are 
1 mg/kg, and it is not clear whether dosages that exceed 1 mg/kg are clinically relevant and whether these dosages will exert toxic effects in humans. We provide clear evidence for statin protection against heart failure in the absence of changes in circulating lipids and no signs of hepatotoxicity. Furthermore, the beneficial effects are dependent on eNOS and abrogated in eNOS–/– mice. Our findings strengthen the foundation for the development of statin therapy in normocholesterolemic subjects afflicted with cardiovascular diseases such as CHF.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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