Simvastatin Acutely Reduces Myocardial Reperfusion Injury In Vivo by Activating the Phosphatidylinositide 3-Kinase/Akt Pathway

Abstract

Long-term pretreatment with statins reduces myocardial injury after acute ischemia and reperfusion by increasing the expression of endothelial nitric oxide synthase (eNOS). We hypothesized that statins may act rapidly enough to protect the myocardium from ischemia/reperfusion injury when given right at the beginning of the reperfusion period and tried to delineate the role of PI 3-kinase/Akt pathway in early eNOS activation. Activated simvastatin was given intravenously 3 minutes before starting the reperfusion after temporary coronary artery occlusion (CAO) in anaesthetized rats. Simvastatin significantly increased myocardial PI 3-kinase activity, Akt_Ser473, and eNOS_Ser1177 phosphorylation and reduced infarct size by 42%. Infarct size reduction as well as activation of PI 3-kinase/Akt/eNOS pathway were not observed in rats co-treated with the PI 3-kinase inhibitor wortmannin. Contribution of eNOS was further delineated using the NOS inhibitor l-NAME, which could completely block cardioprotection by the statin. In summary, simvastatin acutely reduces the extent of myocardial necrosis in normocholesterolemic rats in an NO- dependent manner by activating the PI 3-kinase/Akt pathway. This is the first study demonstrating short-term cardioprotective effects of simvastatin in an in vivo model of ischemia/reperfusion.

HMG-CoA reductase inhibitors, also known as statins, reduce plasma cholesterol levels and improve survival in patients with coronary artery disease.^1,2 Recently, basic and clinical research has focused on mechanisms by which statins exert cardioprotective effects beyond cholesterol reduction.³ Statins acutely reduce inflammatory responses^4,5 and improve endothelial function.^6,7 Improvement of endothelial function by statins is thought to depend, in part, on increased release of nitric oxide (NO) by endothelial NO-synthase (eNOS).^8,9

The mechanisms by which statins increase eNOS activity^10,11 are related to their ability to stabilize eNOS mRNA¹² and/or to activate the phosphatidylinositide (PI) 3-kinase/Akt pathway, which leads to a phosphorylation at serine 1177 or 1179 of human or bovine eNOS, respectively.^13–15 In previous animal studies of ischemia and reperfusion, long-term pretreatment with statins resulted in an up-regulation of eNOS, which essentially contributed to cardiovascular protection achieved by these drugs. For example, up-regulation of eNOS is responsible for the reduction in stroke size after regional cerebral ischemia since no such beneficial effects of statins were observed in eNOS-deficient mice.¹⁶ Furthermore, pretreatment with statins for several days improves cardiac function in ischemic isolated rat hearts^17,18 and in in vivo models of myocardial ischemia/reperfusion.^8,19,20

Several hours of treatment with statins are necessary for an up-regulation of eNOS by stabilizing eNOS mRNA as shown in cell culture systems.¹² Conversely, activation of eNOS by phosphorylation via the PI 3-kinase/Akt pathway occurs within minutes.¹³ This mechanism of eNOS activation raises the possibility that statins can even reduce myocardial infarct size when given right at the beginning of the reperfusion period, which would have therapeutic implications for patients suffering from acute myocardial infarction.

Most recently Bell and Yellon²¹ reported that acute administration of atorvastatin reduced ischemia reperfusion injury in isolated mouse hearts. In this in vitro study cardioprotection was mediated by activation of PI 3-kinase and was absent in eNOS-deficient mice. With the present study we extend previous work and focus on the acute effects of statins in vivo. We tried to determine whether statins can acutely protect the ischemic myocardium in the anesthetized rat, and if so, whether the protective effect is mediated by the PI 3-kinase/Akt pathway. The results of this study seem to be encouraging to investigate cardioprotective effects of statins during myocardial reperfusion in patients.

METHODS

Animal Care

The present study has been carried out in accordance with institutional guidelines for the care and use of laboratory animals as adopted by the “Ministerium für Natur und Umwelt des Landes Schleswig-Holstein, Germany.” Male Wistar rats (220–310 g body weight, Charles River, Sulzfeld, Germany) were fed normal rodent food and water ad libidum.

Surgical Preparations

The surgical protocol was performed according to methods described previously.²² Briefly, rats were anesthetized with pentobarbital (60 mg kg⁻¹, i.p.), intubated, and ventilated with room air (tidal volume: 10 mL kg⁻¹, 50 strokes per minute) enriched with oxygen. The amount of oxygen supply has been shown to adjust arterial oxygen tension to physiological levels between 100 and 120 mm Hg in preliminary studies. Rats were placed on heating plates to maintain core temperature within the normal range (37.0–37.6°C) and the left jugular vein was cannulated to inject drugs. A lateral thoracotomy was performed, and a 6-0 suture was looped under the left descending coronary artery, which was occluded for 30 minutes before the reperfusion period was started.

Experimental Protocols

In a first set of experiments, rats were randomized to achieve either activated (acid form) simvastatin (1 mg kg⁻¹, i.v., n = 15) or vehicle (0.9% NaCl, n = 10) during myocardial ischemia, 3 minutes before starting the 3-hour reperfusion period. The dosage of simvastatin has been used previously and correlates with 75 mg of simvastatin in humans.¹⁹ To delineate the role of NO, a third group was treated with the NOS inhibitor N(ω)-nitro-l-arginine methyl ester (l-NAME, 15 mg kg⁻¹, i.v., applied 15 minutes before CAO, n = 9). One animal died because of severe bleeding in this group. In a second set of experiments, effects of simvastatin were investigated after inhibition of PI 3-kinase with wortmannin (15 μg kg⁻¹, i.v., n = 5) given 15 minutes before starting the reperfusion and compared with wortmannin alone (n = 5). The time course of the experiments is depicted in Figure 1. Previously, doses of l-NAME^20,23 and wortmannin²⁴ have been shown to be effective in similar models.

FIGURE 1.: Experimental protocols. Male Wistar rats were subjected to 30 minutes of coronary artery occlusion followed by 180 minutes of reperfusion. Activated simvastatin (1 mg kg⁻¹, i.v) was applied 3 minutes before reperfusion. In separate experiments the role of nitric oxide as well as the phosphatidylinositide (PI) 3-kinase/Akt/eNOS pathway was explored by co-treatment with the selective inhibitors l-NAME or wortmannin, respectively, given 15 minutes prior to reperfusion.

In a separate sets of experiments, preparation and treatments were performed as described, but after initiation of reperfusion the thorax was closed surgically in 2 layers (5-0 prolene). To establish a reperfusion period of 24 hours the animals were allowed to recover. Mechanical ventilation was stopped as soon as rats started to breathe spontaneously (usually within 1 hour after finishing surgery). Animals were watched frequently and buprenorphine (0.5 mg kg⁻¹, s.c.) was administered to limit pain when rats started to get awake. Two and three animals died within 4 to 6 hours after extubation in the control and simvastatin-treated group, respectively, whereas 8 and 7 animals, respectively, could be included in the study.

At the end of the experiments, 3 mL of blood were obtained, plasma was separated from blood cells by centrifugation and stored at −80°C. For further analysis, hearts were either prepared for infarct size measurement or the tissue was quickly frozen in liquid nitrogen.

Measurement of Area at Risk and Infarct Size

Myocardial infarct size was determined as described previously.²² Briefly, at the end of the reperfusion period, hearts were removed and aortas were quickly cannulated. The coronary artery ligature was retied, and hearts were perfused with black Chinese ink at a constant pressure (80 mm Hg) to stain the perfused myocardium black, whereas the area at risk (AAR) remained unstained. Atria and the right ventricle were removed and the left ventricle (LV) including the septum was cut into slices (1-mm thickness) from apex to base. Slices were then incubated for 30 minutes at room temperature in 2,3,5-triphenyltetrazolium chloride (TTC, 1% in 0.1 mol L⁻¹ phosphate buffer, pH 7.4), which stained viable tissue red but not the infarct area. Areas of LV, AAR, and infarct size (IS) were quantified using computer-assisted planimetry.

Measurement of Total Cholesterol Levels in Plasma

Total cholesterol levels were measured with enzyme assays based on the formulation of Allain et al²⁵ and the modification of Roeschlau et al²⁶ using an Aeroset^TM System (Abbott Labaratories, Wiesbaden, Germany).

Immunoblot Analysis

Tissue samples from rat hearts were homogenized in lysis buffer containing protease and phosphatase inhibitors (Cell Signaling Technology Inc., Beverly, MA). The total protein concentration in the homogenate was quantitated, and 50 μg (Akt) or 100 μg (eNOS) of protein per sample were separated by 7.5% SDS-polyacrylamide gels using standard methods and electrotransferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Proteins were detected with antibodies against Akt, Ser₄₇₃-phosphorylated Akt and Ser₁₇₇₇-phosphorylated eNOS (all from Cell Signaling, Beverly, MA), or total eNOS (BD Bioscience, San Diego, CA), horseradish peroxidase–labeled secondary antibodies (Dako Diagnostics, Hamburg, Germany), and enhanced chemiluminescence reagent (Perkin Elmer, Boston, MA). The antibodies against Akt and phosphorylated Akt were known to detect the α, β, and γ isoforms. Bands were quantified densitometrically with Molecular Analyst software (BioRad).

Measurement of PI 3-kinase Activity

Hearts were homogenized and protein (200 μg) was co-incubated with anti-PI 3-kinase p85 antibody (2 μg, Upstate Biotechnology, Lake Placid, NY) for 4 hours at 4°C. Then protein G Sepharose beads (Pierce, Rockford, IL) were added. After 16 hours at 4°C, the beads were washed 3 times with PBS containing 1% IGEPAL and 0.1 mmol L⁻¹ sodium orthovanadate (pH 7.5), 3 times with 500 mmol L⁻¹ LiCl, 100 mmol L⁻¹ TRIS (pH 7.5), and 2 times in 10 mmol L⁻¹ TRIS, 100 mmol L⁻¹ NaCl, 1 mmol L⁻¹ EDTA, and 0.1 mmol L⁻¹ sodium orthovanadate (pH 7.5). The beads were then suspended in 60 μL of reaction buffer (20 mmol L⁻¹ HEPES, 15 mmol L⁻¹ MgCl₂, 0.4 mmol L⁻¹ EGTA, and 0.4 mmol L⁻¹ NaH₂PO₄) supplemented with phosphatidylinositol (20 μg; Sigma, Deisenhofen, Germany). The phosphorylation reaction was started by the addition of 10 μL of a solution that contained 720 μmol L⁻¹³²P-ATP (25 μCi mmol^-1) (NEN, Dreieich, Germany). After 20 minutes at room temperature, the reaction was stopped by the addition of 40 μL 25% HCl and 190 μL of methanol/chloroform (1:1). The organic phase was extracted and applied to a silica gel thin-layer chromatography plate. Phosphatidylinositol-3 phosphorylation was determined using a phospho imager.

Calculations and Statistics

To compare the results of different gels or activity assays, densitometric (immunoblotting) and phospho-imager-derived values were related to internal standards. All quantitative data are given as means ± SEM of 4 to 15 independent experiments. All data were compared with the corresponding treatment groups using a one-way ANOVA with Dunn’s correction. Differences were considered as being statistically significant at an error level of P < 0.05.

RESULTS

Simvastatin Acutely Reduces Myocardial Infarct Size

To determine whether simvastatin reduces myocardial reperfusion injury in vivo, activated simvastatin was administered 3 minutes before starting the reperfusion period. The ratios between AAR and LV were 57 ± 4%, 57 ± 5% in vehicle or simvastatin treated animals, respectively. There were no significant differences between both groups indicating a constant placement of the coronary ligature. Acute administration of simvastatin reduced the ischemia/reperfusion injury by 42% (Fig. 2) after 3 hours of reperfusion (ratio IS/AAR: 49 ± 6% in controls versus to 28 ± 3% in simvastatin treated animals, P < 0.05).

FIGURE 2.: Myocardial infarct size (IS) was determined by triphenyltetrazolium chloride (TTC) staining after 3 hours of reperfusion and expressed as percentage of the area at risk (AAR). Rats were treated with simvastatin or vehicle. Nitric oxide synthase and PI 3-kinase were inhibited using the selective inhibitors l-NAME and wortmannin, respectively. Data represent mean ± SEM. *P < 0.05 versus vehicle.

Simvastatin Increases Myocardial PI 3-kinase Activity and Akt Phosphorylation

To determine whether the effects of simvastatin on infarct size occur at the level of PI 3-kinase/Akt pathway, we measured PI 3-kinase activity (Fig. 3) and Akt phosphorylation (Fig. 4) using homogenates of hearts that underwent ischemia/reperfusion. Treatment of animals with simvastatin significantly increased PI 3-kinase activity (factor 2.2) and Akt_Ser473 phosphorylation (factor 1.9) compared with vehicle (P < 0.05) whereas total myocardial Akt content remained unchanged. As expected, wortmannin completely blocked the effects of simvastatin on PI 3-kinase activity as well as Akt_Ser473 phosphorylation.

FIGURE 3.: PI 3-kinase activity was determined in myocardial tissue after 30 minutes of coronary artery occlusion and 3 hours of reperfusion. Rats were treated with simvastatin or vehicle. The PI 3-kinase inhibitor wortmannin was applied 15 minutes prior to coronary artery occlusion. Data represent mean ± SEM. *P < 0.05 versus vehicle.

FIGURE 4.: Phosphorylation of Akt_Ser473 (A) was determined in myocardial tissue after 30 minutes of coronary artery occlusion and 3 hours of reperfusion. Rats were treated with simvastatin or vehicle. Wortmannin was applied 15 minutes prior to coronary artery occlusion to inhibit PI 3-kinase. Total myocardial Akt content did not differ between the treatment groups (B). Data represent mean ± SEM. *P < 0.05 versus vehicle.

Inhibition of PI 3-kinase Abolishes Cardioprotective Effects of Simvastatin

To elucidate the functional significance of PI 3-kinase activation to the effects of simvastatin, animals were co-treated with the PI 3-kinase inhibitor wortmannin in a dose that has previously been shown to block insulin-induced cardioprotection in a similar model.²⁴ Again, the ratio between AAR and LV did not differ between wortmannin, and wortmannin + simvastatin-treated animals (56 ± 8% and 56 ± 7%, respectively). However, animals treated with wortmannin alone showed significantly larger infarct sizes (70 ± 2%) than control rats in the first set of experiments. The cardioprotective effect of simvastatin was completely blocked by co-treatment with wortmannin (68 ± 3%), indicating a predominant role of PI 3-kinase in statin-induced cardioprotection (Fig. 2).

Cardioprotection by Simvastatin is Mediated by eNOS

The contribution of eNOS to statin-induced cardioprotection was documented using the NOS inhibitor l-NAME, which completely blocked the infarct size reduction achieved by simvastatin (ratio IS/AAR: 48 ± 5%) (Fig. 2). In a previous study, l-NAME itself did not influence infarct size in the same rat model.²⁰ To further substantiate the hypothesis of a sequential activation of the PI 3-kinase/Akt pathway and eNOS, we measured eNOS phosphorylation at serine 1177 as an indicator of eNOS activation. There was a tendency of increased eNOS phosphorylation by simvastatin whereas total eNOS expression remained unchanged. Activation of eNOS by simvastatin could completely be blocked by co-treatment with wortmannin (Fig. 5).

FIGURE 5.: Phosphorylation of eNOS_Ser1177 was determined in myocardial tissue after 30 minutes of coronary artery occlusion and 3 hours of reperfusion (bars). Rats were treated with simvastatin or vehicle. Wortmannin was applied 15 minutes prior to coronary artery occlusion to inhibit PI 3-kinase. Total myocardial eNOS content did not differ between the treatment groups. Data represent mean ± SEM.

Statin-Induced Cardioprotection is Preserved up to Twenty-Four Hours

In a separate set of experiments we investigated whether cardioprotection by statins prevents or only delays reperfusion injury. As shown in Figure 6, cardioprotection achieved by a single dose of activated simvastatin was still present when the reperfusion period was prolonged up to 24 hours (IS/AAR: 25 ± 2% in simvastatin treated rats versus 40 ± 5% in controls, P < 0.05).

FIGURE 6.: Myocardial infarct size (IS) as determined by TTC staining after 24 hours of reperfusion and expressed as percentage of area at risk (AAR). Rats were treated with simvastatin or vehicle. Data represent mean ± SEM. *P < 0.05 versus vehicle.

Reduction of Infarct Size by Statins is Independent of Plasma Cholesterol Levels

Acute treatment with simvastatin did not alter plasma cholesterol levels. Cholesterol levels were in the lower range without significant differences between all treatment groups after 3 hours of reperfusion (in mmol L⁻¹: Vehicle: 1.57 ± 0.07; simvastatin: 1.35 ± 0.09; wortmannin: 1.34 ± 0.06, and wortmannin + simvastatin: 1.48 ± 0.03) or after 24 hours of reperfusion (in mmol L⁻¹: Vehicle: 1.62 ± 0.09; simvastatin: 1.59 ± 0.07), indicating that reduction of infarct size by simvastatin was present in animals with low plasma cholesterol levels and was independent of any potential plasma cholesterol-lowering effects of the statin.

DISCUSSION

A principal finding of the present study is the marked reduction of myocardial infarct size by statins in acute ischemia/reperfusion injury in vivo. This beneficial effect of statins occurred in the absence of significant changes in plasma cholesterol levels in normocholesterolemic animals. These results extend the evidence that statins have clinical benefits beyond cholesterol lowering in the setting of acute myocardial infarction followed by reperfusion.

Previous studies from our group and others demonstrated in vitro and in vivo, that treatment of animals with statins prior to the onset of myocardial ischemia reduces ischemia/reperfusion injury.^8,18,20,27 While these studies demonstrate the impact of prophylactic therapy, they do not address the question whether patients with acute myocardial infarction might benefit from an initiation of statin therapy before starting the reperfusion. For evaluation of this question, the exact protocol of statin therapy is decisive. In a previous work, Bauersachs et al²⁸ failed to demonstrate a reduction in infarct size when the statin was administered 24 hours after onset of myocardial ischemia similar to the treatment protocol of the MIRACL trial.²⁹ In a study of Jones et al,²⁷ simvastatin failed to protect the mouse heart when given less than 3 hours before myocardial ischemia while a pretreatment of several days was effective.²⁰ In the present study, cardioprotection was achieved when simvastatin was administered only 3 minutes before starting the reperfusion, which simulates the situation of a patient with acute myocardial infarction undergoing percutaneous coronary intervention. Besides species differences, the pharmacokinetics of different simvastatin applications might be responsible for the conflicting results. In the present study, activated simvastatin was administered intravenously and therefore reached its target immediately without a significant resorption period and first pass metabolism, in contrast to an application by intraperitoneal injection, which was used by Jones et al.²⁷ Our results are in accordance with a recent work of Bell and Yellon²¹ that was published during preparation of this manuscript. The authors observed a significant reduction of infarct size by co-perfusion of atorvastatin during reperfusion in isolated mice hearts.²¹ However, there are some limitations of this in vitro study where infarct size was measured by TTC staining after 30 minutes of reperfusion that are cleared out by the present work. Although reperfusion injury starts almost instantly after the onset of reperfusion, necrosis develops within the following hours.³⁰ Tetrazolium salts such as TTC have been shown to accurately detect infarction if sufficient time has been elapsed to wash out dehydrogenases, NADH, and other co-factors from necrotic tissue.³¹ Otherwise, when inadequate reperfusion times are used, infarct size would be underestimated. TTC staining with 3 hours of reperfusion as in our study has been shown to correlate well with examinations of histologic sections.³² Furthermore, pharmacological interventions initiated early in the reperfusion may only delay rather than prevent reperfusion injury. In addition, injury that occurs later in the reperfusion period, such as invasion of neutrophils might overcome the early beneficial effects.³³ Unfortunately, the reperfusion time in isolated hearts is limited and this model lacks of any detrimental effects of leukocytes in reperfusion. Our study is therefore an important extension of Bell and Yellon’s work and it is a therapeutically important finding that the cardioprotective action of simvastatin was even maintained for a reperfusion period of up to 24 hours. This indicates that the pleiotropic actions of statins contribute to long-term protection in vivo and can be activated by one-time therapy.

High cholesterol levels themselves impair endothelial function³⁴; therefore, reduction of plasma cholesterol levels by statins has been proposed to be responsible for restoring endothelial function in patients with coronary artery diseases.³⁵ Statins, however, improve endothelial function also under normocholesterolemic conditions.³⁶ Enhancement of endothelial function due to an interaction with the endothelial NO-synthase (eNOS) has been suggested as one of the effects of statins beyond their lipid-lowering property,¹⁷ since statins stabilize eNOS mRNA and consecutively increase eNOS activity in human endothelial cells.¹² It was reported previously that cardioprotection after pretreatment with statins in vitro and in vivo is mediated by NO.^17–20

Consequently, we focused on eNOS to investigate whether the acute reduction of infarct size is mediated by the same mechanisms. In the present study eNOS activity tended to be increased by short-term treatment with simvastatin as indicated by the state of phosphorylation at serine 1177 although this effect did not reach statistical significance. The functional significance of NOS was elucidated by using the NOS inhibitor l-NAME, which completely blocked the infarct size-limiting effect of simvastatin. In a previous study we could demonstrate that l-NAME itself did not influence infarct size in the same rat model although it completely blocked the increase in eNOS activity expression achieved by statin treatment.²⁰ Together with the recent results from isolated mice hearts, where cardioprotection by atorvastatin increased eNOS phosphorylation and cardioprotection by the statin was absent in eNOS-deficient mice²¹ there is sufficient evidence that rapid effects of statins on reperfusion injury are mediated by NO.

Enhanced NO levels, simulated by application of NO donors, can reduce infarct size.³⁷ Mechanisms of cardioprotection by NO have not yet been fully elucidated. NO might exert beneficial effects by inhibiting the production of nuclear factor kappa B (NFκB),³⁸ an essential transcription factor, which is involved in the expression of several genes with proinflammatory functions. Other possible mechanisms include the antithrombotic³⁹ or radical scavenging properties⁴⁰ of NO.

There are two mechanisms described regarding the mechanism by which statins may increase eNOS activity. Statins have been shown to increase eNOS mRNA stability by inhibiting Rho and Rho kinase.¹² However, the acute effect of simvastatin observed in our study is not likely to be explained by a modification of mRNA stability and a consecutively enhanced protein translation, since endothelial dysfunction as an early marker of reperfusion injury begins within minutes after starting the reperfusion period.⁴¹ As expected, short-term treatment with simvastatin did not increase eNOS protein expression in our study. Therefore, a fast activation of eNOS at the protein level must be responsible for cardioprotection in a model of acute myocardial infarction.

The serine-threonine kinase Akt is an important regulator of various cellular processes including cell metabolism and apoptosis.^42,43 Stimulation of receptor tyrosine kinases and G-protein-coupled receptors leads to activation of PI 3-kinase, the products of which, namely 3′-phopholipids, provoke the phosphorylation and activation of Akt.^44,45 Akt has been shown to modulate several targets, such as glycogen synthase kinase-3β, Bad, and caspase-9, by phosphorylation, and plays a pivotal role in intracellular signaling.^45,46 Recent studies focused on the activation of eNOS via the PI 3-kinase/Akt pathway and demonstrated that eNOS is indeed a substrate of Akt, which phosphorylates eNOS at serine 1179 or 1177.^14,15,47,48 The contribution of PI 3-kinase/Akt pathway to the effects of statins has been reported by Kureishi et al¹³ who described that simvastatin activates Akt within minutes, which leads to an increased NO production, and could be inhibited by the PI 3-kinase inhibitor wortmannin. In addition, these authors observed that simvastatin inhibits apoptosis of endothelial cells in culture, an effect that was abolished in cells overexpressing dominant-negative Akt.¹³

The precise mechanism by which statins activate PI 3-kinase is not yet identified, but there is evidence that they are independent of a reduction in plasma cholesterol levels. Similar to Bell and Yellon²¹ we applied the commonly used inhibitor of PI 3-kinase, wortmannin, to delineate the role of PI 3-kinase in our in vivo model. The dose of wortmannin has previously been shown to inhibit insulin-induced PI 3-kinase activation in a similar model.²⁴ In contrast to the in vitro data,²¹ inhibition of PI 3-kinase itself increased infarct size in our study, indicating that the PI 3-kinase/Akt pathway plays an important role in the pathophysiology of myocardial infarction in a whole animal model. Interestingly, when rats were co-treated with wortmannin the effect of simvastatin on infarct size was completely abolished. Since wortmannin itself increased myocardial infarct size the possibility remains that simvastatin activates other signal pathways than the PI 3-kinase/Akt in parallel, which are overwhelmed by the detrimental effects of wortmannin on PI 3-kinase. Therefore, to substantiate the contribution of the PI 3-kinase/Akt/eNOS pathway to the cardioprotective effect of simvastatin in our in vivo model we measured myocardial PI 3-kinase activity, which was significantly increased under statin therapy. In addition, simvastatin treatment led to phosphorylation of Akt_Ser473, and eNOS_Ser1177 downstream of PI 3-kinase. A limitation of the present study is that inhibition of PI 3-kinase by wortmannin did not reduce PI 3-kinase activity nor Akt_Ser473 phosphorylation below baseline levels. This might be due to the time course of the experiment where PI 3-kinase activity and Akt_Ser473 phosphorylation were measured 3 hours after administration of wortmannin, when its efficiency may already have subsided. However, co-treatment of wortmannin with simvastatin completely blocked the increase in PI 3-kinase activity as well as the Akt_Ser473 and eNOS_Ser1177 phosphorylation achieved by the statin. Taken together, these data provide strong evidence for a PI 3-kinase/Akt and eNOS-mediated cardioprotection by statins in vivo.

In our study, using normocholesterolemic rats, an infarct size reduction was achieved independently of the plasma cholesterol-lowering properties of statins. Most recently, Skaletz-Rorowski et al⁴⁹ have reported that statins rapidly translocate Akt to specific membrane compartments, which might represent ‘lipid rafts.’ This effect as well as phosphorylation of Akt was abolished after loading the endothelial cells with cholesterol. This study indicates that statins may affect the rapidly exchanging pool of cholesterol within the cell membrane of endothelial cells.⁴⁹ One can speculate that such changes in membrane cholesterol levels might not be associated with changes in the plasma cholesterol levels. Using an in vivo model, we cannot exclude that simvastatin affects the cholesterol pool of cell membranes in our study. However, since the incidence of cardiovascular diseases is associated with elevated plasma cholesterol levels, activation of the PI 3-kinase/Akt pathway might still be regarded as one of the ‘pleiotropic’ effects of statins.

In summary, this study demonstrates that statins reduce the extent of myocardial necrosis after acute regional ischemia in rats when given right at the beginning of the reperfusion period. This effect is independent of changes in plasma cholesterol levels. With the present study we extend recent evidence from isolated mouse hearts and provide first evidence in an in vivo model, that activation of the PI 3-kinase/Akt pathway and their downstream target eNOS as a newly identified signal pathway of statins participates in reduction of ischemia/reperfusion injury. These findings imply the potential that statins may also protect the human heart in the setting of acute myocardial infarction.

ACKNOWLEDGMENT

The authors thank Mrs. Cindy Krause and Mrs. Maren Drenckhan for their expert technical assistance with the immunoblottings and PI 3-kinase activity assays.