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Cardiovascular Research 2005 66(3):462-471; doi:10.1016/j.cardiores.2005.02.008
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Copyright © 2005, European Society of Cardiology

Chronic treatment with rosuvastatin modulates nitric oxide synthase expression and reduces ischemia–reperfusion injury in rat hearts

Pericle Di Napolia, Alfonso Antonio Taccardia, Alfredo Grillib, Maria Anna De Lutiisb, Antonio Barsottic, Mario Felacob and Raffaele De Caterinaa,d,*

aLaboratory of Experimental Cardiology, Department of Clinical Sciences and Bioimaging, and Center of Excellence of Aging, G. d'Annunzio University, Chieti, Italy
bDepartment of Biomorphology, Biology Section, "G. d'Annunzio" University, Chieti, Italy
cDepartment of Internal Medicine (DiMI), University of Genoa, Genoa, Italy
dCNR Institute of Clinical Physiology, Pisa, Italy

* Corresponding author. Department of Clinical Sciences and Bioimaging, "G. d'Annunzio" University of Chieti, C/o Ospedale S. Camillo de Lellis, Via Forlanini, 50, 66100 Chieti, Italy. Tel.: +39 0871 41512 fax: +39 0871 402817. Email address: rdecater{at}unich.it

Received 12 October 2004; revised 5 February 2005; accepted 7 February 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Acknowledgments
 References
 
Objective: Due to reported modulatory effects of statins on nitric oxide synthase (NOS) expression, we tested the hypothesis of protective effects of in vivo chronic treatment with rosuvastatin, a novel 3-hydroxy-3-methyl-glutaryl coenzyme A-reductase inhibitor, on ischemia–reperfusion injury, and investigated mechanisms involved.

Methods: After 3 weeks of in vivo treatment with rosuvastatin (0.2–20 mg/kg/day) or placebo, excised hearts from Wistar rats were subjected to 15 min global ischemia and 22–180 min reperfusion. We evaluated creatine-phosphokinase and nitrite levels in the coronary effluent, heart weight changes, microvascular permeability (extravasation of fluoresceine-labeled albumin), ultrastructural alterations, and the expression of endothelial (e) and inducible (i) nitric oxide synthase (NOS) (by reverse-transcription polymerase chain reaction and Western blotting).

Results: Rosuvastatin 0.2 and 2 mg/kg/day significantly reduced myocardial damage and vascular hyperpermeability, concomitant with a reduction in endothelial and cardiomyocyte lesions. At 2 mg/kg/day, rosuvastatin significantly increased eNOS mRNA and protein compared with untreated hearts, and conversely decreased iNOS mRNA and protein, as well as nitrite production after ischemia–reperfusion. The addition of the NOS inhibitor N{varpi}-nitro-L-arginine methylester (L-NAME, 30 µmol/L) significantly reduced cardioprotection against ischemia–reperfusion.

Conclusions: Chronic treatment with rosuvastatin before ischemia reduces ischemia–reperfusion injury and prevents coronary endothelial cell and cardiomyocyte damage by NO-dependent mechanisms.

KEYWORDS Statins; Rosuvastatin; Nitric oxide; Cardiomyocytes; Cardioprotection; Ischemia–reperfusion injury

This article is referred to in the Editorial by U. Laufs and M. Böhm (pages 427–429) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Acknowledgments
 References
 
Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, statins, are currently used in the treatment of hypercholesterolemia for prevention of vascular disease. Cholesterol lowering by statins is associated with decreased cardiovascular morbidity and mortality in both primary [1–5] and secondary [6–9] prevention studies in coronary artery disease (CAD). Part of these favorable effects, and the effects of statins on the incidence of ischemic stroke, the occurrence of which is not related to cholesterol levels in epidemiological studies [10], might however be due to the ability of statins to improve endothelial function [11–14], possibly through direct endothelial effects [10,15]. In particular, statins have been found to modulate nitric oxide(NO) synthase (NOS) activity and NO bioavailability [16–18]. We have previously demonstrated cardioprotective effects of an acutely administered statin, simvastatin, in a model of ischemia–reperfusion in the Langendorff rat heart, and related it to the effects of the drug on nitric oxide synthases(NOS) [18].

Rosuvastatin is a new highly effective synthetic hydrophilic statin [19,20]. Two studies have reported that rosuvastatin exerts, in acute administration, direct vascular and cardioprotective effects in models of ischemia–reperfusion, by increasing endothelial NO production or reducing polymorphonuclear leukocyte adherence [21,22]. It is however unknown whether this drug can modulate cardiac NOS activity and NO production during chronic in vivo treatment, reproducing conditions of its administration in humans. Thus, the aim of this study was to investigate whether rosuvastatin exerts effects on myocardial ischemia–reperfusion damage, endothelial NO production and NOS activity during chronic in vivo treatment of normolipidemic rats.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 5. Conclusions
 Acknowledgments
 References
 
2.1. Drug administration
Rosuvastatin (bis[(E)-7-[4-(4-fluorophenyl)-6-isopropyl-2-[methyl(methylsulfonyl)amino] pyrimidin-5-yl](3R,5S)-3,5-dihydroxyhept-6-enoic acid] calcium salt, AstraZeneca, Mölndal, Sweden) was given to a total of eighty adult male Wistar rats, weighing 250–300 g, at 0.2, 2, or 20 mg/kg/day (in various protocols, see below) intraperitoneally for 3 weeks. The last administration was performed 18 h before the sacrifice. Control rats (n=20) were given an equal volume (2 mL) of 0.9% NaCl saline.

2.2. Heart perfusion technique
Rats were anesthetized with a mixture of ether and air. After injection of 1000 IU sodium heparin into the femoral vein, the hearts were quickly excised and weighed. A modified Krebs–Henseleit (KH) solution (108 mmol/L NaCl, 25 nmol/L NaHCO3, 4.8 mmol/L KCI, 1.2 mmol/L KH2PO4, 1.2 mmol/L MgSO2, 2.5 mmol/L CaCl2, 11 mmol/L glucose, 287 mOsmol/L, pH 7.4, at 37 °C) was used as perfusion medium. The solution was bubbled with 95% O2–5% CO2. Preload and afterload were set at 20 and 72 cm H2O, respectively. Aortic and coronary flows (mL/min), aortic pressure (mm Hg), heart rate (bpm), minute work (mm Hg x mL/min) and coronary resistances (dyn.s.cm–5) were determined as previously described [18]. This investigation conformed to the Guide for the Care and Use of Laboratory Animals (U.S. National Institutes of Health, NIH Publication No. 85-23, revised 1996). The protocol was approved by the local Institutional Ethics Committee.

2.3. Assessment of left ventricular function and myocardial tissue damage
Hearts were all submitted to an identical protocol, comprising a 20 min stabilization followed by 15 min of global ischemia and 180 min of reperfusion (in the Langendorff perfusion mode for the first 10 min; and in the working heart mode for the remaining 170 min) [18]. For the analyses, the hearts were subdivided into 4 groups (n=10/group): A) control group: untreated hearts; B) Rosuvastatin 0.2: hearts from rats pre-treated with rosuvastatin at the dose of 0.2 mg/kg/day; C) Rosuvastatin 2: hearts from rats pre-treated with rosuvastatin at the dose of 2 mg/kg/day; D) Rosuvastatin 20, hearts from rats pre-treated with rosuvastatin at the dose of 20 mg/kg/day.

2.3.1. Heart weight changes
Hearts were weighed before and after the experiment on an analytical balance. Percent weight gain between the end and the beginning of the experiment was calculated in each group.

2.3.2. Creatinephosphokinase (CK) release
CK activity in the coronary effluent was measured by a spectrophotometric assay [18] immediately before ischemia (at 20 min), during the Langendorff perfusion mode (at 35, 37, 39 min), and during the working heart reperfusion mode (at 55, 65, 75, 85, 95, 105 min). Data were reported as IU/mL/g wet weight.

2.3.3. Ultrastuctural analysis
Myocardial tissue specimens were processed by electron microscopy, as described [18]. Histological changes (interstitial space area, mitochondrial damage score) were analyzed using a computerized image analysis system (Image-Pro plus, Media Cybernetics, Silver Spring, MD). To quantify mitochondrial damage, a validated mitochondrial score index was used [23]. Interstitial space changes were quantified as the area of interstitial space with respect to total myocardial area x 100; these data were expressed as percent changes with respect to normal heart specimens (n=5), serving as control.

2.4. Assessment of microvascular permeability changes
After ischemia, additional rat hearts (n=6/group) were perfused with fluoresceine isothyocyanate (FITC)–albumin (Sigma, Milan, I) (75 mg dissolved in 200 mL KH), to assess microvascular permeability, as described [18,24].

2.5. Evaluation of eNOS and iNOS mRNA expression
Semiquantitative multiplex reverse-transcription polymerase chain reaction (RT-PCR) was used to determine mRNA levels of the constitutive (endothelial) (eNOS) and the inducible (iNOS) nitric oxide synthase isoforms in ventricular tissue from myocardial samples (n=7/group, from the groups studied for the assessment of left ventricular function and myocardial tissue damage), as described [18].

2.6. Western blot analysis
Protein extracts were prepared according to Lee et al. [25]. Proteins (50 µg) were separated on 10% SDS-polyacrylamide gels and electroblotted to a nitrocellulose membrane. Endothelial (e)NOS and iNOS were detected by monoclonal antibodies, using β-actin as internal control to correct for variations of different samples, as described [18].

2.7. Nitrite measurements
Samples of coronary perfusate were collected to determine the levels of nitrites (stable metabolites of nitric oxide) spectrophotometrically at 540 nm by the Griess reaction, as described [18]. Nitrites were measured in the coronary effluent before ischemia (at 20 min), and during reperfusion (at 45, 60, 120 and 180 min). Each nitrite assay was determined in duplicate. The detection limit of the assay in our hands is 0.08 µmol/L.

2.8. Assessment of the effects of NOS inhibition
In order to confirm the involvement of nitric oxide in the cardioprotective effects of rosuvastatin, 30 µmol/L N{varpi}-nitro-L-arginine methylester (L-NAME, Sigma, Milan, I), a false substrate acting as specific inhibitor of NOS, was used in the perfusate of control hearts (n=8) or of hearts from rats treated with 2 mg/kg/day rosuvastatin (n=8) in the experimental protocols devoted to assessing left ventricular function and myocardial tissue damage (n=5) or microvascular permeability changes (n=3).

2.9. Statistical analysis
Two-group comparisons (heart weight, ultrastructural data, vascular permeability, changes in gene expressions) were analyzed using the Student's t-test. Multiple-group comparisons (hemodynamic parameters, CK release) were performed by two-way analysis of variance (ANOVA) (variables: drug concentration and time). All such tests were run after the assessment of normality of distribution. Significance level was set at P<0.05. All results are reported as mean ± S.D.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 5. Conclusions
 Acknowledgments
 References
 
3.1. Effects of in vivo administered rosuvastatin on left ventricular function, CK release and heart weight
Rosuvastatin treatment at the doses of 0.2 and, especially, 2 mg/kg/day conferred a significant cardioprotection against mechanical dysfunction compared with both control and rosuvastatin 20 mg/kg/day. Rosuvastatin 20 mg/kg/day determined no significant cardioprotection (Fig. 1). Concomitant with protection from mechanical dysfunction, rosuvastatin 0.2 and 2 mg/kg/day caused a significant improvement in coronary flow and resistances (Fig. 1). A significant reduction of early-reperfusion CK release was detected in hearts from rats treated with rosuvastatin 0.2 and, mainly, 2 mg/kg/day (Fig. 2). In the same groups, a significant reduction of reperfusion edema occurred (heart weight gain, group A (control): 30 ± 5%; group B (rosuvastatin 0.2 mg/kg/day): 22 ± 3%; group C (rosuvastatin 2 mg/kg/day): 16 ± 4%, P<0.001 vs. group A; group D: 19 ± 6, P<0.001 vs. group A).


Figure 1
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  		Fig. 1 Time course of minute work (mm Hg x mL/min), coronary flow (mL/min), pressure-rate product (mm Hg x bpm) and coronary vascular resistances (dyn.s.cm–5) in the control and rosuvastatin experimental groups. Data are expressed as mean ± S.D., with n=10 in each group. R: rosuvastatin.

 

Figure 2
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  		Fig. 2 The effects of in vivo rosuvastatin treatments on creatinephosphokinase (CK) release in the coronary effluent (n=10, top left panel), microvascular permeability (FITC-albumin extravasation) (n=6, top right panel), ultrastructural mitochondrial damage score index (n=7, bottom left panel) and interstitial space area (n=7, bottom right panel) in isolated–perfused hearts. Data are expressed as mean ± S.D. R: rosuvastatin; IOI: Integrated Optical Intensity units.

 
3.2. Effects of rosuvastatin on microvascular permeability
Ischemia–reperfusion induced a marked FITC-albumin extravasation in the peri-vascular and peri-myocytic space in the hearts of rats in the control group. In hearts obtained from the 0.2 and 2 mg/kg/day rosuvastatin groups, a significant reduction of FITC-albumin diffusion was detected (P<0.001 vs. control) (Fig. 2); at these concentrations, albumin extravasation was limited to the perivascular space, and no significant diffusion of the tracer was observed in the perimyocytic space. At the highest rosuvastatin dose used, post-ischemic hyperpermeability was similar to controls.

3.3. Effects of rosuvastatin on ultrastructural morphometry
We detected a moderate degree of intracellular and interstitial edema, as well as swollen and disrupted mitochondria in the control group as the result of ischemia–reperfusion. Here, myofibrils appeared focally disrupted or contracted. The coronary endothelium showed enlargement of tight junctions and interruption of the basal membrane, associated with a swelling of endothelial cells and intraluminal membrane-bound vesicles. In hearts obtained from rats pretreated with rosuvastatin at the doses of 0.2 and 2 mg/kg/day, signs of endothelial cell and myocyte damage, including changes in CK release, vascular permeability, mitochondrial damage score index and interstitial space area were all reduced. At rosuvastatin 20 mg/kg/day, ultrastructural signs of ischemia–reperfusion damage were, conversely, similar to control (Fig. 2).

3.4. Effects of rosuvastatin on eNOS and iNOS mRNA expression
Ischemia–reperfusion induced a significant (P<0.01) reduction in eNOS mRNA. Rosuvastatin 0.2 and, particularly, 2 mg/kg/day treatment produced a significant increase (by 50%, P<0.05; and 112%, P<0.02, respectively) of eNOS mRNA levels compared with control hearts. Ischemia–reperfusion also induced a significant (P<0.01) increase of iNOS mRNA levels vs. controls, which was significantly attenuated (by 47% and 71%, respectively) in 0.2 and 2 mg/kg/day rosuvastatin-treated hearts (P<0.05 and P<0.01, respectively) (Fig. 3).


Figure 3
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  		Fig. 3 The effects of in vivo rosuvastatin treatment on eNOS and iNOS mRNA expression after ischemia–reperfusion. Top panels: representative examples of results of NOS and GAPDH RT-PCR amplifications of mRNA in different experimental conditions. GAPDH m-RNA levels are used for the densitometric normalization of NOS mRNA levels. GAPDH levels closely match those of total RNA in the same experimental conditions (not shown). Position and size of markers in base pairs (bp) are shown on the right. 1: control heart in non-ischemic conditions; 2: heart subjected to ischemia–reperfusion; 3: heart subjected to ischemia–reperfusion from a rat treated with 0.2 mg/kg/day rosuvastatin; 4: heart subjected to ischemia–reperfusion from a rat treated with 2 mg/kg/day rosuvastatin; 5: heart subjected to ischemia–reperfusion from a rat treated with 20 mg/kg/day rosuvastatin. Lower panels: mRNA steady state levels are calculated as the densitometric ratio of eNOS to GADPH and iNOS to GAPDH mRNAs, with GAPDH serving as internal control. Data are expressed as mean ± S.D., with n=7 in each group. I/R: ischemia–reperfusion; R: rosuvastatin.

 
3.5. Immunoblot analysis of eNOS and iNOS proteins
eNOS protein was detectable in all samples. After ischemia–reperfusion, eNOS protein levels were significantly decreased (P<0.02). In hearts from rats treated in vivo with rosuvastatin 0.2 and, mostly, 2 mg/kg/day, a significant (P<0.05 and P<0.02, respectively) re-increase of eNOS protein levels compared with untreated hearts submitted to ischemia–reperfusion was observed (Fig. 4). iNOS was undetectable in control hearts and was dramatically induced after ischemia–reperfusion. In vivo treatment with rosuvastatin 0.2 and, particularly, 2 mg/kg/day significantly blunted this induction (Fig. 4). In hearts from rats treated with rosuvastatin 20 mg/kg/day, eNOS and iNOS protein levels were similar to controls (Fig. 4).


Figure 4
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  		Fig. 4 The effects of in vivo rosuvastatin treatment on eNOS and iNOS protein expression after ischemia–reperfusion. Original representative Western blots are reported in the upper panels and results of densitometry on lower panels. Levels of β-actin are shown for comparison. 1: control heart in non-ischemic conditions; 2: heart subjected to ischemia–reperfusion; 3: heart subjected to ischemia–reperfusion from rats treated with 0.2 mg/kg/day rosuvastatin; 4: heart subjected to ischemia–reperfusion from a rat treated with 2 mg/kg/day rosuvastatin; 5: heart subjected to ischemia–reperfusion from a rat treated with 20 mg/kg/day rosuvastatin. Data are expressed as mean ± S.D., with n=7 in each group. I/R: ischemia–reperfusion; R: rosuvastatin.

 
3.6. Effects of rosuvastatin on nitrite levels in the coronary effluent
Consistent with increased iNOS expression, increased nitrite levels after ischemia were observed in control-untreated hearts (at 20 min: 0.28 ± 0.09 µmol/L; at 45 min: 0.60 ± 0.12 µmol/L; P<0.01). Nitrite levels quickly decreased during reperfusion. In the hearts from rats treated with rosuvastatin 0.2 and 2 mg/kg/day, nitrite levels were significantly lower in the early phases of reperfusion compared with hearts from untreated animals (at 45 min from rats treated with rosuvastatin 2 mg/kg/day: 0.41 ± 0.05 µmol/L; P<0.02 vs. controls), while in hearts from the group of rats treated with rosuvastatin 20 mg/kg/day they were similar to control (0.55 ± 15 µmol/L, P=NS).

3.7. Effects of nitric oxide synthase inhibition on rosuvastatin effects
Perfusions of the excised heart with 30 µmol/L L-NAME (a false substrate inhibitor of all NOS) alone did not significantly affect any of the parameters investigated (data not shown). The addition of 30 µmol/L L-NAME to KH in rosuvastatin-treated hearts reversed the improvement in cardiac function, with a significant reduction of post-ischemic functional recovery (Fig. 5) and a significant increase of the heart weight gain and of CK release in the coronary effluent (Fig. 6). The addition of L-NAME also increased FITC-albumin extravasation both in the perivascular and the perimyocytic space (Fig. 6). Electron microscopy here confirmed a significant attenuation of rosuvastatin-related amelioration in indices of ultrastructural damage.


Figure 5
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  		Fig. 5 The effects of L-NAME on minute work (mm Hg x mL/min), coronary flow (mL/min), pressure-rate product (mm Hg x bpm) and coronary vascular resistances (dyn.s.cm–5) in control and the 2 mg/kg/day rosuvastatin-treated experimental animals. Data are expressed as mean ± S.D., with n=10 in each group. R: rosuvastatin.

 

Figure 6
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  		Fig. 6 The effects of L-NAME, rosuvastatin (R) and their combination on creatinephosphokinase (CK) release in the coronary effluent (n=5, top left panel), microvascular permeability (FITC-albumin extravasation) (n=3, top right panel), ultrastructural mitochondrial damage score index (n=5, bottom left panel) and interstitial space area (n=5, bottom right panel) in hearts from the control and the rosuvastatin 2 mg/kg/day-treated groups. Data are expressed as mean ± S.D. R: rosuvastatin; IOI: Integrated Optical Intensity units.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
5. Conclusions
 Acknowledgments
 References
 
In the present study, the chronic in vivo pre-ischemic administration of rosuvastatin before myocardial ischemia reduced myocardial dysfunction, vascular endothelial damage, as well as myocardial enzyme release and morphologically detectable injury occurring after ischemia–reperfusion, in normocholesterolemic rats. The rat model used by us has been already extensively characterized in the past as (a) exhibiting low levels of cholesterol (in the order of 90 mg/dL in this very same strain of rats [26]); (b) being resistant to the cholesterol-lowering effect of statins in general [27,28], and of rosuvastatin, in particular, up to the maximum dose used by us (20 mg/kg/day) [29]. Therefore, all effects of statins found in our model have to be defined as cholesterol-independent. Concurrent with these beneficial effects, rosuvastatin partially prevented eNOS mRNA and protein reduction induced by ischemia–reperfusion and reversed the majority of ischemia–reperfusion-related iNOS induction. Particularly, the action of rosuvastatin on eNOS appears to be causally linked to the described beneficial effects, since these were totally prevented by the additional acute treatment of the heart with the non-selective NOS inhibitor L-NAME.

Myocardial damage induced by ischemia–reperfusion in the isolated perfused rat heart has been extensively characterized by previous work [30–32]. Some reports have focused on the role of neutrophils in mediating this process [30,33,34]. Despite evidently being magnified by the presence of neutrophils in the system, it is however clear that ischemia–reperfusion damage also occurs in their total absence, such as in our experimental condition [18]. The main thrust of our current report is therefore the demonstration that statin-mediated cardioprotection, in this cell-free perfusion mode of the isolated–perfused working rat heart, also occurs with prior in vivo chronic drug administration, and not only in the previously reported less physiological situation of the acute addition of the drug to the cardiac perfusate [18].

The results with prior in vivo administration here fully confirm the existence of a complex dose–response curve: in our experiments, the chronic administration of rosuvastatin resulted in a concentration-dependent cardioprotective response at low doses (0.2 mg/kg/day, and, more pronounced, 2 mg/kg/day). A higher dose (20 mg/kg/day) resulted in a reversal of such cardioprotection, with non-significant differences in myocardial damage compared with control conditions. Although we did not observe the occurrence of statin-related cardiac damage with very high doses, as previously shown with in vitro administrations [18], the directional trend of this dose–response curve is consistent with those previous data. The reduction of cardioprotection shown at the highest dose, not investigated further in this study, might be due to the loss of the beneficially effects of rosuvastatin on e- and iNOS expression associated with the greater mitochondrial damage and coronary microcirculatory dysfunction occurring after ischemia–reperfusion. From a clinical standpoint, this effect appears anyhow irrelevant because only the doses found of cardioprotective efficacy in our system, similar to those previously validated in other experimental studies [21,22,29], are in the therapeutic range proposed in the preclinical and clinical studies with rosuvastatin [20].

Previous literature has indicated NO as a possible mediator of cardioprotection by statins. NO is known to modulate endothelial permeability [35], reduces post-ischemic hyperpermeability [36], decreases platelet adhesion and aggregation [37], and leukocyte adherence and emigration [38]. The administration of NO donors prevents reperfusion injury in an animal model [39]. Conversely, removal of NO by pharmacological inhibition of NOS [18,40,41] or in transgenic iNOS and eNOS null mice [41] exacerbates ischemia–reperfusion injury. Induction of iNOS and consequent NO overproduction might also, on the one hand, exert beneficial effects, by inhibiting the generation of inositol-1,4,5-trisphosphate, reducing calcium overload that mediates protein kinase-C translocation, and inhibiting neutrophil-associated injury [42]. On the other hand, excess NO production also leads to the reaction of NO with superoxide anion to form peroxynitrite, which is a strong cytotoxic agent [43]. Previous research has shown that the induction of iNOS (by tumor necrosis factor-{alpha}, IL-1 and interferon-{gamma}) exerts negative inotropic effects with deterioration of myocardial performance and the induction of myocardial damage [44–46], due to NO free radical action, likely involving the production of peroxynitrite [43,47]. In our study the nitrite content in the coronary effluent was significantly reduced in rosuvastatin-treated hearts, consistent with the reduction of iNOS overexpression, demonstrated at the level of mRNA and protein. An attenuation of this increase by statins would also promote cardioprotection during ischemia–reperfusion. It is therefore overall more likely that the biological role of eNOS and iNOS are different in ischemia–reperfusion injury [44,47,48]. Here a moderate increase of the basal NO production in the picomolar range, by an augmentation of eNOS, would restore endothelial function in the coronary microcirculation and prevent or delay vascular and cardiomyocyte damage. Conversely, the burst of NO production in the nanomolar range that occurs during reperfusion through increased iNOS activity, would promote lipid peroxidation and cell damage [44,47,48]. In our experimental model, rosuvastatin increases eNOS and decreases iNOS expression, with a net decrease in the mass production of NO as measured by the nitrite assay. Since however the cardioprotective effects of rosuvastatin are abolished (and not mimicked) by a concomitant treatment with L-NAME, they appear to be most likely mediated through the increased expression of eNOS. On the basis of the experiments with the NOS inhibitor, the causal role of limiting excess NO in rosuvastatin-induced myocardial and vascular protection in our system appears to be therefore less relevant than the restoration of "normal" eNOS activity.

This is the first investigation in such experimental model to address the issue of cardioprotection after chronic pretreatment with statins. Ikeda et al. administered rosuvastatin in rats for only 18 h before ischemia–reperfusion in an isolated–perfused heart model in the presence of polymorphonucleates, documenting that the drug limits the contractile dysfunction through an NO-mediated mechanism [22]. Jones et al. have also administered rosuvastatin for 18 h in mice before ischemia–reperfusion induced by the reversible ligature of the left anterior descending coronary artery, documenting increased NO production and attenuated myocardial necrosis [21]. Our report therefore differs from these two in that we used a chronic administration of the statin, resembling the human use, and we deliberately used a system of crystalloid perfusion, able to investigate a mechanism of cardioprotection independent from that mediated by effects on neutrophils or other circulating cells.

Two other studies used a chronic statin administration, similar in duration to the one used by us, but in pig models. Lazar et al. administered atorvastatin for 21 days before the occlusion of the second and third diagonal arteries in pigs for 90 min followed by 45 min of blood cardioplegic arrest and 180 min of reperfusion. Atorvastatin-treated pigs required fewer cardioversions, and had a smaller infarct size than untreated pigs [49]. Contrary to these findings, Rendig et al. treated pigs with atorvastatin, pravastatin, simvastatin or no statin, in a single daily dose for each drug, for 3 weeks. Parameters of myocardial performance and coronary resistance were measured invasively during coronary artery occlusion and reperfusion. Percent recovery of either regional or global ventricular function did not differ among groups, despite some differences in bradykinin and acetylcholine-induced relaxation of conductance coronary arteries [50]. Reasons for the discrepancy between these two reports are unclear, but may be quantitative rather than qualitative. Our study in the rat suggests that cardioprotection is not species-specific and is independent of cholesterol-lowering. Compared with these latter reports [49,50], our study additionally provides a mechanistic explanation–in terms of eNOS induction–for the benefit.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
Acknowledgments
 References
 
We have shown that rosuvastatin, chronically administered in vivo, in a condition mimicking the use of statins in humans, but in a species, the rat, resistant to the cholesterol-lowering effects of these drugs, exerts NO-dependent and neutrophil-independent cardioprotection from ischemia–reperfusion injury. These results support the clinical relevance of favorable, cholesterol-independent myocardial effects of HMG-CoA reductase inhibition in humans [51].


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Acknowledgments
References
 
This work was supported by an unrestricted grant from AstraZeneca, which is gratefully acknowledged. We also thank the specific important collaboration for this study of Dr. Teodoro Piliego, Dr. Fergus McTaggart, and Dr. Maren White from AstraZeneca.


   Notes
 
Time for primary review 22 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Acknowledgments
 References
 

  1. Shepherd J., Cobbe S.M., Ford I., Isles C.G., Lorimer A.R., MacFarlane P.W., et al. Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. West of Scotland Coronary Prevention Study Group. N Engl J Med (1995) 333:1301–1307.[Abstract/Free Full Text]
  2. Downs J.R., Clearfield M., Weis S., Whitney E., Shapiro D.R., Beere P.A., et al. Primary prevention of acute coronary events with lovastatin in men and women with average cholesterol levels: results of AFCAPS/TexCAPS. Air force/texas coronary atherosclerosis prevention study. JAMA (1998) 279:1615–1622.[Abstract/Free Full Text]
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