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Cardiovascular Research 2001 51(2):283-293; doi:10.1016/S0008-6363(01)00306-6
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Copyright © 2000, European Society of Cardiology

Simvastatin reduces reperfusion injury by modulating nitric oxide synthase expression: an ex vivo study in isolated working rat hearts

Pericle Di Napolia, Alfonso Antonio Taccardia, Alfredo Grillib, Rita Spinaa, Mario Felacob, Antonio Barsottia,1 and Raffaele De Caterinaa,*

aLaboratory of Experimental Cardiology, Department of Clinical Sciences and Bioimaging, "G. d'Annunzio" University of Chieti, Ospedale S. Camillo de Lellis, Via Forlanini, 50, 66100 Chieti, Italy
bDepartment of Biomorphology, Biology Section, "G. d'Annunzio" University of Chieti, Chieti, Italy

* Corresponding author. Tel.: +39-0871-41512; fax: +39-0871-402817 rdecater{at}ifc.cnr.it

Received 17 January 2001; accepted 4 April 2001


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: We tested the hypothesis of beneficial effects of the 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA)-reductase inhibitor simvastatin in a model of ischemia–reperfusion, and investigated potential mechanisms. Methods: Isolated working rat hearts were subjected to 15 min global ischemia and 22–180 min reperfusion in the presence or absence of simvastatin (10–100 µM). We evaluated creatinephosphokinase and nitrite levels in 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-transcribed polymerase chain reaction and Western blotting) in the presence or absence of the transcriptional inhibitor actinomycin-D. Results: Simvastatin (25 µM) significantly reduced myocardial damage and vascular hyperpermeability, concomitant with a reduction in endothelial and cardiomyocyte lesions. Protection became less evident at 50 µM and reverted to increased damage at 100 µM. At 25 µM, simvastatin significantly increased eNOS mRNA and protein compared with untreated hearts, probably due to a post-transcriptional regulation since unaltered by animal pretreatment with actinomycin D. Simvastatin also significantly 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 µM) to 25 µM simvastatin-treated hearts significantly reduced cardioprotection against ischemia–reperfusion. Conclusions: In this model, in the absence of perfusing granulocytes, the acute administration of a pharmacologically relevant simvastatin concentration reduces ischemia–reperfusion injury and prevents coronary endothelial cell and cardiomyocyte damage by cholesterol-independent, NO-dependent mechanisms.

KEYWORDS Endothelial function; Nitric oxide; Ischemia; Myocytes; Reperfusion; Statins


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Recent studies have suggested that cholesterol lowering with 3-hydroxy-3-methyl-glutaryl-Coenzyme A (HMG-CoA) inhibitors is associated with decreased cardiovascular morbidity and mortality in both primary and secondary prevention studies in coronary artery disease (CAD) [1–5]. This occurs to a magnitude unprecedented in previous trials with other cholesterol lowering strategies [6]. Statins also appear to reduce the incidence of stroke [7], the occurrence of which is not clearly related to cholesterol levels in epidemiological studies [8]. These favorable effects of statins have therefore been claimed to occur on top of cholesterol reduction [9]. Statins have also been reported to improve coronary endothelial function [10,11], which is impaired in hypercholesterolemia [12]. Although the mechanism for this restoration of endothelial function is to some extent attributable to the reduction of plasma low density lipoprotein (LDL) and subsequent atherosclerosis, direct effects of these drugs on endothelial function have also been recently reported [13]. In hypercholesterolemic subjects, indeed, the deterioration of endothelium-dependent vasodilation was attenuated after six [10], three [14] or even 1 month of statin therapy [15], therefore much earlier than any detectable improvement in coronary atherosclerosis. Recent reports have then demonstrated a preventive effect of statins on hypoxia [16] and oxidized LDL [17] -induced down-regulation of endothelial NO production and endothelial nitric oxide synthase (eNOS) activity, thus providing an experimental basis for the understanding of cholesterol-independent effects of statins on endothelial function.

We therefore hypothesized cholesterol-independent effects of a widely used statin, simvastatin, on myocardial damage, cardiac function and vascular permeability induced by ischemia–reperfusion. We investigated such effects in a model of myocardial damage in isolated working hearts of normolipidemic rats.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Animals and perfusion technique
One-hundred and forty-two adult male Wistar rats (250–300 g) were anesthetized with a mixture of ether and air. After injection of 1000 IU heparin in the femoral vein, the hearts were quickly excised and weighed. A modified Krebs–Henseleit (KH) solution (108 mM NaCl, 25 nM NaHCO3, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO2 2.5 mM CaCl2, 11 mM glucose, 287 mOsm, 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) were measured collecting aortic chamber overflow and heart chamber effluent into graded cylinders. Aortic pressure (mmHg) was monitored through a membrane transducer (TNF-R, Viggo-Spectramed, Oxnard, CA) connected to the aortic cannula. Heart rate (bpm) was determined with an epicardial electrocardiogram (Cardioline 350/1, Milan, Italy). Minute work (mmHgxml/min) was calculated as the product of cardiac output (aortic+coronary flow) and peak aortic systolic pressure. Coronary resistances (dynes·s·cm–5) were also calculated as mean aortic pressurex80/coronary flow. This investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No.85-23, revised 1996).

2.2 Assessment of left ventricular function and myocardial tissue damage
Hearts were submitted to 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). The hearts were subdivided into five main groups (n=10/group): (A) control group, untreated hearts perfused with KH solution; hearts treated with (B) 10 µM simvastatin; (C) 25 µM simvastatin; (D) 50 µM simvastatin; (E) 100 µM simvastatin. Simvastatin (Merck Sharp and Dohme Italy, Rome, Italy) was chemically activated by alkaline hydrolysis [18] and added to KH solution 20 min before ischemia.

2.2.1 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.

2.2.2 Creatinephosphokinase (CK) release
CK activity in the coronary effluent was measured by a spectrophotometric commercial assay (Boehringer Mannheim, Milan, Italy) at fixed times: immediately before ischemia (at 20 min), during Langendorff mode perfusion (at 35, 37, 39 min), and during working heart mode reperfusion (at 55, 65, 75, 85, 95, 105 min). Data were reported as IU/ml per g wet wt.

2.2.3 Ultrastructural analysis
Myocardial tissue specimens were fixed in 2.5% phosphate-buffered-saline glutaraldehyde (pH 7.4), post-fixed in 1.33% osmic acid, dehydrated in ethanol and embedded in Epon 812 resin. Thin sections (30–40 sections/specimen) were cut, stained with uranyl acetate and lead citrate, and studied by electron microscopy. An average of 30 fields in each section were examined and photographed at 4500x magnification. 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 mitochondrial score index was used [19]. Interstitial space changes were quantified as the area of interstitial space with respect to total myocardial areax100; these data were expressed as percent changes with respect to normal heart specimens (n=5), serving as control.

2.3 Assessment of microvascular permeability changes
After ischemia, rat hearts (n=6/group) were perfused with fluoresceine isothyocyanate (FITC)-albumin (Sigma, Milan, Italy) (75 mg dissolved in 200 ml KH) in order to assess microvascular permeability [20,21]. After FITC-albumin perfusion (mean perfusion time: 20 min), hearts were reperfused for 2 min with KH (in the Langendorff mode) to eliminate intravascular fluorescence, and re-weighed. Microvascular permeability changes were determined on FITC-albumin perfused-hearts by fluorescence microscopy. Ventricles were cut transversely into 4–5 blocks. Tissue blocks were immediately fixed in O.C.T. (Miles, Elkhart, IN) and stored at –80°C. Tissue blocks were mounted on a specimen holder in a Slee microtome-cryostat maintained at –35°C and oriented in order to cross-section capillaries and muscle fibers. Ten-micrometer sections were obtained from each time block, placed on a pre-warmed dark box for at least 1 h and then photographed at 40x magnification under UV light (488 nm). FITC-albumin accumulation was quantified using a computerized image analysis system. Results were expressed as integrated optical intensity (IOI) units [22].

2.4 Evaluation of eNOS and iNOS mRNA expression
For this purpose, hearts were subdivided into four groups (n=7/group):

• Group F: Hearts quickly excised, fixed in liquid nitrogen, then stored at –80°C and used for biochemical and immunohistochemical analyses as control;
• Group G: non-treated hearts submitted to 15 min global ischemia and 180 min reperfusion in the working heart mode;
• Group H: 25 µM simvastatin-treated hearts, submitted to ischemia–reperfusion;
• Group I: Hearts treated with the transcriptional inhibitor actinomycin D (1.5 mg/kg given intraperitoneally 3 h before heart excision) and then submitted to ischemia–reperfusion in the presence of 25 µM simvastatin.

Quantitative multiplex reverse-transcribed 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 rat ventricular tissue. Myocardial samples (n=7/group), fixed in liquid nitrogen and stored at –80°C, were homogenized in 800 µl of RNA Fast Solution (Celbio, Milan, Italy). Total RNA was isolated as recommended by the manufacturer. RNA was dissolved in diethyl pyrocarbonate (DEPC)-treated water and quantified spectrophotometrically at 260 nm. First-strand cDNA was generated by adding RNA (0.1 µg) to a mixture containing 1 mM deoxy-nucleoside-tri-phosphates (d-NTP), 1 U/µl RNase inhibitor, 2.5 U/µl Moloney murine leukemia virus reverse transcriptase, 2.5 µM random examers, 5 mM MgCl2, 10x PCR buffer in a final volume of 20 µl. Reverse transcription was performed at 42°C for 1 h followed by heat inactivation of reverse transcriptase at 99°C for 5 min. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified from the same amount of RNA to correct for variation of different samples. The PCR solution contained 10 µl of first-strand cDNA, 4 µl 10x PCR buffer, 2 mM MgCl2, 0.15 mM of both sense (5'-ACC ACA GTC CAT GCC ATC AC-3') and antisense (5'-TCC ACC ACC CTG TTG CTG TA-3') GAPDH primers, 0.15 mM of both sense (5'-CGA GAT ATC TTC AGT CCC AAG C-3') and antisense (5'-GTG GAT TTG CTG CTC TCT AGG-3') eNOS, or 0.15 mM of both sense (5'-TCT GTG CCT TTG CTG ATG AC-3') and antisense (5'-CAT GGT GAA CAC GTT CTT GG-3') iNOS primers, 2 U Termophylus Acquaticus (Taq) DNA polymerase (Celbio, Milan, Italy), and water to a final volume of 50 µl. These samples were overlaid with mineral oil and subjected to 35 cycles at 95°C for 60 s, 60°C for 60 s, and to one cycle at 72°C for 7 min. PCR products were run on 2% agarose gel electrophoresis and photographed after ethidium bromide staining under UV light. Bands on the gel were scanned and quantified using a computerized densitometric system (Bio Rad Gel Doc 1000, Milan, Italy).

2.5 Western Blot analysis
Ventricular samples from the hearts of groups F, G, H and I were used for this purpose. Protein extracts were prepared according to Lee et al. [23]. Proteins (50 µg) were separated on 10% SDS–polyacrylamide gels (BIO-RAD, Hercules, CA) and electroblotted to a nitrocellulose membrane. eNOS and iNOS were detected by monoclonal antibodies (Santa Cruz Biotech, Inc. Santa Cruz, CA). β-Actin was used as internal control to correct for variations of different samples. Protein bands were visualized by a chemiluminescence detection system and quantified determining the change in Integrated Optical Intensity (IOI) using a computerized system (Kodak ISO Transmission Density, Eastman Kodak Company, Rochester, USA).

2.6 Nitrite measurement
In the hearts of groups F, G, H and I, samples of coronary perfusate were collected to determine nitrite (stable metabolite of nitric oxide) level. Nitrite concentrations were quantified spectrophotometrically at 540 nm in the coronary perfusate by the Griess reaction to estimate the total amount of NO production according to Kanno et al. [24]. 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. Detection limit of the assay is 0.08 µmol/l.

2.7 Assessment of the effects of nitric oxide synthase inhibition
In order to confirm the involvement of nitric oxide in the cardioprotective effects of simvastatin, 30 µM N{varpi}-nitro-L-arginine methylester (L-NAME, Sigma, Milan, Italy), a specific inhibitor of nitric oxide synthase, was used alone (group J, n=8) or in 25 µM simvastatin-treated hearts (group K, n=8) in the experimental protocols devoted to assessing left ventricular function and myocardial tissue damage (n=5) or microvascular permeability changes (n=3).

2.8 Statistical analysis
Statistical analysis was performed using Student's t-test for two-group comparisons (heart weight, ultrastructural data, vascular permeability, changes in gene expressions) and two-way analysis of variance (ANOVA) (variables: drug concentration and time) for multiple-group comparisons (haemodynamic parameters, CK release), after the assessment of normality of distribution. The probability of null hypothesis <5% (P<0.05) was considered statistically significant. All results are reported as mean±S.D.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effects of simvastatin on left ventricular function, CK release and heart weight
Simvastatin treatment gave a significant cardioprotection against mechanical dysfunction at 25 µM compared to both control and simvastatin 10 µM, and a significant detrimental effect at higher concentrations (Fig. 1). At 50 µM simvastatin (group D), functional recovery was still significantly improved compared to controls, but less evident compared to 25 µM simvastatin. At 100 µM simvastatin (group E), a significant worsening of ventricular function was evident, with cardiac arrest occurring within 75 min. Parallel to these changes, 25 and 50 µM simvastatin caused a significant improvement in coronary flow and resistances (Fig. 1). A significant reduction of early-reperfusion CK release was detected at simvastatin 50 µM and, particularly, 25 µM (Fig. 2). In the same groups, a significant reduction of reperfusion edema occurred (heart weight gain group A: 30±5%; group B: 24±5%; group C: 11±4%, P<0.001 vs. group A; group D: 14±5, P<0.001 vs. group A; group E: 28±4.6%).


Figure 1
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  		Fig. 1 Time course of minute work (mmHgxml/min), coronary flow (ml/min), pressure–rate product (mmHgxbpm) and coronary vascular resistances (dynes·s·cm–5) in the control and simvastatin experimental groups. Data are expressed as mean±S.D., with n=10 in each group. SIM, simvastatin.

 

Figure 2
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  		Fig. 2 The effects of simvastatin, at different concentrations, 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). Data are expressed as mean±S.D. SIM, simvastatin; IOI, Integrated Optical Intensity units.

 
3.2 Effects of simvastatin on microvascular permeability
In the control group, ischemia induced a marked FITC-albumin extravasation in the perivascular and perimyocytic space. In 25 and 50 µM simvastatin-treated hearts, a significant reduction of FITC-albumin diffusion was detected (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. In 10 and 100 µM simvastatin-treated hearts, post-ischemic hyperpermeability was similar to controls.

3.3 Effects of simvastatin 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. Coronary endothelium showed enlargement of tight junctions, reduction of micropinocitosis vesicles, interruption of the basal membrane, associated with a swelling of endothelial cells and intraluminal membrane-bound vesicles. At 10 µM simvastatin, no significant ultrastructural changes were noticed compared to controls. At 25 and 50 µM simvastatin, signs of endothelial cell and myocyte damage were significantly reduced. At 100 µM simvastatin, ultrastructural changes were similar to controls, although a significant increase in indices of mitochondrial damage were detected (Fig. 2).

3.4 Effects of simvastatin on eNOS and iNOS mRNA expression
Ischemia–reperfusion induced a significant (P<0.02) reduction in eNOS mRNA, and 25 µM simvastatin treatment produced a significant (P<0.04) increase in eNOS mRNA levels compared with control hearts. Ischemia–reperfusion also induced a significant (P<0.01) increase in iNOS mRNA levels versus controls, which was significantly reduced in 25 µM simvastatin-treated hearts (P<0.02), and unchanged by actinomycin D pre-treatment (Fig. 3).


Figure 3
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  		Fig. 3 The effects of simvastatin 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 densitometric normalization of NOS mRNA levels. Position and size of markers in base pairs (bp) are shown on the left. (a) Control heart in non-ischemic conditions; (b) heart subjected to ischemia–reperfusion; (c) heart subjected to ischemia–reperfusion treated with 25 µM simvastatin; (d) hearts pre-treated with the transcriptional inhibitor actinomycin D (1.5 mg/kg) and then submitted to ischemia–reperfusion in the presence of 25 µM simvastatin. 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.

 
In 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). Simvastatin (25 µM) significantly (P<0.05) re-increased eNOS protein levels compared to untreated hearts submitted to ischemia–reperfusion. The concomitant treatment with actinomycin D did not significantly modify eNOS protein. iNOS was undetectable in control hearts and was dramatically induced after ischemia–reperfusion. Simvastatin (25 µM) treatment significantly blunted this induction (Fig. 4).


Figure 4
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  		Fig. 4 The effects of simvastatin on eNOS and iNOS protein expression after ischemia–reperfusion. Original Western blots are reported in the upper panels and results of densitometry on lower panels. Levels of β-actin are shown for comparison. (a) Control heart in non-ischemic conditions; (b) heart subjected to ischemia–reperfusion; (c) heart subjected to ischemia–reperfusion treated with 25 µM simvastatin; (d) hearts pre-treated with the transcriptional inhibitor actinomycin D (1.5 mg/kg) and then submitted to ischemia–reperfusion in the presence of 25 µM simvastatin. Data are expressed as mean±S.D., with n=7 in each group.

 
3.5 Effects of simvastatin on nitrite levels in the coronary effluent
Consistent with increased iNOS expression, an increase in nitrite levels after ischemia was observed in control-untreated hearts (controls=20 min: 0.28±0.09 µmol/l; 45 min: 0.60±0.12 µmol/l; P<0.01). Nitrite levels quickly decreased during reperfusion. In 25 µM simvastatin-treated hearts, nitrite levels were significantly lower in the early phases of reperfusion compared to untreated-hearts (at 45 min simvastatin 25 µM: 0.38±0.06 µmol/l; P<0.02 vs. controls). The concomitant treatment with actinomycin D did not significantly modify nitrite levels in 25 µM simvastatin-treated hearts (simvastatin 25 µM+actinomycin D: 0.34±0.07 µmol/l).

3.6 Effects of nitric oxide synthase inhibition on simvastatin effects
L-NAME perfusion (30 µM) alone (group K) did not significantly affect any of the parameters investigated (data not shown). The addition of 30 µM L-NAME to KH in 25 µM simvastatin-treated hearts (group J) induced a deterioration of cardiac function, with a significant reduction of post-ischemic functional recovery (Fig. 5) and a significant increase in heart weight gain (+24±4%; P<0.01 vs. group C) and CK release in the coronary effluent (Fig. 6). Addition of L-NAME to 25 µM simvastatin (group J) also increased FITC-albumin extravasation both in the perivascular and the perimyocytic space (Fig. 6). Electron microscopy confirmed a significant attenuation of simvastatin-related amelioration in indices of ultrastructural damage.


Figure 5
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  		Fig. 5 The effects of L-NAME on minute work (mmHgxml/min), coronary flow (ml/min), pressure–rate product (mmHgxbpm) and coronary vascular resistances (dynes·s·cm–5) in control and 25 µM simvastatin experimental groups. Data are expressed as mean±S.D., with n=10 in each group. SIM, simvastatin.

 

Figure 6
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  		Fig. 6 The effects of L-NAME 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 the control and 25 µM simvastatin experimental groups. Data are expressed as mean±S.D. SIM, simvastatin; IOI, Integrated Optical Intensity units.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
This study shows that the acute administration of the HMG-CoA reductase inhibitor simvastatin before myocardial ischemia reduces myocardial dysfunction (deterioration in hemodynamic performance and cardiac enzyme leakage), vascular endothelial and myocardial damage (post-ischemic increase in microvascular permeability, ultrastructural changes in endothelial cells and cardiomyocytes, and changes in microcirculatory resistances) occurring after ischemia–reperfusion in the isolated working rat heart. Concurrent with these beneficial effects, simvastatin partially prevents eNOS reduction induced by ischemia–reperfusion, likely occurring through post-transcriptional mechanisms since it is prevented by actinomycin-D. Simvastatin also prevents most of the ischemia–reperfusion-related iNOS induction. The action of simvastatin on the enzymes catalyzing NO production appears to be causally linked to the beneficial effects reported here, since it is totally abolished by the simultaneous treatment of the heart with the NOS inhibitor L-NAME.

Myocardial damage induced by ischemia–reperfusion in the isolated perfused rat heart has already been extensively characterized by previous work [25–28]. Some previous reports have focused on the role of neutrophils in mediating this process [26,28]. Despite evidently being magnified by the presence of neutrophils in the system, it is however clear that ischemia–reperfusion damage also occurs in the total absence of neutrophils, such as in our experimental system. In our experiments, the acute application of simvastatin, added in active form to the perfusion medium, resulted in a concentration-dependent biphasic response, with protection exerted up to a concentration of 25 µM, and potentiation of damage at higher concentrations. Since pharmacologically achievable plasma concentrations of simvastatin in humans are up to 0.5–10 µM [29], it is likely that in vivo relevant plasma concentrations may exert some protective effects against damage induced by ischemia–reperfusion. These were evident on multiple endpoints, including myocardial enzyme leakage, myocardial performance, vascular permeability, and a variety of ultrastructural changes occurring in cardiomyocytes and endothelial cells. Since the administration of simvastatin was acute and unable to affect cholesterol levels in our plasma-free system, these effects are also clearly independent of plasma and even, due to the rapidity of onset, of intracellular cholesterol levels [30]. They therefore appear to fully qualify as some of the ‘pleiotropic’ effects of statins, which have received increasing attention in recent years [6]. Such effects might be implicated in the preservation of endothelial function in early and advanced atherosclerosis, as well as in preventing the acute myocardial and vascular damage accompanying ischemia and/or reperfusion.

NO release is an important factor in the regulation of endothelial function. Besides its well known vasodilatory effects, NO is known to modulate endothelial permeability under basal and ischemic conditions [31], it reduces post-ischemic hyperpermeability [32], decreases platelet adhesion and aggregation [33], and leukocyte adherence and emigration [34].

The role of NO in ischemia–reperfusion damage and myocardial dysfunction remains controversial. Several investigations have reported that the administration of NO donors prevents reperfusion injury [35], but approaches to remove NO by pharmacological inhibition of NOS and transgenic iNOS and eNOS knockout mice have shown an exacerbation of reperfusion injury [24,36]. Comparisons between these studies are difficult because of differences in agents and study design, and of the multiple effects of NO during ischemia and reperfusion. On the one hand, NO has been reported to exert beneficial effects by inhibiting inositol-1,4,5-trisphosphate, reducing calcium overload, mediating protein kinase C translocation, and inhibiting neutrophil-associated injury [24]. On the other hand, NO also reacts with superoxide to form peroxynitrite, which is considered a strong cytotoxic agent. It is conceivable that the biological roles of eNOS and iNOS are different in ischemia–reperfusion conditions: an increase in the basal NO production in the picomolar range by an augmentation of eNOS would prevent and/or restore endothelial function in coronary microcirculation. Conversely, the burst of NO production in the nanomolar range, that occurs during reperfusion by the increase in iNOS activity, would promote lipid peroxidation and cell damage [37–39]. An attenuation of this increase by statins also would promote cardioprotection.

In our experimental model, simvastatin modulates NO synthesis, by increasing eNOS and decreasing iNOS expression. The first such effects appeared to be due to post-transcriptional mechanisms, likely mRNA stabilization, since abolished by the pretreatment with the transcriptional inhibitor actinomycin-D. This was used at pharmacologically effective concentrations, as shown by the inhibition of iNOS induction detected in our experiments. The increase in eNOS expression due to mRNA stabilization agrees with in vitro findings on cultured endothelial cells [16,17]. On theoretical grounds, it is likely that the eNOS increased expression by simvastatin has positive effects after ischemia–reperfusion because of the host of endothelial protective effects of NO. It is also conceivable, on the other hand, that a simvastatin-related decrease in iNOS induction after ischemia–reperfusion also could exert beneficial effects, due to the negative inotropic effects of the massive NO production occurring in conditions of iNOS induction [37–39]. Several studies have indeed demonstrated that the induction of iNOS activity (by various factors such as tumor necrosis factor-{alpha}, IL-1 and interferon-{gamma}) could induce myocardial damage or deterioration of myocardial performance [40–42]. One previous study has also shown that ischemia–reperfusion may induce and increase reperfusion damage [38], due to the free radical action of NO or of its byproduct peroxynitrite [37] promoting peroxidative damage of cell membranes [43]. In our study, the nitrite content in the coronary effluent was significantly reduced in simvastatin-treated hearts, likely due to the reduction of iNOS overexpression. The causal role of NO in simvastatin-induced myocardial and vascular protection in our system is proven by the loss of these favorable effects in the presence of the NO synthase inhibitor L-NAME.

Many of the various described pleiotropic effects of statins are currently ascribed to inhibition of production of metabolites distal to HMG-CoA reductase, such as farnesol [44–46], geranyl-geraniol [46], dolichol or ubiquinone and the isoprenylation of the {gamma}-subunit of certain G-proteins may be involved in the modulation of Ca2+ entry [30], an important step in reperfusion injury [47], and is likely implicated in the stabilization of eNOS mRNA [16], due to the role of rho proteins in accelerating eNOS mRNA degradation [16,17,48]. Recently, a potential role of non-genomic activation of eNOS due to modulation of signaling transmission in caveolae has also been suggested [49]. Inhibition of ubiquinone synthesis may itself induce mitochondrial damage by impairing mitochondrial respiration [50–52]. Inhibition of ubiquinone synthesis may be responsible for the mitochondrial ultrastructural damage and for the mechanical ventricular dysfunction observed at high (100 µM) simvastatin concentrations. The time elapsing between the administration of simvastatin and the peak effect (around 30 min) in our experimental system is indeed compatible with the inhibition of isoprenoid synthesis.

Our experiments were performed in isolated and buffer-perfused rat hearts, and therefore differ substantially from those performed by Lefer et al. [28] in a similar model, but in the presence of neutrophils. Together with ours, these data indicate that simvastatin exerts myocardial protective effects mediated by a reduction of neutrophil accumulation and neutrophil-independent effects. One possible candidate cardiac target for simvastatin-related cardioprotection independent of effects on circulatory neutrophils might be the population of resident mast cells [53], which are capable of degranulating and releasing preformed tumor necrosis factor (TNF)-{alpha}, initiating further cytokine release, iNOS expression and myocardial damage during ischemia–reperfusion [54]. This hypothesis awaits further investigation.

Limitations intrinsic in our experimental set-up may question the in vivo transferability of our results: (1) the oxygen content in our perfusion buffer is low, with consequent unphysiologically high baseline coronary flow; (2) glucose is practically the only energy source in our model, contrary to the preferential fatty acid utilization of the intact heart; (3) the absence of measurement of metabolites distal to HMG-CoA reductase (e.g. farnesol, geranyol) limits the evaluation of the effects of high dose of simvastatin; (4) simvastatin was administered acutely, different from most clinical uses of statins; (5) similar to most cholesterol-independent effects reported, active concentrations of simvastatin for these beneficial effects are at the upper end of the pharmacologically achievable range in humans, and with a narrow therapeutic window. Simvastatin concentrations of this order of magnitude are however likely achieved by more recent intense efforts at cholesterol reduction now attempted in some ongoing clinical trials, making our observations of potential clinical interest with the use of high statin doses.

In conclusion, we have shown that simvastatin exerts direct, cholesterol-independent, NO-dependent cardioprotection against ischemia–reperfusion injury in the isolated rat heart. These results expand the spectrum of potentially favorable effects of HMG-CoA reductase inhibition in CAD.

Time for primary review 29 days.


   Acknowledgements
 
This work was supported by Institutional funds of the University of Chieti (MURST 60%).


   Notes
 
1 Present address: Department of Internal Medicine, University of Genoa, Genoa, Italy. Back


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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