Introduction
Oxidative stress has been implicated in the pathophysiology of several types of cardiovascular disease (CVD), including ischemic stroke, myocardial ischemia, myocardial stunning, ischemia-reperfusion injury, hypertension and atherosclerosis. It is also considered to play a role in the progression of atherosclerosis (1–4). Previous studies have demonstrated that the majority of patients with CVD are likely to have chronic oxidative stress and that this is associated with their diagnosed disease state (5–7). H2O2 is an important byproduct of oxidative metabolism and is a major contributor to oxidative stress-induced functional and metabolic dysfunction.
The β-hydroxy-β-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are widely used to inhibit the progression of atherosclerosis and reduce the incidence of CVD. As well as their cholesterol-lowering effects, statins improve endothelial function in normocholesterolemia (8,9). Certain over-the-counter (OTC) products, including resveratrol, also exhibit similar effects to statins. Resveratrol (trans-3,5,4-trihydroxystilbene) is a polyphenol (phytoalexin) that naturally occurs in red wine and in a variety of therapeutic plants. In vitro experiments have revealed that the cardiovascular protective effects of resveratrol may occur through a number of mechanisms. Resveratrol inhibits the proliferation of smooth muscle cells, platelet aggregation and the oxidation of low-density lipoprotein cholesterol, and reduces the synthesis of lipids and eicosanoids, which promote inflammation and atherosclerosis (10). These multiple protective effects of resveratrol increase its demand as an OTC product, even for those undergoing treatment with statins.
A number of studies have demonstrated the aggravating effects of statins on oxidative stress in organisms (11,12). Such aggravating effects of statins on the myocardium have already been shown (13,14,15,16). The aim of the present study was to evaluate the effects of atorvastatin, resveratrol and resveratrol + atorvastatin (R+A) pretreatment on myocardial contractions and endothelial function in the presence of H2O2 as an experimental model of oxidative stress in rats.
Materials and methods
Animals and experimental procedure
A total of 28 male Wistar albino rats, aged 8 weeks and weighing 260–280 g, obtained from the Animal Care Facility of Meram Medical Faculty (Konya, Turkey) were used in the present study. Animals were housed identically in cages in an air-conditioned room under a 12-h light/dark cycle. Temperature and relative humidity were controlled within the limits of 21±2°C and 55±15%, respectively. All animals were acclimated for ≥7 days prior to the onset of the study. A standard diet and tap water were provided ad libitum. The experimental procedures were approved by the Animal Ethics Committee of the Meram School of Medicine (Konya, Turkey). All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and stored according to the manufacturers’ instructions unless otherwise specified. For 14 days, the control group (n=8) received 1.5 ml drinking water by oral gavage and 1 ml 10% v/v dimethyl sulfoxide [DMSO; intraperitoneal (i.p.)], the vehicle for resveratrol. The atorvastatin group (n=6) received 40 mg/kg atorvastatin (Lipitor®), which was prepared daily and dissolved in drinking water, by oral gavage and 1 ml 10% v/v DMSO i.p. for the same period of time. The resveratrol group (n=6) was treated with 30 mg/kg i.p. resveratrol and 1.5 ml drinking water by oral gavage for the 14 days, and the R+A group (n=8) was treated with 40 mg/kg atorvastatin by oral gavage and 30 mg/kg i.p. resveratrol. Rats were weighed every five days for adjustments to the dosing schedule and observed every day or as necessary. On day 15, the rats were anesthetized with an i.p. injection of 50 mg/kg body weight sodium pentobarbital. The heart and thoracic aorta were removed from each rat and placed immediately into fresh, oxygenated, ice-cold Krebs-Henseleit solution (KHS) composed of 119 mmol/l NaCl, 4.7 mmol/l KCl, 1.5 mmol/l MgSO4, 1.2 mmol/l KH2PO4, 2.5 mmol/l CaCl2, 25 mmol/l NaHCO3 and 11 mmol/l glucose.
Detection of myocardial contraction
Myocardial strips (10–13 mm long and 2–3 mm wide) were prepared from the left ventricle using a previously described method (17). The strips were placed in a 10-ml chamber containing oxygen-enriched (0.5 l/min) KHS at 37°C. One end of the strip was attached to a force transducer (MP30; Biopac Systems, Inc., Santa Barbara, CA, USA) by a thin silk thread and the other end was attached to a hook in the tissue bath. The strips were left for 30 min for stabilization. Following the stabilization period, the maximum contractions to electrical stimulation (ES) were recorded [frequency, 0.5 Hz; duration, 5 msec; and voltage, 30–40 V (20% above threshold)]. The effects of H2O2 on myocardial contractions were then evaluated at concentrations of 1×10−7, 1×10−6 and 1×10−5 M. Following each dose, the organ bath was washed with KHS and the next dose was applied after 20 min resting time. The same procedure was conducted for all four groups. Contractions are given as a percentage of the initial contractions.
Detection of thoracic aorta responses
Thoracic aorta rings (2–3 mm wide) were placed into a 10-ml chamber containing oxygen-enriched (0.5 l/min) KHS at 37°C. One end of the strip was attached to a force transducer (MP30; Biopac Systems, Inc.) by a thin silk thread, while the other end was pinned to a hook in the tissue bath. The strips were left for 15 min to spontaneously recover their isometric tension, following which they were gradually stretched to a resting force of 1 × g. The tissues were allowed to equilibrate for 30 min with repeated washing every 10 min with KHS. Following the equilibration period, the thoracic rings were contracted with 80 mM KCl. After the 30-min wash-out period in which the tissues were repeatedly washed every 10 min with KHS, 1×10−8, 1×10−7, 1×10−6, 1×10−5 and 1×10−4 M H2O2 were cumulatively added to the organ bath. Once the contractions reached a plateau, the tissues were washed twice every 15 min and incubated with 1×10−4 M N (G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) formation, for 30 min to evaluate the effect of the vascular endothelium on the H2O2 results. Following incubation, 1×10−8, 1×10−7, 1×10−6, 1×10−5 and 1×10−4 M H2O2 were cumulatively added to the organ bath once more. All results are expressed as a percentage of the previous contraction induced by 80 mM KCl.
Statistical analysis
Data are expressed as the mean ± standard error of the mean. The statistical significance of differences between the groups was analyzed by one-way analysis of variance or the Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.
Results
Intragroup myocardial results
H2O2 was applied to the organ bath at doses of 1×10−7, 1×10−6 and 1×10−5 M. To observe the effects of increasing doses of H2O2, an intragroup comparison of the myocardial results was carried out for each group. In the control group, H2O2 significantly reduced the contractions induced by ES at all doses (1×10−7 vs. 1×10−6 M and 1×10−6 vs. 1×10−5 M, P<0.01). The results were 94.16±5.94, 80.35±5.66 and 62.61±8.28% for doses of 1×10−7, 1×10−6 and 1×10−5 M, respectively. In the rats treated with atorvastatin, H2O2 caused a significant dose-dependent decrease in myocardial contractions (72.09±3.80, 66.59±3.14 and 48.96±8.93% for H2O2 doses of 1×10−7, 1×10−6 and 1×10−5 M, respectively; 1×10−5 vs. 1×10−7 M and 1×10−6 M, P<0.01). In the resveratrol group, no significant changes in contraction were observed following H2O2 application at all doses (87.91±2.33, 89.66±14.91 and 79.77±17.33% for H2O2 doses of 1×10−7, 1×10−6 and 1×10−5 M, respectively; P>0.05). In the R+A group, the contraction results following H2O2 application were 76.57±1.40, 66.34±5.91 and 66.55±11.10% for 1×10−7, 1×10−6 and 1×10−5 M H2O2, respectively; H2O2 application did not significantly decrease the contractions when its concentration was increased.
Intergroup myocardial results
Intergroup comparisons of the myocardial results were evaluated for all doses of H2O2. At 1×10−7 M H2O2 the atorvastatin group showed a significantly lower contraction percentage when compared with the control and resveratrol groups (P<0.01). The R+A group also demonstrated a significant decrease in contraction percentage when compared with the control group (P<0.01). However, no significant difference was observed between the contraction percentages of the resveratrol and control groups (Fig. 1A). Following a 20-min washing period, the organ bath was adjusted to 1×10−6 M H2O2. The myocardial contractions of the atorvastatin and R+A groups were significantly lower than those control group (P<0.05). The resveratrol group tissues showed a significantly higher percentage contraction than those of the atorvastatin and R+A groups (P<0.01). No significant difference was observed between the myocardial contractions in the resveratrol and control groups (Fig. 1B). At the final dose of H2O2 (1×10−5 M), the atorvastatin group exhibited a significant decrease in contraction compared with all the other groups (atorvastatin versus control and R+A groups, P<0.05; atorvastatin versus resveratrol group; P<0.01). The results of the present study demonstrated that resveratrol treatment alone attenuated the decrease in contraction percentage and that this effect was significant compared with all groups at 1×10−5 M H2O2 (P<0.01 vs. atorvastatin and control and P<0.05 vs. R+A). In the R+A group, the contraction percentages were higher than those in the atorvastatin group but significantly lower than those in the resveratrol group (P<0.05) (Fig. 1C).
Results of thoracic aorta responses
Fig. 2 shows the cumulative dose responses to H2O2 (between 1×10−8 and 1×10−4 M) in aortic segments for the control, resveratrol, atorvastatin and R+A groups, with and without 1×10−4 M L-NAME incubation. In all groups, H2O2 caused vasoconstriction and the contraction responses increased in a concentration-dependent manner. The aortic rings reached their maximum contraction at 1×10−4 M H2O2, and the maximum contraction responses were 16.14±1.09, 7.50±0.75, 6.82±1.33 and 5.58±1.37% for the control, atorvastatin, resveratrol and R+A groups, respectively. The H2O2 responses were significantly lower in the treatment groups than those in the control group at 1×10−4 and 1×10−5 M H2O2 (control versus atorvastatin and resveratrol groups, P<0.05; control versus R+A group, P<0.01). When the maximum vasoconstriction responses of the treatment groups were examined, no statistical differences were identified among the resveratrol, atorvastatin and R+A groups (Fig. 2A). Following incubation with 1×10−4 M L-NAME for 30 min, the contraction responses of the tissues to H2O2 significantly increased when compared with their previous responses (Fig. 2B). In all groups, L-NAME incubation significant augmented the H2O2 response at 1×10−5 and 1×10−4 M when compared with the response without L-NAME incubation (Fig. 3). The maximum responses were 25.56±3.32, 18.06±1.74, 26.19±3.17 and 23.24±3.24% for the control, atorvastatin, resveratrol and R+A groups, respectively.
The thoracic aorta responses demonstrated that treatment of rats with resveratrol, atorvastatin and R+A resulted in a significantly lower vascular contraction response to H2O2 at a concentration 1×10−4 M when compared with the control group. However, this result was eliminated with 1×10−4 M L-NAME incubation.
Discussion
The cardiac results of the present study revealed that oral administration of 40 mg/kg atorvastatin for 14 days resulted in a more sensitive myocardial response to H2O2 in rats. Treatment with 30 mg/kg i.p. resveratrol showed a cardioprotective effect against atorvastatin-aggravated and H2O2-induced contractile dysfunction in the rat myocardium. Furthermore, the study revealed that atorvastatin and resveratrol exhibited a protective effect against H2O2-induced vasoconstriction and that this protective effect was mediated by NO.
Vasoconstriction induced by cumulative concentrations of H2O2 was augmented with L-NAME incubation. These results indicated that the vasoconstriction elicited by high concentrations of H2O2 was negatively modulated by the endothelium. NO exerted a protective effect to counteract the oxidative effect of H2O2 in the groups without L-NAME incubation. The protective effect of NO on H2O2 in endothelial monolayer permeability has been previously demonstrated by McQuaid et al (18). In their study, it was concluded that, although lower levels of NO may only give a small amount of cytoprotection, the barrier dysfunction in the endothelium caused by H2O2 may be partially reversed by NO (13). It may be concluded from the results of the present study that the protective effect of resveratrol and/or atorvastatin treatment may be attributed to increased NO production in the vascular endothelium.
Under normal conditions, the H2O2 concentrations in human plasma, blood and vascular cells are likely to be in the lower micromolar ranges or below. However, in pathological states, including myocardial ischemia and heart failure, it has been demonstrated that H2O2 concentrations can increase to millimolar levels (19–21). Atorvastatin impairs cholesterol production by inhibiting the synthesis of mevalonate. In addition to cholesterol-lowering effects, statins inhibit the biosynthesis of the major natural antioxidants ubiquinone (ubiphenol) Q10 and glutathione peroxidase (22–24). As a result of this, statins may aggravate oxidative stress in the organism. Such aggravating effects of statins on the myocardium have previously been demonstrated (16). These changes may modulate myocardial contractility. A previous study revealed that an increase in reactive oxygen species (ROS) in the myocardium resulted in ischemia, reperfusion injury and myocardial damage (25).
Studies on the antioxidant effects of statins have been performed using oxidative stress markers (OSMs) in body fluids (26–29). Although OSMs are accepted to reflect the levels of oxidative stress within tissues, Argüelles et al (30) demonstrated that OSMs were not correlated with the tissue levels of oxidative stress; furthermore, they suggested that OSMs did not reflect the local oxidative stress status of individual organs. This is consistent with the results of the present study, in which atorvastatin treatment aggravated the H2O2 response in the myocardial tissue and showed a protective effect on H2O2-induced vascular contractions. Although the current study did not assess the antioxidant capacity of the myocardium, the diminished myocardial response may be attributed to decreased levels of antioxidant agents in the myocardium, including ubiquinone Q10, whose production is HMG-CoA reductase-dependent (31). The protective effect of resveratrol can be attributed to its inhibitory effect on ROS production in the myocardium (32).
Increased levels of pro-oxidants have been associated with vascular diseases and they are considered to be an important initial step in the development of vascular diseases, including atherosclerosis and hypertension (33). The present study demonstrated that atorvastatin treatment disrupted ES-induced myocardial function in the presence of H2O2, but that its co-treatment with resveratrol recovered this effect. R+A treatment also exhibited a protective effect on H2O2-induced vascular responses. From these results, resveratrol appears to be a promising treatment for the improvement of myocardial function in diseases associated with the development of oxidative stress. Resveratrol has been a popular choice in OTC products. Resveratrol is frequently used for the prevention of atherosclerosis; thus, the indications for its administration appear to be similar to those for the administration of statins. The combined treatment of R+A provides a superior treatment for CVD compared with treatment with either agent alone.