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1 From the Department of Pharmacology and First Department of Internal Medicine, School of Medicine, University of Debrecen, Debrecen, Hungary (TP, IB, PK, and AT); InterHealth Nutraceuticals, Benicia, CA (DB); and the Department of Surgery, School of Medicine, University of Connecticut Health Center, Farmington, CT (DKD).
2 Supported by grants from the Hungarian and German Academies of Sciences (MOB-DAAD 33); the Hungarian Basic Research Foundation (ETT T062/20 and OTKA T-032008); the National Institutes of Health (NIH HL 22559); and the NATO American-Hungarian Cooperative Project, Brussels (LST.CLG.977254). 3 Address reprint requests to A Tosaki, Department of Pharmacology, School of Medicine, University of Debrecen, Nagyerdei krt. 98, 4032-Debrecen, Hungary. E-mail: tosaki{at}king.pharmacol.dote.hu.
ABSTRACT
Background: Increasing evidence shows that red wine consumption has cardioprotective effects. These effects have been attributed to the polyphenolic compounds in grapes.
Objective: We studied the effects of red grape seed proanthocyanidins on the recovery of postischemic function in isolated rat hearts.
Design: Two groups of rats were fed different doses of proanthocyanidin-rich extract for 3 wk and another group was untreated and served as controls. The animals were then anesthetized and the hearts were isolated and subjected to 30 min of ischemia followed by 2 h of reperfusion. Coronary effluents were collected during the third minute of reperfusion for measurement of oxygen free radicals by using electron spin resonance spectroscopy.
Results: In rats treated with 50 and 100 mg grape seed proanthocyanidins/kg, the incidence of reperfusion-induced ventricular fibrillation was reduced from its control value of 92% to 42% and 25%, respectively (P < 0.05 for both). The incidence of ventricular tachycardia showed the same pattern. In rats treated with 100 mg proanthocyanidins/kg, the recovery of coronary flow, aortic flow, and developed pressure after 60 min of reperfusion was improved by 32% ± 8%, 98% ± 8%, and 37% ± 3%, respectively (P < 0.05 for all) compared with untreated control rats. Electron spin resonance studies indicated that proanthocyanidins significantly inhibited the formation of oxygen free radicals. In rats treated with 100 mg proanthocyanidins/kg, free radical intensity was reduced by 75% ± 7% (P < 0.05) compared with the control rats.
Conclusion: Grape seed proanthocyanidins have cardioprotective effects against reperfusion-induced injury via their ability to reduce or remove, directly or indirectly, free radicals in myocardium that is reperfused after ischemia.
Key Words: Grape seeds • proanthocyanidins • postischemic cardiac function • free radicals • electron spin resonance • antioxidants • wine • grapes • polyphenols • rats • ventricular arrhythmia
INTRODUCTION
It is known that free radicals play an important role in reperfusion-induced cardiac arrhythmias (1,2). Garlick et al (3) and Zweier (4) showed that abrupt reperfusion of the ischemic myocardium led to massive formation of oxygen free radicals. Thus, direct evidence suggests that formation of oxygen free radicals could be important in the development of reperfusion-induced ventricular arrhythmias. Agents known to scavenge or inhibit the formation of free radicals could prevent reperfusion-induced arrhythmias, including ventricular tachycardia (VT) and ventricular fibrillation (VF), and improve the recovery of myocardium that is reperfused after ischemia. Ventricular arrhythmias remain an important cause of death in ischemic heart diseases; therefore, every effort must be made to enhance the recovery of cardiac function and minimize sudden cardiac death caused by VF.
An inverse relation between moderate alcohol consumption and risk of cardiac disorders was found in several studies (5–7). Considerable controversy has arisen regarding the mechanisms that may be responsible for alcohol-induced cardiac protection; several different mechanisms have been suggested. These include inhibition of platelet aggregation (5), inhibition of neointimal hyperplasia (8), antithrombogenesis (9), increased concentrations of HDL (6), and effects on free radical production (10,11).
Flavonoids, which increase the antioxidant capacity of cells and tissues (12–14), are probably responsible for the antioxidant property of red wine. As a consequence, red wine drinkers have reduced risk of cardiovascular disease or death from coronary artery disease (15). Thus, the beneficial effect of red wine has been attributed to the antioxidants present in its polyphenol fraction (16); these antioxidants include resveratrol, catechin, and proanthocyanidins. In vitro studies reported that proanthocyanidins are potent scavengers of peroxyl and hydroxyl radicals that are generated in the reperfused myocardium after ischemia. This suggests that the cardioprotective effects could be attributed, at least in part, to the ability of proanthocyanidins to scavenge hydroxyl and peroxyl radicals (10,11).
We hypothesized that grape seed proanthocyanidins could play an important role in the scavenging of free radicals and could thereby reduce the incidence of reperfusion-induced arrhythmias in isolated rat hearts. This proanthocyanidin treatment was selected because it resulted in effective cardiac protection in rats during a previous study (11). Our study was designed to address 2 objectives: 1) to determine whether treatment with proanthocyanidins would reduce the incidence of reperfusion-induced VF and VT and improve postischemic cardiac function and 2) to determine whether proanthocyanidins could attenuate the formation of oxygen free radicals, as measured by electron spin resonance (ESR), in myocardium that was reperfused after ischemia.
MATERIALS AND METHODS
Animals
Sprague-Dawley rats (age: 17–19 wk; body weight: 320–340 g; Charles River Corp, Budapest) were used for all studies. Animals were housed in wire-bottomed cages (2 rats in each) for 3 d before the study began. Throughout the study, the rats were maintained on a 12-h dark-light cycle with free access to standard laboratory nonpurified diet (SSNIFF, rat/mouse-ZH; Soest ZH, Soest, Germany) and tap water. All animals received humane care in compliance with the National Academy of Sciences guidelines. The studies were conducted and approved by the Hungarian National Research Council.
Grape seed proanthocyanidin extract
We used a commercially available IH636 grape seed proanthocyanidin extract (ActiVin; InterHealth Nutraceuticals, Benicia, CA). Gas chromatography–mass spectrometry analyses in conjunction with HPLC showed that this novel IH636 grape seed proanthocyanidin extract contains 54% dimeric proanthocyanidins, 13% trimeric proanthocyanidins, 7% tetrameric proanthocyanidins, and small amounts (<5% each) of monomeric and high-molecular-weight oligomeric proanthocyanidins and flavonoids. Grape seed proanthocyanidins were homogenized in 2 mL 1% methylcellulose solution and then diluted with 0.9% NaCl to 10 mL.
Treatment
The rats were given 50 or 100 mg·kg-1·d-1 of the grape seed proanthocyanidins in the form of an extract for 3 wk. The extract was given orally by gavage at a rate of 10 mL·kg-1·d-1. An untreated group of age-matched control rats received a methylcellulose-saline solution daily for 3 wk.
Heart isolation
At the end of the treatment period, rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (60 mg/kg) and then given intravenous heparin. The hearts were excised and perfused with a drug-free buffer according to the Langendorff method (17) for a 10-min washout period at a constant perfusion pressure equivalent to 100 cm water (10 kPa). During the washout period, the pulmonary vein was cannulated and the Langendorff preparation was switched to the working mode for an additional 5 min of perfusion as described previously (18). The perfusion medium consisted of a modified Krebs-Henseleit bicarbonate buffer: 118 mmol sodium chloride, 4.7 mmol potassium chloride, 1.7 mmol calcium chloride, 25 mmol sodium bicarbonate, 0.36 mmol potassium biphosphate, 1.2 mmol magnesium sulfate, and 10 mmol glucose. After oxygenization, the pH was 7.4 at 37°C.
Exclusion criteria were decided in advance for the present studies. Working hearts were excluded if 1) ventricular arrhythmias occurred during the period before induction of global ischemia, or 2) coronary flow and aortic flow were <19 and 35 mL/min, respectively, before the initiation of ischemia.
Ischemia and reperfusion
To determine whether the proanthocyanidin treatment would reduce the incidence of reperfusion-induced VF and VT and improve postischemic cardiac function, hearts (n = 12 in each group) were subjected to 30 min of global ischemia followed by 2 h of reperfusion. The left atrial inflow and aortic outflow lines were clamped during ischemia at a point close to their origin, and reperfusion was initiated by unclamping the atrial inflow and aortic outflow lines. To prevent the myocardium from drying out during global ischemia, the thermostated glassware was covered and the humidity was kept constant at 90–100%.
To determine whether proanthocyanidins could attenuate the formation of oxygen free radicals in the myocardium, as measured by ESR, we collected heart effluents during the third minute of reperfusion. This time point was chosen for sampling because it gave a maximum signal intensity of free radical production in our model system (19) and because of our previous finding that a prominent ESR spectrum consisting of a 1:2:2:1 signal quartet [5,5-dimethyl-pyrroline-N-oxide (DMPO)-OH adduct] was observed after 3 min of reperfusion (20).
Electron spin resonance
Spin trap studies were performed by infusing the spin trap with DMPO through the side arm located just proximal to the end of the heart perfusion cannula. To prevent light-induced degradation of the DMPO solution, the infusion syringe was covered with aluminum foil. During the first 3 min of the Langendorff reperfusion period, DMPO (100 mmol/L stock solution) was infused directly into the untreated or proanthocyanidin-treated hearts at a rate of 1 mL/min. This resulted in a final perfusate DMPO concentration of 10–12 mmol/L, depending on the coronary flow of each heart. To prevent spin-adduct decay, the effluent was immediately frozen in liquid nitrogen as it flowed from the myocardium with an effluent sampling time of 30 s. ESR spectra were recorded in a flat quartz cell with a Bruker ECS106 spectrometer (Bruker Medical Instruments, Billerica, MA) operating at X band (9.3 MHz) with a 100-kHz modulation frequency. The microwave power was maintained at 10 mW to avoid saturation. Scans were traced with 0.2 mT of modulation amplitude with 2 min of scan time and 300 ms of response time. Hyperfine coupling constants were measured directly from the field scan by using Mn2+ as a marker for calibration.
Indexes measured
An epicardial electrocardiogram was recorded with a polygraph throughout the experimental period; 2 silver electrodes were attached directly to the heart. Each electrocardiogram was analyzed to determine the incidence of VF and VT (21). A heart was considered to be in VF if an irregular undulating baseline was apparent on the electrocardiogram. VT was defined as 5 consecutive premature ventricular complexes. This classification included repetitive monomorphic VT, which is difficult to dissociate from rapid VT. The heart was considered to be in sinus rhythm if normal sinus complexes occurring in a regular rhythm were apparent on the electrocardiogram. The aortic flow rate was measured with an in-line flow rotameter. The coronary flow rate was measured with a timed collection of the coronary effluent that dripped from the heart. Before ischemia and during reperfusion, the heart rate, coronary flow rate, and aortic flow rate were registered. The left ventricular developed pressure (LVDP) was also recorded with a catheter inserted into the left ventricle via the left atrium and mitral valve. The hemodynamic parameters were registered by using a Hemosys computer acquisition system, EXP COUPSYS SYSTEM (Experimetria, Budapest).
Statistical analyses
For the statistical analyses, we used a general calculator and a computer system described by Wallenstein et al (22). The data for heart rate, coronary flow rate, aortic flow rate, LVDP, and signal intensity of the DMPO-OH adduct are expressed as means ± SEMs. One-way analysis of variance was carried out to test for differences between the mean values of the groups. If differences were found, the values of the proanthocyanidin-treated groups were compared with those of the untreated control group by using a multiple t test followed by Bonferroni correction. For the distribution of discrete variables, such as the incidences of VF and VT, which follow a nonparametric distribution, an overall chi-square test for a 2 x n table was constructed. This was followed by a sequence of 2 x 2 chi-square tests to compare individual groups. A difference of P < 0.05 between the untreated control group and the treated groups was considered statistically significant.
RESULTS
Treatment with 50 and 100 mg proanthocyanidins/kg resulted in a stepwise reduction in oxygen free radical production in isolated rat hearts (Figure 1, top). In the group treated with 100 mg proanthocyanidins/kg (spectrum D), the production of oxygen free radicals, calculated from the ESR signal intensity of the DMPO-OH adduct, was reduced by 75% in comparison with the untreated control group (spectrum B). The quantitative ESR results (n = 6 hearts in each group) are also shown in Figure 1 (bottom). In both proanthocyanidin-treated groups (50 and 100 mg/kg), a significant reduction in the intensity of the DMPO-OH adduct was observed [Figure 1, bottom and top (spectra C and D)].
FIGURE 1. . Electron spin resonance (ESR) spectra of hydroxyl radical formation (top) and the signal intensity of 5,5-dimethyl-pyrroline-N-oxide (DMPO) adduct during the third minute of reperfusion (bottom) in untreated (control) and proanthocyanidin-treated hearts that were reperfused after ischemia. DMPO was infused during the first 3 min of reperfusion and effluent samples were frozen or immediately used for the ESR studies. ESR spectra were recorded in cells operating at 9.3 MHz with a 100-kHz modulation frequency. Scans were traced with 0.2 mT of modulation amplitude with 2 min of scan time and 300 ms of response time. The 4 spectra were as follows: A, typical spectrum recorded from buffer after passage through nonischemic heart; B, coronary effluent collected after 30 min ischemia followed by 3 min reperfusion in the untreated control heart; C and D, coronary effluents collected after 30 min ischemia followed by 3 min reperfusion in hearts from rats treated with 50 or 100 mg proanthocyanidins/kg, respectively. *Significantly different from the untreated control, P < 0.05 (n = 6 in each group).
This reduction in DMPO-OH signal intensity was reflected in a striking reduction in the incidence of reperfusion-induced VF and VT (Figure 2). The incidence of reperfusion-induced VF was reduced from its control value of 92% to 42% and 25% in rats treated with 50 and 100 mg proanthocyanidins/kg, respectively (P < 0.05 for both). The incidence of reperfusion-induced VT was reduced from its control value of 93% to 42% (P < 0.01) in rats treated with the higher dose (100 mg/kg) of proanthocyanidins.
FIGURE 2. . The incidences of ventricular fibrillation (VF) and ventricular tachycardia (VT) in rats (n = 12 in each group) given a daily dose of 0 (untreated control group), 50, or 100 mg proanthocyanidins/kg for 3 wk. Hearts were isolated and subjected to 30 min of global ischemia followed by 120 min of reperfusion. *Significantly different from the untreated control group, P < 0.05.
Treatment with proanthocyanidins did not change the heart rate significantly during reperfusion in comparison with control values (Figure 3). However, we observed a significant recovery in coronary flow rate, aortic flow rate, and LVDP in hearts obtained from proanthocyanidin-treated rats compared with the corresponding values in the untreated control rats.
FIGURE 3. . Mean (±SEM) heart rate, coronary flow rate, aortic flow rate, and left ventricular developed pressure (LVDP) in rats given a daily dose of 0 (untreated control group; ), 50 (), or 100 () mg proanthocyanidins/kg for 3 wk; n = 12 in each group. Hearts were isolated and subjected to 30 min of ischemia followed by 120 min of reperfusion. *Significantly different from the untreated control group, P < 0.05.
After the 3-wk treatment period, body weight and food intake did not differ significantly between the control group and the groups treated with 50 mg and 100 mg proanthocyanidins/kg (body weight: 332 ± 5, 328 ± 6, and 331 ± 4 g, respectively; food intake on the last day of treatment: 21.1 ± 0.9, 19.8 ± 1.0, and 22.1 ± 1.8 g/d, respectively).
DISCUSSION
Ischemic heart disease continues to be the most common cause of cardiac death, and VF is often responsible for the development of sudden cardiac death. Considerable evidence suggests that oxygen-derived free radicals are involved in the cardiac injury caused by ischemia and reperfusion. Increased postischemic susceptibility to damage caused by free radicals results from the build-up of a strongly reducing environment during ischemia along with a decreased antioxidant defense capacity. The mechanism of cellular damage involves the stepwise reduction of molecular oxygen to the relatively inactive superoxide and hydrogen peroxide, with subsequent metal ion–catalyzed formation of the highly reactive hydroxyl radical, the ultimate tissue toxicant (23).
Recently, many studies have sought to elucidate the role of free radicals in the pathophysiology of injury during myocardial ischemia and reperfusion. Indirect evidence of the involvement of free radicals in ischemia- and reperfusion-induced injury derives from studies in which antioxidant enzymes, organic antioxidants, or agents that inhibit the production of oxygen free radicals appeared to have certain effects. These effects were to 1) reduce the vulnerability of the myocardium to reperfusion-induced arrhythmias, 2) limit infarct size, 3) attenuate myocardial stunning, and 4) enhance postischemic recovery in models of surgical global ischemia (24).
Therapeutic strategies are designed to reduce free radical– induced damage, either by intervening in the process by which free radicals are formed or by scavenging the free radicals that have already been formed. Different degrees of protection were obtained with hydroxyl radical scavengers (25,26), chelators of iron and copper (27,28), antioxidant enzymes such as superoxide dismutase and catalase (26), and redox-metal displacement by zinc (29). The clinical application of these compounds is limited, however, by 1) their toxicity and side effects; 2) their solubility, persistence, and membrane penetration, 3) the requirement for preischemic loading of the tissue; and 4) the incomplete protection they provide even under ideal conditions.
Because epidemiologic evidence indicates that consumption of red wine is beneficial in the prevention of coronary artery disease (30), and this beneficial effect could be attributed to antioxidants present in the polyphenol fraction of red wine (16), we investigated the effects of grape seed proanthocyanidins. In the present study, we examined the effects of proanthocyanidins on hydroxyl radical formation, incidence of arrhythmias, and cardiac function in myocardium that was reperfused after ischemia.
We showed that the myocardium of rats fed proanthocyanidins was more resistant to injury caused by ischemia and reperfusion than was the myocardium of untreated control rats. The proanthocyanidin-fed group consistently showed a better postischemic ventricular recovery and a reduced incidence of reperfusion-induced VF and VT compared with the control group. The results suggest that the reduced incidence of reperfusion-induced VF and VT led to significantly better recovery of postischemic cardiac function (Figure 3), including coronary flow, aortic flow, and LVDP, in the proanthocyanidin-treated groups in comparison with the untreated control group. It is of interest to note that rats treated with 100 mg proanthocyanidins/kg had a pronounced advantage in the recovery of cardiac function compared with rats treated with 50 mg proanthocyanidins/kg.
Our study also showed that the development of reperfusion-induced arrhythmias and cardiac cell damage could contribute, at least in part, to the reactive hydroxyl radical formation in the reperfused isolated myocardium. One of the major functions of antioxidants is to interfere with free radical formation. It is well known that antioxidant reserve and antioxidant enzyme capacity are significantly reduced after ischemia and reperfusion. The loss of the key antioxidant enzymes and antioxidants reduces the overall antioxidant reserve of the myocardium and makes the heart susceptible to damage caused by ischemia and reperfusion. This reduced antioxidative defense is likely to be incapable of providing complete protection against increased activities of the reactive oxygen species.
Although the results of our study attribute the protective effects of proanthocyanidins to their ability to eliminate hydroxyl radicals, other possibilities should not be overlooked. Proanthocyanidins comprise a group of polyphenolic bioflavonoids ubiquitously found in fruit and vegetables. In this context, the effect of proanthocyanidins on intracellular calcium concentrations and the possible biological effects of other components of the extract merit discussion. Flavonoids may interact with intracellular calcium ions, leading to a reduction in the ionized calcium content. By this mechanism, flavonoids may increase the binding affinity of a substrate or improve the electron transfer efficacy between NADPH–ferrihemoprotein reductase and the P-450 enzyme (31), thereby providing further protection against reperfusion-induced calcium overload. Thus, we believe that any simple effect on intracellular calcium is likely to be relevant and important in the protection observed in our model system.
The observation that proanthocyanidins are active when given as a 3-wk pretreatment is of particular interest. Antioxidants, for example those involved in the glutathione peroxidase system, can reduce hydroxyl radicals to water. Thus, proanthocyanidins may not bind to the myocardium, but may instead remain active for several days or weeks and act as a sink for hydroxyl radicals. Furthermore, proanthocyanidins may act as a regenerator of other antioxidants, keeping the concentrations of other antioxidants high enough to affect the formation of hydroxyl radicals. It was shown by Cestaro et al (32) that red wine increases concentrations of ascorbic acid and -tocopherol, 2 major antioxidants, in the body. In addition, Roig et al (33) showed that red wine consumption by rats enhanced catalase glutathione peroxidase activity and lowered the ratio of reduced to oxidized glutathione.
The ability of proanthocyanidins to improve the functional recovery of the heart and reduce the incidence of arrhythmias after a period of global ischemia could have valuable applications during routine cardiac surgery and might also be useful in cardiac transplantation. This statement is supported by Feng et al (8), who found that long-term consumption of red and white wine decreases intimal thickening after balloon injury in cholesterol-fed rabbits. The ethanol content, as well as the phenolic antioxidants, in red wine might be responsible for these favorable effects. Because antioxidants were shown to effectively inhibit neointimal thickening and macrophage accumulation in animals (34) and to reduce restenosis after balloon angioplasty in humans (35), it would be worthwhile to determine whether red wine might alter restenosis after coronary angioplasty. Finally, the extrapolation of our results, obtained in isolated rat hearts, to an actual clinical situation should be viewed with some caution because of the absence of blood and its elements in our model system.
REFERENCES