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Oxidative Stress Developed During Open Heart Surgery Induces Apoptosis: Reduction of Apoptotic Cell Death by Ebselen, a Glutathione Peroxidase Mimic

Maulik, Nilanjana; Yoshida, Tetsuya

Journal of Cardiovascular Pharmacology: November 2000 - Volume 36 - Issue 5 - p 601-608
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Apoptosis, a genetically controlled programmed cell death, has been found to play a role in ischemic reperfusion injury in several animal species including rats and rabbits. To examine whether this also is true for other animals, a surgically relevant model was established using an isolated in situ swine heart. Hearts were subjected to 15 min of normothermic regional ischemia by left anterior descending artery (LAD) occlusion followed by 30 min of normothermic cardioplegic arrest and 3 h of reperfusion. Oxygen free radicals have been shown to be the inducers of apoptosis and because reperfusion of ischemic myocardium is associated with the generation of free radicals, an additional group of hearts was preperfused with three different doses (5, 10, and 25 nM) ebselen, a glutathione peroxidase mimic, for 15 min before 15 min of LAD occlusion. Hearts were then subjected to 30 min of normothermic cardioplegic arrest followed by 3 h of reperfusion at normothermia. Control experiments were performed by perfusing the hearts for 4 h at normothermia. Two other groups of hearts were subjected to either 30 or 60 min of LAD occlusion followed by 30 min of cardioplegic arrest without subjecting them to reperfusion. At the end of each experiment, hearts were processed for the evaluation of apoptosis and DNA laddering. The in situ end-labeling (ISEL) technique was used to detect apoptotic cardiomyocyte nuclei while DNA laddering was evaluated by subjecting the DNA obtained from the cardiomyocytes to 1.8% agarose gel electrophoresis followed by photographing under UV illumination. The apoptotic cells appeared only after 90 min of reperfusion, as demonstrated by the intense fluorescence of the immunostained genomic DNA when observed under fluorescence microscopy. None of the ischemic hearts showed any evidence of apoptosis. These results were corroborated with the findings of DNA fragmentation showing increased ladders of DNA bands in the same reperfused hearts. The presence of apoptotic cells and DNA fragmentation in the myocardium was abolished by preperfusing the hearts in the presence of 10 nM ebselen, which also moderated the oxidative stress developed in the heart. Apoptotic cells and DNA ladders were completely absent in the hearts subjected to either 30 or 60 min of LAD occlusion. The results demonstrate that reperfusion of the ischemic heart induces apoptosis, which can be reduced with ebselen by reducing the oxidative stress associated with ischemia/reperfusion.

Department of Surgery, University of Connecticut School of Medicine, Farmington, Connecticut, U.S.A.

Received April 3, 2000; revision accepted August 8, 2000.

Address correspondence and reprint requests to Dr. N. Maulik at Department of Surgery, University of Connecticut, School of Medicine, Farmington, CT 06030-1110, U.S.A.

A growing body of evidence indicates that cardiomyocytes undergo apoptotic cell death in a variety of coronary diseases including heart failure (1), myocardial infarction (2), and ischemia/reperfusion (3-11). Characteristic signs of apoptosis appear in the ischemic myocardium only after several hours of ischemia. In the case of the rat heart, apoptosis first occurs after 2 h of ischemia, and significant number of cells undergo apoptotic cell death after prolonged ischemia (3,4). In contrast, reperfusion even after a brief period of ischemia results in apoptosis (5-11). Recent studies from our laboratory also demonstrated that in the rat heart, apoptosis does not occur with up to 2 h of ischemia, but significant numbers of myocytes are subject to apoptotic cell death and DNA fragmentation after 15 min of ischemia and 90 min of reperfusion (5-7). These results suggest that although apoptosis occurs during prolonged ischemia or in frankly infarcted myocardium, reperfusion triggers a distinct signal for apoptosis, which would contribute to the pathophysiology of surgical ischemic reperfusion injury in which the heart is frequently subjected to cardioplegic arrest, ischemia, and reperfusion.

Most of the studies on the contribution of apoptosis in myocardial ischemic reperfusion injury so far have been conducted using the rat heart or rabbit heart (5-7,9-11). To examine whether apoptosis contributes to myocardial reperfusion injury in other animals, we developed a more clinically relevant model using the isolated in situ swine heart subjected to 15 min of left anterior descending artery (LAD) regional ischemia followed by 30 min of normothermic cardioplegic arrest and 3 h of reperfusion. Additional studies were performed using 30 and 60 min of LAD occlusion followed by 30-min cardioplegic arrest without reperfusion and a control group with no ischemia whatsoever. The results of our study indicate that cardiomyocytes do not die by apoptosis even after 60 min of ischemia, but a significant number of myocytes undergo apoptotic cell death after only 90 min of reperfusion after 15 min of ischemia and 30 min of cardioplegic arrest. Because oxidative stress and changes in the redox system are believed to be an important regulator of apoptosis, (12), a group of hearts was pretreated with ebselen, a glutathione peroxidase mimic, before subjecting the hearts to ischemia/reperfusion protocol.

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MATERIALS AND METHODS

Animal protocol

Forty-four Yorkshire pigs of either sex weighing 20 to 25 kg were used in this study. Animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal resources and published by the National Institutes of Health (NIH publication 85-23, revised 1985). The pigs were tranquilized with ketamine (20 mg/kg) and anesthetized with sodium pentobarbital (25 mg/kg). Endotracheal intubation was performed, and ventilation was maintained by a volume ventilator with room air. Pigs were randomly divided into five groups: group I, perfused with blood for 4 h to serve as control (n = 6); group II, perfused with blood for 15 min and then subjected to LAD occlusion (just distal to first diagonal coronary artery) for 15 min followed by 30 min of global normothermic cardioplegic arrest and up to 3 h of reperfusion (n = 24: experiments terminated at 1, 1.5, 2, and 3 h of reperfusion, n = 6 per group); and groups III-V, hearts were preperfused with ebselen [5 nM (group III), 10 nM (group IV), 25 mM (group V)] for 15 min followed by 15 min of LAD occlusion followed by 30 min of cardioplegic arrest and 3 h of reperfusion (n = 6). The experimental protocol is depicted in Fig. 1. Additional experiments were performed by subjecting the hearts to 30 min (n = 4) and 60 min (n = 4) of LAD occlusion followed by 30 min of cardioplegic arrest in each case, but without reperfusion.

FIG. 1

FIG. 1

A median sternotomy was performed. The azygos vein was ligated. The pericardium was incised and suspended in a pericardial cradle. After heparinization with sodium heparin (300 units/kg), an arterial cannula was placed in the ascending aorta through the right carotid artery, and a venous cannula was placed in the right atrium through the right atrial appendage. Another cannula was placed in the left atrium through the appendage to control preload of the left heart. A catheter-tip pressure transducer (Millar Instruments, Inc., Houston, TX, U.S.A.) was inserted into the apex of the left ventricle for pressure recording. Cardiopulmonary bypass with a membrane oxygenator was used, and after collecting blood into the reservoir, the isolated heart was completed by cross-clamping the ascending aorta just distal to the right brachiocephalic artery, ligating both superior and inferior vena cavae, and cross-clamping the main pulmonary trunk. This means the complete cessation of systemic circulation except coronary perfusion with a cardiopulmonary bypass pump system. Coronary perfusion was maintained at 75 mm Hg. After stabilization, baseline measurements were made. The LAD was then occluded with a snare just distal to the first diagonal branch. After 15, 30, or 60 min of regional ischemia, coronary perfusion was stopped, and normothermic cardioplegic arrest was instituted. Initial high-potassium blood cardioplegic solution (K+, 20-24 mEq/L, 10 ml/kg) was injected through the arterial cannula. Additional low-potassium cardioplegic solution (K+, 8-12 mEq/L, 5 ml/kg) at normothermia was administered every 15 min. The snare on the LAD was released immediately after the induction of cardioplegic arrest to simulate surgical revascularization. Sonometric dimension crystals (diameter, 6 mm), which were made of 3-MHz piezoelectrc crystals (Triton Technologies, Inc., San Diego, CA, U.S.A.) were placed at the endocardial surface across the anteroposterior minor axis, septal-free wall minor axis, and base-apex major axis of the left ventricle. The anteroposterior crystals were placed adjacent to the anterior and posterior descending coronary arteries. The septal-free crystals were located one-half of the distance from the apex to the base. The base crystal was placed into the left ventricle adjacent to the origin of the left circumflex coronary artery, and the apex crystal was placed into the left ventricular apex. After 30 min of cardioplegic arrest, the heart was reperfused on a cardiopulmonary bypass pump. Normothermia was maintained with a heat exchanger, and reperfusion was continued for 180 min. Defibrillation was applied if the heart developed ventricular fibrillation during reperfusion. The heart was paced at 120 beats/min using an electronic pacer (Phillips & Bird, Inc., Richmond, VA, U.S.A.) when necessary. No cardiotonic or antiarrhythmic drugs were administered during the experiment. Blood samples were taken through the venous cannula at 60, 120, and 180 min of reperfusion for creatine kinase (CK) measurement.

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Measurement of myocardial function

Functional data were obtained by adding and withdrawing up to 60 ml of saline through the LA cannula to raise the left ventricular end-diastolic pressure (LVEDP) to create pressure-volume loops at 30, 60, 90, 120, and 180 min of reperfusion (13). These data were digitized and recorded in real time with a 12-bit AD converter sampling at 200 Hz using the Cordat II Data Acquisition, Analysis, and Presentation System (Data Integrated Scientific Systems, Pinckney, MI; Triton Technologies, Inc., San Diego, CA, U.S.A.). The digitized data were later analyzed using the CV Auto Report Cardiovascular Data Analysis Program (Scitelligence, Inc., Brighton, MI, U.S.A.).

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Measurement of creatine kinase release

CK was quantified from 0.5 ml of plasma obtained before LAD occlusion, and every 60 min during reperfusion. CK was analyzed by the enzymatic assay method using a CK assay kit (Sigma Diagnostics, St. Louis, MO, U.S.A.). The absorbance was read at 340 nm using a Beckman DU-8 spectrophotometer (Columbia, MD, U.S.A.).

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Evaluation of infarct size

For infarct-size determination, frozen hearts were sliced perpendicular to the long axis from apex to base in 1-cm-thick sections. Sections were then fixed in 2% paraformaldehyde. Thin heart cross sections were placed between two cover slips and digitally imaged using an IBM-compatible PC and a Microtek ScanMaker 600z, a 600 dot per inch, flat-bed, full-color scanner. The cross section was imaged at the maximal scaling and dot resolution that the scanner would allow. The digitized image was stored in Adobe TIFF file format by the software package, PhotoStyler, v.1.0.3 (U-Lead Systems, Inc., San Jose, CA, U.S.A.) (14). For analysis of infarct areas, some enhancement of the image was necessary at times to visualize more clearly the areas of staining by Corel Photo-Paint 4.0 (Corel Corp. Ltd., Dublin2, Ireland). Corel was also used to mark the stained areas. To quantitate the areas of interest in pixels, an NIH Image 5.1 (a public-domain software package) was used. The entire area of risk was quantified in pixels using the computer software, and the measured infarct areas were compared with the entire area at risk in a blinded fashion.

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Evaluation for apoptosis

Apoptotic cell death was evaluated by in situ end labeling (ISEL) of cardiomyocyte nuclei. In short, cryostat sections of heart biopsies were fixed with glutaraldehyde (1%) followed by permeabilization with methanol/acetone (1:1 vol/vol) (4,6,7). After incubating with proteinase K (20 μg/ml) for 20 min at ambient temperature, the sections were stained with Hoechst 33258 for 30 min in the dark. After repeated washings with phosphate-buffered saline (PBS) buffer, the sections were incubated in a solution containing potassium cacodylate (200 mM), CoCl2 (2 mM), bovine serum albumin (0.25 mg/ml), Tris-HCl, pH 6.6 (25 mM), biotin-16-dUTP (10 μM), and terminal transferase (25 units) for 1 h at 37°C. For control, sections were treated with DNAase I (10 U/ml) before adding terminal transferase. The reaction was terminated by repeated washing with PBS, and then the sections were incubated with a staining solution containing avidin-FITC (2.5 μg/ml), saline/sodium citrate buffer (4×), triton X-100 (0.1%), and powdered milk (5%) for 30 min in the dark. The sections were washed again with PBS, and the intensely fluorescent nuclei were observed by fluorescence microscopy, and ISEL-positive myocytes were counted.

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DNA fragmentation

Apoptosis is best characterized biochemically by the cleavage of genomic DNA into nucleosomal fragments of 180 bp or multiples thereof that are readily detected as a DNA ladder by gel electrophoresis. DNA was isolated by standard techniques (6,7). To perform DNA laddering, 10 μg of DNA obtained from control, ischemic, and reperfused myocardium was subjected to electrophoresis on 1.8% agarose gel containing 0.5 μg/ml ethidium bromide, and photographed under UV illumination.

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Oxidative stress

Malonaldehyde (MDA) was estimated in heart muscle to determine the development of oxidative stress and free radical generation, as described previously (15). In short, weighed heart biopsies were homogenized in 2 ml of 20% trichloroacetic acid, 5.3 mM sodium bisulfite, kept on ice for 10 min, centrifuged at 3,000 g for 10 min, and then supernatants were collected, derivatized with 2,4-dinitrophenylhydrazine (DNPH), and extracted with pentane. Aliquots of 25 μl in acetonitrile were injected onto a Beckman Ultrasphere C18 (3 mm) column (Fullerton, CA. U.S.A.). The products were eluted isocratically with a mobile phase containing acetonitrile/water/acetic acid (40:60:0.1, vol/vol/vol) and measured at three different wavelengths (307, 325, and 356 nm) using a Waters M-490 multichannel UV detector (Millford, MA, U.S.A.). The peak for malonaldehyde was identified by co-chromatography with DNPH derivative of the authentic standard, peak addition, UV pattern of absorption at the three wavelengths, and by GC-MS.

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Statistical analysis

For statistical analysis, a two-way analysis of variance (ANOVA) followed by Scheffé's test was first carried out using Primer Computer Program (McGraw-Hill, Blacklick, OH, U.S.A.) to test for any differences between groups. If differences were established, the values were compared using Student's t test for paired data. The values were expressed as mean ± SEM. The results were considered significant if p was <0.05.

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RESULTS

Effects of ebselen on myocardial function

Hemodynamic parameters with a load of 60 ml of saline in control and treatment groups are shown in Fig. 2. As expected, in group II, left ventricular function was significantly reduced during reperfusion as compared with the control group, demonstrating myocardial stunning associated with ischemia and reperfusion. Thus, both left ventricular developed pressure (LVDP) and the maximal first derivative of LV (LVmaxdp/dt) were decreased after 60 min of reperfusion, and further reduced during subsequent reperfusion periods. Ebselen, at 10 and 25 nM concentrations, showed significantly higher recovery of postischemic LVDP and LVmaxdp/dt at 1, 2, and 3 h of reperfusion, suggesting that in these concentrations, ebselen improved postischemic ventricular function. There was no difference in functional recovery between the ebselen concentrations of 10 and 25 nM. At lower concentration (5 nM), ebselen had no effect on functional recovery.

FIG. 2

FIG. 2

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Effects of ebselen on tissue injury

Release of CK in the coronary effluent is considered as a presumptive marker for tissue injury. As shown in Fig. 3, CK release increased steadily and progressively in both the experimental groups as compared with the control group. However, the rise in CK was significantly lower at 1, 2, and 3 h of reperfusion in the ebselen-treated groups (10 and 25 nM) compared with the untreated group and 5 nM ebselen-treated group.

FIG. 3

FIG. 3

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Effects of ebselen on myocardial infarction

Infarct size for each heart was expressed as (∑ infarct area of each slice/∑ Total ventricular area of each slice) × 100. The white area that was not stained by triphenyl tetrazolium indicated irreversible ischemic injury (Fig. 4A). Infarct size was significantly lower for both 10 and 25 nM ebselen groups compared with the control group (Fig. 4A and B). There was, however, no significant difference in infarct size between the 5 nM ebselen− treated and untreated groups at the end of 3 h of reperfusion.

FIG. 4

FIG. 4

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Induction of apoptosis by ischemia/reperfusion

We were unable to detect apoptotic cells in the control and in the ischemic hearts that were not subjected to reperfusion. Even 60 min of LAD occlusion could not induce apoptosis; there was no sign of fragmented nuclear DNA in these biopsies. Apoptotic cells were identified only in the reperfused hearts. The extent of apoptosis increased with the progression of reperfusion time. Apoptotic cells were first evidenced after 90 min of reperfusion (Fig. 5). The number of apoptotic cells increased after 2 and 3 h of reperfusion, as evidence from the immunohistochemical staining of the extended DNA in these hearts. The number of apoptotic cells was sharply reduced in the ebselen (10 nM)-treated hearts (Fig. 5).

FIG. 5

FIG. 5

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DNA fragmentation induced by ischemia/reperfusion

Like apoptotic cell death, even a prolonged (60-min) period of ischemia did not cause DNA laddering. DNA fragmentation became obvious after 90 min of reperfusion after 15 min of LAD occlusion and 30 min of cardioplegic arrest (not shown). Hearts reperfused for 2 and 3 h demonstrated more clear DNA fragmentation as compared with 90-min reperfused hearts. The amount of DNA fragmentation was dramatically reduced in the ebselen (10 nM)-treated hearts (Fig. 6).

FIG. 6

FIG. 6

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Effect of ebselen on MDA content of the hearts

The production of MDA is an indicator of the development of oxidative stress. We have estimated the level of MDA formation to monitor the extent of lipid peroxidation. As shown in Fig. 7, ebselen contributed to a significant reduction in MDA content throughout the reperfusion period. At the end of 3-h reperfusion, the MDA content in the ebselen (10 and 25 nM)-treated hearts showed significant reduction in MDA concentration compared with the nonebselen group and 5 nM ebselen-treated groups. There were no differences in the MDA concentration between the nontreated and 5 nM ebselen-treated groups.

FIG. 7

FIG. 7

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DISCUSSION

The results of our study clearly demonstrated that, similar to rat and rabbit hearts, up to 60 min of normothermic ischemia does not induce apoptotic cell death in swine hearts. To the contrary, cardiomyocytes undergo apoptosis after 90 min of reperfusion after as short as 15 min of ischemia in conjunction with 30 min of cardioplegic arrest. Additionally, our results indicated that oxidative stress developed during the reperfusion of ischemic myocardium plays a crucial role in the development of apoptosis. Ebselen reduced both apoptotic and necrotic cardiomyocyte cell death. However, ebselen was not able to inhibit apoptosis 100%, the fraction of apoptotic nuclei were still present (5% after 120 min R and 8% after 180 min R) in the treated group during reperfusion (Fig. 5). Evidence is rapidly accumulating to support the notion that both necrosis and apoptosis contribute to the pathophysiology of ischemic and reperfusion injury. Most of the studies so far have been reported for rat hearts in which no evidence of apoptosis were found in hearts subjected to up to 2 h of ischemia. For example, recent studies from our laboratory found no evidence for apoptosis in the rat hearts subjected to 2 h of ischemia, but apoptosis became evident when these hearts were subjected to 15 min of ischemia followed by 90 min of reperfusion (5-7). Another related study showed that apoptotic and necrotic myocyte cell deaths have been shown to contribute independently to infarct size in rats, but apoptosis did not become evident for up to 2 h of ischemia. In a more recent study, characteristic signs of apoptosis were shown to appear only after 2.25 h of ischemia. To the contrary, hearts subjected to relatively short periods of ischemia followed by reperfusion showed apoptosis, and this was independently shown by our group (5-7,10,11) and two other groups (4,9). In the present study, like rat and rabbits hearts, cardiomyocytes from swine heart also did not show any signs of apoptosis and DNA fragmentation for up to 1 h of LAD occlusion in conjunction with 30 min of cardioplegic arrest, but these cells underwent apoptosis only after 15 min of LAD occlusion followed by 30 min of arrest and 90 min of reperfusion. Taken together, this raises the interesting possibility that reperfusion of ischemic myocardium triggers some distinct signal for apoptosis, which is not mediated by up to 2 h of ischemia.

Substantial evidence exists to support the notion that reperfusion of the ischemic heart generates oxygen free radicals, which contribute to the pathogenesis of reperfusion injury (16-19). Interestingly, free radicals and/or oxidative stress are also common mediators of apoptosis, perhaps through the formation of lipid peroxidation and lipid hydroperoxides (8,20-21). A direct role of oxygen free radicals also has been found in the pathogenesis of apoptosis. For example, SOD or an expression vector containing SOD cDNA was found to delay apoptotic cell death (12) and free radical-mediated apoptosis was found to be modulated by protooncogene expression (22). In fact, reactive oxygen species and prooxidants may act through mobilization of Ca2+(23). In cultured sympathetic neurons, when injected with SOD or with an expression vector containing SOD cDNA, apoptosis was delayed by a considerable amount (12). Injection of antisense SOD expression vector into the neurons decreased the amount of SOD, simultaneously delaying apoptosis. This study also demonstrated that if SOD was injected after the development of oxidative stress, it had no effect on apoptosis. The results of the present study clearly demonstrated that a seleno peroxide mimic, ebselen, could reduce apoptotic cell death and DNA fragmentation in concert with reduction of myocardial ischemic reperfusion injury. The precise mechanism by which ebselen reduced programmed cell death was not clearly understood. Ebselen, a synthetic selenium-containing heterocycle, is known to function as an antioxidant by its ability to reduce lipid hydroperoxides (24). These authors demonstrated that ebselen suppresses free radical formation from hydroperoxides and hydrogen peroxide, which have been implicated in the pathogenesis of ischemic reperfusion injury. Indeed, this compound was also shown to reduce ischemia reperfusion injury (25). Recently, 2-(methylseleno)benzanilide, the main metabolite of ebselen in vivo, was shown to possess antioxidant activity against peroxynitrite, which may be formed in vivo during pathologic conditions (26). More recently, ebselen was found to inhibit apoptosis and DNA fragmentation in mouse thymocytes, with simultaneous inhibition of lipid peroxidation (27). These reports led us to use ebselen in our study to block apoptotic cell death in the ischemic reperfused hearts. The results of our study confirmed these previous findings (28) and further demonstrated that removal of oxidative stress developed during reperfusion of ischemic myocardium can reduce apoptosis in concert with the amelioration of reperfusion injury.

In this study, ebselen was found to be cardioprotective at 10 and 25 nM concentrations, whereas there was no effect at 5 nM concentration. Interestingly, no additional benefits were achieved at 25 nM ebselen compared with 10 nM ebselen concentration. At 10 nM, ebselen inhibited both apoptotic and necrotic cell death, reduced oxidative stress as evidenced by the decrease in MDA formation, and improved ventricular recovery, suggesting that this glutathione peroxidase mimic is an ideal cardioprotective compound in this particular concentration.

In summary, our results confirmed that the cardiomyocytes of swine hearts undergo apoptosis during reperfusion after even a short duration of ischemia. We also showed for the first time that the oxidative stress developed in the reperfused heart is one of the causative factors for the development of apoptosis. The model used in our study is relevant to open heart surgery in which reperfusion of the heart after brief ischemic insult and not prolonged ischemia (more than 2 h) contributes to ischemic reperfusion injury. The results also suggested that free radical scavengers/antioxidants previously known to reduce reperfusion injury may, at least in part, function by abrogating apoptotic cell death. It is tempting to speculate that a different mechanism may be instrumental for cardiomyocytes undergoing apoptosis after prolonged ischemia (>2 h).

Acknowledgment: This study was supported in part by NIH HL 22559, HL 33889, HL 34360, and HL 56803 as well as a Grant-in-Aid from the American Heart Association.

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Keywords:

Apoptosis; DNA laddering; Ischemia; Reperfusion; Free radicals; Oxidative stress; Ebselen; Glutathione peroxidase; Oxidative stress

© 2000 Lippincott Williams & Wilkins, Inc.