© 1996 American Heart Association, Inc.
Articles |
Apoptosis in Ischemic and Reperfused Rat Myocardium
the Department of Physiology, Faculty of Medicine, University of Ottawa (Canada).
Correspondence to Henry Fliss, PhD, Department of Physiology, Faculty of Medicine, University of Ottawa, 451 Smyth Rd, Ottawa, Ontario K1H 8M5, Canada. E-mail hfliss@labsun1.med.uottawa.ca.
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Abstract |
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Apoptosis has been observed previously in hearts subjected to either continuous ischemia or ischemia followed by reperfusion. The purpose of this study was to compare the timing and extent of apoptosis in both continuously ischemic and reperfused myocardium. We show that rats subjected to continuous coronary artery occlusion display characteristic signs of apoptosis solely in the ischemic myocardium after only 2.25 hours of ischemia, as illustrated by positive in situ end labeling (ISEL) of apoptotic cardiomyocyte nuclei in tissue sections and/or the presence of DNA "ladders" in agarose gels. In contrast, reperfusion after a 45-minute occlusion accelerated the process, with apoptosis becoming evident solely in the reperfused myocardium after only 1 hour of reperfusion. ISEL and DNA ladder intensity increased with duration of ischemia or reperfusion. The volume of myocardium in which ISEL was observed was smaller in the reperfused hearts, and the ISEL-stained nuclei represented 23% and 33% of the total nuclei in the reperfused and permanently occluded myocardium, respectively. Therefore, the data suggest that reperfusion lowers the extent of apoptosis in ischemic myocardium but, paradoxically, accelerates the residual apoptosis, possibly because of reperfusion injury. A large accumulation of neutrophils was observed in both the permanently occluded and reperfused myocardium, suggesting that the inflammatory response may have contributed to apoptosis in both settings. This study therefore confirms that both ischemic and reperfused rat myocardium can undergo apoptotic cell death. However, the data suggest that although reperfusion lowers the number of myocytes undergoing apoptosis, it accelerates apoptosis in the nonsalvageable cells.
Key Words: apoptosis • myocardial ischemia • reperfusion injury • neutrophil
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Introduction |
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Prolonged periods of myocardial ischemia can cause tissue injury and cell death. Early reperfusion, although essential for tissue salvage, can, paradoxically, also cause increased cell mortality, possibly as a result of the inflammatory response and the associated neutrophil accumulation and oxidant production.1 2 3 4 The precise mechanisms of ischemic or reperfusion injury remain to be elucidated. However, it is clear that cell death remains the most important clinical consequence of both types of injury and that the success of any therapeutic intervention will depend heavily on a clear understanding of the mechanisms of this cell loss.
Although a large portion of the cell loss during cardiac ischemia and reperfusion occurs through necrosis,5 there is presently increasing interest in the possibility that myocardial cell death may also occur through apoptosis.6 Unlike necrosis, apoptosis proceeds through a genetically programmed series of biochemical and morphological steps designed to avoid the indiscriminate release of cytosolic contents and the ensuing inflammatory response.7 The salient features of this type of cell death are chromatin condensation, endonuclease fragmentation of internucleosomal DNA to multiples of 180- to 200-bp fragments, and cell fragmentation into small membrane-bound vesicles. The involvement of apoptosis in ischemic injury has been examined in a number of tissues. For example, several recent studies have shown that apoptotic cell death can occur in brain tissues subjected either to prolonged periods of ischemia8 or to transient ischemia followed by reperfusion.9 Similar studies have also provided evidence for apoptotic effects in kidney tissue during prolonged ischemia or after ischemia and reperfusion.10
A recent series of studies has also demonstrated cardiomyocyte apoptosis in a number of injurious settings. For example, apoptosis has been observed in isolated rat cardiomyocytes subjected to hypoxia11 or in isolated rat heart papillary muscle exposed to sustained stretching.12 In vivo studies have demonstrated cardiomyocyte apoptosis during postnatal maturation13 and spontaneous hypertension14 in rats and after rapid ventricular pacing15 and microembolization-induced cardiac failure16 in dogs. Significantly, clear evidence of apoptosis has also been documented recently in ischemic rat myocardium beginning after 
2 hours of ischemia.17 Extensive cardiomyocyte apoptosis was also observed in reperfused rabbit18 and rat19 hearts as well as in myocardial autopsy tissue after death from acute myocardial infarction.20 Therefore, it appears likely that apoptosis contributes significantly to myocardial ischemic injury.
The causes of apoptosis in ischemic or reperfused myocardium remain obscure. Moreover, it is not yet clear whether myocardial apoptosis is triggered consistently during both ischemia and reperfusion. The rabbit study suggests that reperfusion, but not ischemia alone, causes apoptosis.18 In contrast, the rat study clearly demonstrated apoptosis after ischemia but did not examine the effects of reperfusion.17 The principal objective of the present study was therefore to determine whether periods of ischemia, with or without reperfusion, can cause apoptosis in rat myocardium and whether the extent and timing of apoptosis is altered by reperfusion. We show that apoptosis can, in fact, be detected in both permanently ischemic and reperfused rat myocardium and that the onset of apoptosis is accelerated by reperfusion.
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Materials and Methods |
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Coronary Artery Occlusion and Reperfusion
The technique was a modification of a previously published protocol21 and was in compliance with institutional guidelines on the care and use of experimental animals. Briefly, male Sprague-Dawley rats (250 to 300 g) were anesthetized with 5% halothane/100% oxygen. The animals were then intubated and ventilated (10 mL/kg, 70 breaths per minute) with 1.5% halothane/100% oxygen using a rodent respirator (model 683, Harvard Instruments). An incision was made in the skin on the left side of the chest, and the pectoral muscles were gently retracted to expose the ribs. An incision was made through the fourth intercostal space, and the ribs were gently spread to expose the heart. A 6-0 silk ligature was placed under the left main coronary artery and was tied using a "shoestring" knot. The chest was briefly compressed to expel intrapleural air, and the pectoral muscles were returned to their original position to seal the thoracic incision. The skin incision was then closed using surgical clips, leaving one end of the coronary suture protruding from the chest. The animals were then ventilated with room air, and they regained consciousness within 5 to 10 minutes. After 45 minutes of occlusion, the coronary artery was reperfused by pulling on the exteriorized suture to release the knot and remove the suture, and reperfusion was continued for varying periods of time (1 to 4 hours). Another group of rats was subjected to varying periods of permanent occlusion only (from 1 hour and 45 minutes to 4 hours and 45 minutes). Sham-operated rats were subjected to identical treatment without tying the coronary ligature.
At the end of the reperfusion or permanent occlusion, the rats were anesthetized with sodium pentobarbital (65 mg/kg IP), the abdomen was opened, and 1 mL of Evans blue dye (5% in saline) was injected into the vena cava to stain the area of the myocardium perfused by the patent coronary arteries, thereby delineating the ischemic region by negative staining.22 For analysis of neutrophil content or DNA fragmentation, freshly excised hearts were immediately chilled in ice-cold saline, and the unstained and normal myocardial tissues were rapidly isolated at 0°C, weighed, and homogenized using the protocols below. For in situ end labeling (ISEL), hearts were rapidly excised and immediately chilled in saline at 0°C. They were then frozen over dry ice and sectioned (10 µm) in a cryostat. With sham-operated rats, the left ventricular free wall was separated from the remainder of the myocardium and analyzed as above.
Microsphere Injection
To demonstrate myocardial reperfusion, rats were anesthetized, and a cannula (PE-50 tubing) was advanced through the right carotid artery into the left ventricle. The coronary artery was then occluded for 45 minutes and reperfused as described above. After 5 minutes of reperfusion, black nonradioactive microspheres (10 µm, 3x106 in 1 mL saline, New England Nuclear) were injected through the carotid cannula over the span of 1 minute. Reperfusion was then continued for 4 hours, followed by Evans blue infusion as described above. Cryosections were prepared as described above, and the presence of microspheres was established in the normal and ischemic regions of the myocardium using light microscopy. To demonstrate the relationship between the regions of myocyte apoptosis and areas of hypoperfusion, rats were subjected to permanent occlusion (4.75 hours) or 45-minute ischemia followed by 4-hour reperfusion as described above. Evans blue dye (1 mL) containing 8x1010 fluorescent latex microspheres (FluoSpheres, 0.5 µm, blue fluorescent, Molecular Probes) and 2 mol/L KCl was injected, and cryosections were prepared as described above and examined for microsphere distribution using a Zeiss Axiophot microscope.
ISEL
The ISEL protocol was based on previously published procedures.23 24 25 Unless otherwise specified, all reagents were products of Sigma Chemical Co or BDH. Frozen cryostat sections were thawed and fixed in 1% glutaraldehyde for 15 minutes at room temperature (RT) and then washed twice (5 minutes each) with PBS. The sections were subsequently permeabilized with methanol/acetone (1:1) for 10 minutes at RT and washed twice with PBS. They were then incubated with 20 µg/mL proteinase K in 25 mmol/L Tris-HCl (1 mL per section), pH 6.6, for 15 minutes at RT, washed twice (15 minutes each) with water, stained with Hoechst 33258 (0.05 µg/mL) for 30 minutes at RT, protected from light, and washed three times (1 minute each) with PBS. The sections were then incubated in 75 µL of a buffer solution containing 200 mmol/L potassium cacodylate, 2 mmol/L CoCl2, 0.25 mg/mL bovine serum albumin, 25 mmol/L Tris-HCl, pH 6.6, 10 µmol/L biotin-16-dUTP (Boehringer Mannheim Canada), and 25 U terminal transferase (Boehringer) for 1 hour at 37°C in a humidified chamber.
The reaction was terminated by washing the sections three times (1 minute each) with PBS at RT. The sections were then incubated with 1 mL of a staining solution containing 2.5 µg/mL avidin-FITC, 4x saline–sodium citrate buffer, 0.1% Triton X-100, and 5% powdered milk for 30 minutes at RT and protected from light. The sections were washed three times with PBS and coverslipped in "antifade" solution containing 1 mg/mL p-phenylenediamine and 90% glycerol in PBS, and histofluorescence was monitored with a Zeiss Axiophot microscope. Positive control samples were prepared by incubating sections with 10 U/mL DNAse I for 20 minutes at 37°C before treatment with terminal transferase.
Agarose Gel Electrophoresis of DNA
The protocol was based on previously published procedures.26 27 Freshly isolated or frozen myocardium (200 to 500 mg) was minced in an equal volume of homogenization buffer (10 mmol/L Tris-HCl, 25 mmol/L EDTA, and 100 mmol/L NaCl, pH 8.0) at 0°C and was homogenized for 30 seconds using a Polytron homogenizer at 10 000 rpm. A 100 µL aliquot of the homogenate was then mixed with 1.25 mL of lysis buffer (10 mmol/L Tris-HCl, 25 mmol/L EDTA, 100 mmol/L NaCl, and 1.0% SDS, pH 8.0), and the suspension was incubated for 15 minutes at RT. The suspension was then centrifuged at 13 000g for 15 minutes at RT, and the supernatant, which is enriched for soluble fragmented DNA, was poured off and collected, taking care to leave behind the viscous pellet containing the intact DNA. The supernatant was treated with proteinase K (100 µg/mL) for 30 minutes at 50°C. Ethanol (final concentration, 60%) and NaCl (final concentration, 0.5 mol/L ) were then added, and the DNA was precipitated overnight at -20°C. The DNA was collected by centrifugation at 13 000g for 15 minutes at 4°C, dissolved in 500 µL of TE buffer (10 mmol/L Tris-HCl and 1 mmol/L EDTA, pH 8.0), and extracted once with phenol/chloroform saturated with TE buffer. The DNA solution was washed once with chloroform and was precipitated in 60% ethanol and 0.5 mol/L NaCl at -20°C for 1 hour. The DNA was collected by centrifugation, dried, dissolved in 50 µL TE buffer, treated with RNAse (100 µg/mL) for 30 minutes at 37°C, and subjected immediately to electrophoresis on agarose gels (1.5%) in TAE buffer (40 mmol/L Tris-HCl, 30 mmol/L acetic acid, and 2 mmol/L EDTA, pH 8.0).
Determination of Neutrophil Content
The concentration of myeloperoxidase (MPO) was determined as described by us28 and others21 previously and was used as an index of neutrophil accumulation. Freshly isolated myocardium (200 to 500 µg) was minced in 3 vol of buffer containing 50 mmol/L potassium phosphate, pH 6.0, 0.5% phenylmethylsulfonyl fluoride, and 0.5% hexadecyltrimethylammonium bromide and homogenized for 30 seconds with a Polytron homogenizer at 10 000 rpm at 0°C. The homogenate was kept on ice for 15 minutes and centrifuged at 36 000g for 15 minutes at 4°C. The supernatant was collected, glycerol was added to a final concentration of 10%, and the solution was frozen and kept at -80°C until assayed with o-dianisidine as the substrate.
Protein concentration was determined with Bio-Rad protein assay solution using bovine serum albumin as standard.
Statistical analyses were performed with Student's t test, and all values are presented as mean±SE.
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Results |
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Coronary Artery Occlusion and Reperfusion
To examine the effects of myocardial reperfusion, the left main coronary artery was occluded for 45 minutes, followed by reperfusion for 4 hours. Subsequent perfusion with Evans blue dye delineated a region in the left ventricle (311±25 mg, n=7) that did not stain with the dye. To confirm that this region had, in fact, been reperfused immediately after removal of the coronary artery suture, microspheres were injected directly into the left ventricle of test rats (n=3) within 5 minutes of the initiation of reperfusion, and reperfusion was continued for the usual 4-hour period. Subsequent microscopic examination of tissue sections revealed the presence of microspheres throughout the "unstained reperfused region" (data not shown), confirming that this region had in fact been thoroughly reperfused. Therefore, it appears likely that the appearance of nonperfused myocardium after 4 hours of reperfusion was caused by a postreperfusion gradual decrease in flow (progressive "no reflow"), probably induced by neutrophil accumulation and capillary plugging.29 The weight of the unstained reperfused region, herein referred to simply as "reperfused myocardium," decreased with shorter periods of reperfusion (not shown). The region in the permanently occluded rats that did not stain with Evans blue (412±47 mg, n=7) represented the area at risk and was significantly greater than the reperfused myocardium (P<.05).
ISEL
ISEL staining of histological sections from hearts subjected to coronary occlusion and 4 hours of reperfusion revealed intensely fluorescent nuclei, which were located primarily in the myocytes and only occasionally in endothelial cells or leukocytes (Fig 1
). The ISEL-positive nuclei were evenly distributed and were confined to regions that were generally well demarcated from the areas not containing ISEL staining (Fig 2A). Treatment of ISEL-stained sections with Hoechst 33258, a general nuclear stain, showed an even distribution of nuclei in the normal and reperfused myocardium (Fig 2B), thereby eliminating the possibility that the lack of ISEL staining in the normal myocardium may have been caused by the absence of nuclei. The possibility that the absence of ISEL staining in the normal myocardium was due to an artifact, such as the presence of Evans blue, or to the inaccessibility of nuclei to the ISEL stain was also excluded by the fact that sections treated with DNAse I before ISEL staining showed a similar density of fluorescent nuclei in both the normal and reperfused myocardium (Fig 2C). ISEL staining of sections from hearts subjected to continuous coronary occlusion for a period of 4 hours and 45 minutes also showed fluorescence in numerous nuclei in generally well-demarcated regions (Fig 2D). No ISEL staining was observed in sections obtained from sham-operated hearts after 4.75 hours.
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The regions of ISEL staining correlated well with the nonperfused regions of the myocardium, in both permanently ischemic and reperfused hearts. An example of this correlation is illustrated in Fig 3, which shows apoptotic myocytes solely in the nonperfused region of a permanently occluded heart. No nuclear fluorescence was detected in the normal (Evans blue–stained) myocardium of continuously occluded or reperfused hearts. The fraction of the Hoechst-stained nuclei in the area at risk, which showed ISEL fluorescence after 4.75 hours of continuous ischemia (32.8±1.8%, n=4), was significantly greater than that observed in reperfused myocardium after 4 hours of reperfusion (23±1.0%, n=3, P<.05).
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ISEL staining could be detected as early as 1 hour after reperfusion (Table). The intensity of fluorescence was generally lower after short periods of reperfusion (Fig 4) and increased with the duration of reperfusion. However, the fraction of nuclei showing fluorescence at shorter periods of reperfusion remained relatively constant and did not differ significantly from that observed after 4 hours of reperfusion (data not shown). As with reperfused myocardium, the fluorescence intensity in permanently ischemic myocardium also increased with an increasing duration of ischemia, and the fraction of nuclei showing fluorescence did not change significantly with changing times of ischemia (not shown). Clearly distinguishable nuclear fluorescence could first be observed only after 2 hours and 45 minutes of permanent ischemia (Table and Fig 5).
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Agarose Gel Electrophoresis of DNA
DNA "ladders," indicative of apoptotic internucleosomal DNA fragmentation, were clearly visible in agarose gels of DNA from the reperfused myocardium, but not the normal regions, of hearts subjected to 45 minutes of coronary occlusion followed by 4 hours of reperfusion (Fig 6). Ladders were also present in the DNA from ischemic myocardium of hearts exposed to 4 hours and 45 minutes of permanent occlusion (Fig 6). No ladders were observed with DNA from normal myocardium of permanently occluded or reperfused hearts or from myocardium of hearts from sham-operated rats (Fig 6). The intensity of the ladders diminished progressively with decreasing duration of reperfusion (Fig 6) as well as with decreasing duration of permanent occlusion (not shown). In general, clearly distinguishable DNA ladders appeared less consistently and somewhat later than ISEL-positive nuclei (Table
). The lanes containing DNA from the continuously ischemic or reperfused myocardium also showed a slight background smear, which is indicative of random DNA fragmentation and is therefore suggestive of necrotic cell death (Fig 6). The intensity of the smear appeared to increase with the duration of ischemia or reperfusion (Fig 6).
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Myocardial Neutrophil Content
The MPO activity in the reperfused myocardium of hearts subjected to 45 minutes of coronary occlusion followed by 4 hours of reperfusion increased significantly in comparison with the normal myocardium from the same hearts (Fig 7). However, a significant increase in MPO content was also observed in the ischemic myocardium compared with normal myocardium, from hearts subjected to 4 hours and 45 minutes of permanent occlusion (Fig 7). The MPO activity in the ischemic myocardium was not significantly different from that in the reperfused myocardium. Sham-operated rats did not show an increase in myocardial MPO activity. Evans blue did not interfere with MPO activity in our assays, as was also shown previously by others.30
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Discussion |
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The present study shows that either continuous ischemia or ischemia followed by reperfusion can cause apoptosis in the rat model of coronary artery occlusion. We show that rat myocardium exposed to as little as 2.25 hours of continuous ischemia alone undergoes apoptotic cell death. However, we show for the first time that myocardium that had been subjected to 45 minutes of ischemia followed by reperfusion appears to undergo accelerated apoptosis, displaying the characteristic signs of cardiomyocyte apoptosis as early as 1 hour after reperfusion. Our data therefore confirm the findings of two recent studies that showed apoptotic cell death in reperfused rabbit hearts (30-minute ischemia/4-hour reperfusion)18 and rat hearts (20-minute ischemia/24-hour reperfusion)19 but suggest that reperfusion-induced apoptosis can occur more rapidly than had previously been suspected. Our data also confirm the findings of a recent study that demonstrated early apoptosis in continuously ischemic rat myocardium.17 However, they conflict with a previous rabbit study that found no evidence of apoptosis after 4.5 hours of myocardial ischemia.18 The apparent absence of apoptosis in the ischemic rabbit heart is surprising in view of the well-documented apoptosis in other tissues exposed to continuous ischemia, such as the brain8 and kidney,10 and the fact that the reperfused rabbit myocardium did display apoptosis after 4.5 hours. In view of our data, a possible explanation for this anomaly may be that 4.5 hours of continuous ischemia may be insufficient to effect apoptosis in the rabbit but that reperfusion accelerated this form of cell death.
At the present time, definitive identification of apoptosis in differentiated tissue such as the myocardium remains problematic. Although a number of characteristic morphological changes are known to accompany apoptosis,31 32 their appearance in any given tissue may be dependent on the type or duration of injury. For example, cardiomyocytes in infarcted human myocardium do not show a number of the characteristic morphological markers,20 whereas cardiomyocytes in dogs with chronic heart failure do.16 In contrast, the biochemical tests for apoptosis appear to be more reproducible. ISEL labeling alone is suggestive of apoptotic DNA fragmentation33 but is not yet generally considered to be a definitive marker. Nevertheless, ISEL staining has been used recently to tentatively identify myocardial apoptosis in settings where the total number of apoptotic cells is low.13 16 34 However, the presence of both ISEL staining and DNA ladders is presently generally accepted as a strong indicator of apoptotic DNA fragmentation in tissues in general31 32 and in the myocardium in particular.12 15 18 20 Recent data show that cardiomyocytes contain significant amounts of endonucleases capable of internucleosomal DNA fragmentation.35 36
The present data show both intense ISEL staining and pronounced DNA ladders solely in the regions of the myocardium that were hypoperfused at the termination of either continuous ischemia or reperfusion, as indicated by the absence of Evans blue stain. In the reperfused hearts, the unstained region (reperfused myocardium) consisted of myocardium that had been successfully reperfused immediately upon removal of the coronary occlusion, as verified with microsphere injection, but subsequently reoccluded, presumably as a result of neutrophil accumulation in the microvasculature.29 ISEL staining was confined principally to the cardiomyocytes, suggesting that the observed DNA fragmentation was not associated with other cell types, such as invading neutrophils. An absence of neutrophil apoptosis has been reported previously in reperfused myocardium of cats and dogs by use of morphological criteria37 and in infarcted human myocardium by use of ISEL.20 Therefore, the present data clearly show that apoptosis occurs in both permanently ischemic and reperfused myocardium but not in control myocardium and is confined to the cardiomyocytes.
Clearly distinguishable DNA ladders appeared somewhat later than ISEL staining in each experimental group, suggesting that DNA end labeling may be a considerably more sensitive assay for the early phases of apoptosis. It is intriguing that although the intensity of the DNA ladders increased progressively with the duration of reperfusion or continuous ischemia, the fraction of nuclei undergoing apoptosis in either experimental group remained constant with time, as indicated by ISEL. This suggests that the myocytes that commit to apoptosis do so at an early stage after either ischemia or reperfusion and that the progressive increase in DNA ladder intensity simply reflects the time-dependent increase in DNA fragmentation in the committed cells.
Some aspects of the apoptosis observed in the reperfused myocardium were intriguingly different from the apoptosis observed in the permanently ischemic myocardium. The weight of the apoptotic myocardium (Evans blue negative) was significantly lower in reperfused hearts than in continuously ischemic hearts, and the fraction of nuclei undergoing apoptosis was also significantly smaller in the reperfused myocardium. Consequently, the total number of nuclei undergoing apoptosis was smaller in the reperfused myocardium than in the ischemic myocardium. These data confirm that early reperfusion can salvage ischemic myocardium in this model. Paradoxically, though, the fact that reperfusion accelerated the appearance of apoptotic nuclei in the reperfused myocardium suggests that reperfusion may actually increase the activity of the apoptotic mechanisms in cells committed to this form of cell death. The reason for this acceleration of apoptosis is not clear at the present time but may be attributable to the enhanced inflammatory response in reperfused myocardium, particularly the early sequestration of neutrophils. The reocclusion of the vasculature in the previously reperfused myocardium is indicative of "progressive no-reflow," a form of reperfusion injury probably caused by neutrophil sequestration.29 The accumulated neutrophils may cause injury through capillary plugging or oxidant production.1 2 3 4 Therefore, it seems possible that reperfusion may decrease the overall extent of apoptosis by salvaging ischemic myocardium while concomitantly promoting apoptosis through neutrophil-mediated reperfusion injury.
The precise role of neutrophils in myocardial apoptosis remains unclear at the present time. Recent studies that have examined the contribution of neutrophils to apoptosis in reperfused myocardium in vivo have failed to resolve this issue. Whereas one study has suggested that neutrophils do not potentiate apoptosis in rabbit heart,18 another has shown that the accumulation of neutrophils in reperfused rat myocardium is associated with increased apoptosis.19 The present data show that rat myocardium subjected to 4 hours of reperfusion shows a threefold increase in neutrophil content compared with normal tissue. However, continuously ischemic myocardium also showed a significant accumulation of neutrophils after 4 hours and 45 minutes. Our data are therefore in agreement with previous studies that have reported neutrophil accumulation in both continuously ischemic and reperfused rat myocardium.21 38 However, our study did not examine the rate of neutrophil accumulation in both models. It is possible that the faster rate of neutrophil sequestration in the reperfused myocardium potentiates the rate of apoptosis.
One important unresolved aspect of the present study is the relative contribution of apoptosis and necrosis to the myocardial injury sustained in our models of continuous ischemia or reperfusion. A number of studies have suggested that the relative proportion of apoptotic to necrotic cells in any given tissue may be dependent on the type of insult39 or the intensity and duration of the insult.40 Since both necrotic and apoptotic cells can be observed in ischemic human myocardium,20 it appears likely that the severity and duration of the ischemic insult dictate the relative occurrence of each type of cell death. A recent study has shown for the first time that in the rat model, sustained myocardial ischemia results in initial apoptotic cell death, which is followed several hours later by necrosis. The present study, which shows a time-dependent increase in DNA smearing in the agarose gels, provides support for a progressive increase in necrosis.
In summary, the present data show that continuous ischemia as well as ischemia followed by reperfusion can result in early myocyte apoptosis in rat myocardium. We demonstrate that the total volume of myocardium in which apoptosis can be detected, as well as the fraction of cardiomyocytes undergoing apoptosis, is smaller in the reperfused heart than in the continuously ischemic heart. However, this apparent protective effect of reperfusion is accompanied by a paradoxical acceleration of the residual apoptosis in the reperfused myocardium, possibly as a result of the reperfusion-associated inflammatory response.
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Acknowledgments |
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This study was supported by the Ontario Heart and Stroke Foundation. The technical contributions of Douglas A. Hubatsch, Michel Menard, and Ute Davis are gratefully acknowledged.
Received March 15, 1996; accepted August 27, 1996.
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P. Liu, B. Xu, T. A Cavalieri, and C. E Hock Age-related difference in myocardial function and inflammation in a rat model of myocardial ischemia-reperfusion Cardiovasc Res, December 1, 2002; 56(3): 443 - 453. [Abstract] [Full Text] [PDF]
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D. Pruefer, U. Buerke, M. Khalil, M. Dahm, H. Darius, H. Oelert, and M. Buerke Cardioprotective effects of the serine protease inhibitor aprotinin after regional ischemia and reperfusion on the beating heart J. Thorac. Cardiovasc. Surg., November 1, 2002; 124(5): 942 - 949. [Abstract] [Full Text] [PDF]
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 P A J Krijnen, R Nijmeijer, C J L M Meijer, C A Visser, C E Hack, and H W M Niessen Apoptosis in myocardial ischaemia and infarction J. Clin. Pathol., November 1, 2002; 55(11): 801 - 811. [Abstract] [Full Text] [PDF]
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 L. A. Kubasiak, O. M. Hernandez, N. H. Bishopric, and K. A. Webster Hypoxia and acidosis activate cardiac myocyte death through the Bcl-2 family protein BNIP3 PNAS, October 1, 2002; 99(20): 12825 - 12830. [Abstract] [Full Text] [PDF]
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H. L. Maddock, M. M. Mocanu, and D. M. Yellon Adenosine A3 receptor activation protects the myocardium from reperfusion/reoxygenation injury Am J Physiol Heart Circ Physiol, October 1, 2002; 283(4): H1307 - H1313. [Abstract] [Full Text] [PDF]
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K. Suzuki, B. Murtuza, I. A. Sammut, N. Latif, J. Jayakumar, R. T. Smolenski, Y. Kaneda, Y. Sawa, H. Matsuda, and M. H. Yacoub Heat Shock Protein 72 Enhances Manganese Superoxide Dismutase Activity During Myocardial Ischemia-Reperfusion Injury, Associated With Mitochondrial Protection and Apoptosis Reduction Circulation, September 24, 2002; 106(12_suppl_1): I-270 - I-276. [Abstract] [Full Text] [PDF]
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C. Stamm, I. Friehs, D. B. Cowan, H. Cao-Danh, Y.-H. Choi, L. F. Duebener, F. X. McGowan, and P. J. del Nido Dopamine Treatment of Postischemic Contractile Dysfunction Rapidly Induces Calcium-Dependent Pro-Apoptotic Signaling Circulation, September 24, 2002; 106(12_suppl_1): I-290 - I-298. [Abstract] [Full Text] [PDF]
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A. Abbate, R. Bussani, G. G.L. Biondi-Zoccai, R. Rossiello, F. Silvestri, F. Baldi, L. M. Biasucci, and A. Baldi Persistent Infarct-Related Artery Occlusion Is Associated With an Increased Myocardial Apoptosis at Postmortem Examination in Humans Late After an Acute Myocardial Infarction Circulation, August 27, 2002; 106(9): 1051 - 1054. [Abstract] [Full Text] [PDF]
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Z.-Q. Zhao and J. Vinten-Johansen Myocardial apoptosis and ischemic preconditioning Cardiovasc Res, August 15, 2002; 55(3): 438 - 455. [Abstract] [Full Text] [PDF]
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T. Date, A. J Belanger, S. Mochizuki, J. A Sullivan, L. X Liu, A. Scaria, S. H Cheng, R. J Gregory, and C. Jiang Adenovirus-mediated expression of p35 prevents hypoxia/reoxygenation injury by reducing reactive oxygen species and caspase activity Cardiovasc Res, August 1, 2002; 55(2): 309 - 319. [Abstract] [Full Text] [PDF]
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 G.D. Dispersyn, L. Mesotten, B. Meuris, A. Maes, L. Mortelmans, W. Flameng, F. Ramaekers, and M. Borgers Dissociation of cardiomyocyte apoptosis and dedifferentiation in infarct border zones Eur. Heart J., June 1, 2002; 23(11): 849 - 857. [Abstract] [Full Text] [PDF]
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G. Yaniv, M. Shilkrut, R. Lotan, G. Berke, S. Larisch, and O. Binah Hypoxia predisposes neonatal rat ventricular myocytes to apoptosis induced by activation of the Fas (CD95/Apo-1) receptor: Fas activation and apoptosis in hypoxic myocytes Cardiovasc Res, June 1, 2002; 54(3): 611 - 623. [Abstract] [Full Text] [PDF]
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 B. ZINGARELLI, P. W. HAKE, Z. YANG, M. O'CONNOR, A. DENENBERG, and H. R. WONG Absence of inducible nitric oxide synthase modulates early reperfusion-induced NF-{kappa}B and AP-1 activation and enhances myocardial damage FASEB J, March 1, 2002; 16(3): 327 - 342. [Abstract] [Full Text] [PDF]
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 S. Ruiz-Santana, A. Lopez, S. Torres, A. Rey, A. Losada, L. Latasa, J. L. Manzano, and B. N. Diaz-Chico Prevention of Dexamethasone-Induced Lymphocytic Apoptosis in the Intestine and in Peyer Patches by Enteral Nutrition JPEN J Parenter Enteral Nutr, November 1, 2001; 25(6): 338 - 345. [Abstract] [PDF]
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J. Shiraishi, T. Tatsumi, N. Keira, K. Akashi, A. Mano, S. Yamanaka, S. Matoba, J. Asayama, T. Yaoi, S. Fushiki, et al. Important role of energy-dependent mitochondrial pathways in cultured rat cardiac myocyte apoptosis Am J Physiol Heart Circ Physiol, October 1, 2001; 281(4): H1637 - H1647. [Abstract] [Full Text] [PDF]
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K. Suzuki, B. Murtuza, R. T. Smolenski, I. A. Sammut, N. Suzuki, Y. Kaneda, and M. H. Yacoub Overexpression of Interleukin-1 Receptor Antagonist Provides Cardioprotection Against Ischemia-Reperfusion Injury Associated With Reduction in Apoptosis Circulation, September 18, 2001; 104 (2009): I-308 - I-313. [Abstract] [Full Text] [PDF]
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B. Stadler, J. Phillips, Y. Toyoda, M. Federman, S. Levitsky, and J. D. McCully Adenosine-enhanced ischemic preconditioning modulates necrosis and apoptosis: effects of stunning and ischemia-reperfusion Ann. Thorac. Surg., August 1, 2001; 72(2): 555 - 563. [Abstract] [Full Text] [PDF]
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T. Scarabelli, A. Stephanou, N. Rayment, E. Pasini, L. Comini, S. Curello, R. Ferrari, R. Knight, and D. Latchman Apoptosis of Endothelial Cells Precedes Myocyte Cell Apoptosis in Ischemia/Reperfusion Injury Circulation, July 17, 2001; 104(3): 253 - 256. [Abstract] [Full Text] [PDF]
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H. Liu, B. C. McPherson, and Z. Yao Preconditioning attenuates apoptosis and necrosis: role of protein kinase C{epsilon} and -{delta} isoforms Am J Physiol Heart Circ Physiol, July 1, 2001; 281(1): H404 - H410. [Abstract] [Full Text] [PDF]
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E. Palojoki, A. Saraste, A. Eriksson, K. Pulkki, M. Kallajoki, L.-M. Voipio-Pulkki, and I. Tikkanen Cardiomyocyte apoptosis and ventricular remodeling after myocardial infarction in rats Am J Physiol Heart Circ Physiol, June 1, 2001; 280(6): H2726 - H2731. [Abstract] [Full Text] [PDF]
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G.-W. Wang, Z. Zhou, J. B. Klein, and Y. J. Kang Inhibition of hypoxia/reoxygenation-induced apoptosis in metallothionein-overexpressing cardiomyocytes Am J Physiol Heart Circ Physiol, May 1, 2001; 280(5): H2292 - H2299. [Abstract] [Full Text] [PDF]
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Z. Chen, C. C. Chua, Y.-S. Ho, R. C. Hamdy, and B. H. L. Chua Overexpression of Bcl-2 attenuates apoptosis and protects against myocardial I/R injury in transgenic mice Am J Physiol Heart Circ Physiol, May 1, 2001; 280(5): H2313 - H2320. [Abstract] [Full Text] [PDF]
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P. S. RAY, J. L. MARTIN, E. A. SWANSON, H. OTANI, W. H. DILLMANN, and D. K. DAS Transgene overexpression of {alpha}B crystallin confers simultaneous protection against cardiomyocyte apoptosis and necrosis during myocardial ischemia and reperfusion FASEB J, February 1, 2001; 15(2): 393 - 402. [Abstract] [Full Text] [PDF]
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Y.-P. Wang, H. Xu, K. Mizoguchi, M. Oe, and H. Maeta Intestinal ischemia induces late preconditioning against myocardial infarction: a role for inducible nitric oxide synthase Cardiovasc Res, February 1, 2001; 49(2): 391 - 398. [Abstract] [Full Text] [PDF]
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J.-F. Wang, X. Ren, J. DeAngelis, J. Min, Y. Zhang, T. G. Hampton, I. Amende, and J. P. Morgan Differential Patterns of Cocaine-Induced Organ Toxicity in Murine Heart versus Liver Experimental Biology and Medicine, January 1, 2001; 226(1): 52 - 60. [Abstract] [Full Text]
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 A. Bergh, O. Collin, and E. Lissbrant Effects of Acute Graded Reductions in Testicular Blood Flow on Testicular Morphology in the Adult Rat Biol Reprod, January 1, 2001; 64(1): 13 - 20. [Abstract] [Full Text]
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 Y. Xu, A. S. Clanachan, and B. I. Jugdutt Enhanced Expression of Angiotensin II Type 2 Receptor, Inositol 1,4,5-Trisphosphate Receptor, and Protein Kinase C{epsilon} During Cardioprotection Induced by Angiotensin II Type 2 Receptor Blockade Hypertension, October 1, 2000; 36(4): 506 - 510. [Abstract] [Full Text] [PDF]
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K. Yasui, K. Kada, M. Hojo, J.-K. Lee, K. Kamiya, J. Toyama, T. Opthof, and I. Kodama Cell-to-cell interaction prevents cell death in cultured neonatal rat ventricular myocytes Cardiovasc Res, October 1, 2000; 48(1): 68 - 76. [Abstract] [Full Text] [PDF]
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E. A. W. J. Dumont, L. Hofstra, W. L. van Heerde, S. van den Eijnde, P. A. F. Doevendans, E. DeMuinck, M. A. R. C. Daemen, J. F. M. Smits, P. Frederik, H. J. J. Wellens, et al. Cardiomyocyte Death Induced by Myocardial Ischemia and Reperfusion : Measurement With Recombinant Human Annexin-V in a Mouse Model Circulation, September 26, 2000; 102(13): 1564 - 1568. [Abstract] [Full Text] [PDF]
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S. Mustapha, A. Kirshner, D. De Moissac, and L. A. Kirshenbaum A direct requirement of nuclear factor-kappa B for suppression of apoptosis in ventricular myocytes Am J Physiol Heart Circ Physiol, September 1, 2000; 279(3): H939 - H945. [Abstract] [Full Text] [PDF]
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P. M. Kang, A. Haunstetter, H. Aoki, A. Usheva, and S. Izumo Morphological and Molecular Characterization of Adult Cardiomyocyte Apoptosis During Hypoxia and Reoxygenation Circ. Res., July 21, 2000; 87(2): 118 - 125. [Abstract] [Full Text] [PDF]
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 Yi Xu, V. Menon, and B. I Jugdutt Cardioprotection after angiotensin II type 1 blockade involves angiotensin II type 2 receptor expression and activation of protein kinase C-{varepsilon} in acutely reperfused myocardial infarction in the dog: Effect of UP269-6 and losartan on AT1- and AT2-receptor expression and IP3 receptor and PKC{varepsilon} proteins Journal of Renin-Angiotensin-Aldosterone System, June 1, 2000; 1(2): 184 - 195. [Abstract] [PDF]
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S. W. Schaffer, C. B. Croft, and V. Solodushko Cardioprotective effect of chronic hyperglycemia: effect on hypoxia-induced apoptosis and necrosis Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H1948 - H1954. [Abstract] [Full Text] [PDF]
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