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Early apoptosis in human myocardial infarcts
Early Apoptosis in Human Myocardial Infarcts JOHN P. VEINOT, MD, DEBORAH A. GATTINGER, BSc, AND HENRY FLISS, PHD Myocardial apoptosis has previously been observed in h u m a n acute myocardial infarcts. We examined the time of appearance and extent of apoptosis in h u m a n acute myocardial infarcts, and compared these with necrotic cell death. Because nuclear internudeosomal DNA fragmentation is a hallmark of apoptosis, autopsied tissue from cases of acute myocardial infarct of varying histological ages was subjected to two tests that identify such fragmentation: in siva end-labeling (ISEL) and DNA electrophoresis on agarose gels. Both tests showed widespread apoptosis in infarcts only a few hours in age before the appearance of coagulative necrosis. No apoptosis was detected in normal myocardium. ISEL ha recent infarcts was visible primarily in myocytes containing contraction bands, which occur predominantly in regions of reperfused myocardium. During the next 1 to 2 days, ISEL remained extensive but increasingly appeared in ceils with morphological features of coagulative necrosis, representative
Myocardial apoptosis, or p r o g r a m m e d cell death, has been shown recently in several injurious settings. It has been observed in isolated rat cardiomyocytes subjected to hypoxia 1 and in isolated rat heart papillary muscles exposed to sustained sn'etching. 2 In vivo studies have shown cardiomyocyte apoptosis during postnatal maturation, 3 spontaneous hypertension in rats, 4 as well as after rapid ventricular pacing, ° or microembolization-induced cardiac failure in dogs. 6 Unlike necrosis, apoptosis proceeds through a genetically p r o g r a m m e d series of biochemical and motphological steps 78 ' designed to a void t he indiscriminate release of cytosolic contents and the ensuing inflammatory response. 9 In the heart, as in other organs, the salient morphological features of apoptosis appear to be chromatin condensation, cytoplasm shrinkage, nuclear fragmentation, sarcolemmal invaginations without loss of m e m b r a n e integrity, and ultimate engulfment by macrophages. 6 However, these defining morphological features can be difficult to observe in the heart. 1° In contrast, a biochemical marker of apoptosis, fragmentation of the internucleosomal DNA in apoptotic cells to multiples of 180 to 200 base-pair fragments, seems to be more reproducible. Recent data show that cardiomyocytes contain significant amounts of endonucleases capable of internucleosomal DNA fragmentation.11'~-~ Two tests are presently commonly used to establish From the Department of Laboratory Medicine, Ottawa Civic Hospital, University of Ottawa Heart Institute; and the Department of Physiology', Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada. Accepted for publication September 7, 1996. Supported by Heart and Stroke Foundation of Ontario, and the Medical Research Council of Canada (HF). Address correspondence and reprint requests to Henry Fliss, PhD, Department of Physiology, Faculty of Medicine, University of Oltawa, 451 Smyth Rd, Ottawa, Ontario K1H 8M5, Canada. Copyright © 1997 by W.B. Saunders Company 0046-8177/97/2804-001255.00/0
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of noureperfused myoeardittm, ha older infarcts, the incidence of apoptosis declined in myocytes, but increased in invading inflammatory cells. These data suggest that apoptosis is the early and predominant form of cell death in infarcted httman myocardium, and that its appearance is accelerated in reperfused myocardium. Therapies directed at early rescue of apoptotic myocytes may, therefore, prove valuable. HUM PATHOL 28:485--492. Copyright © 1997 byW.B. Satmders Company Key words: apoptosis, human myocardium, infarct, necrosis, DNA fragmentation. Abbreviations: ISEL, in siva end labeling; PBS, phosphate-buffered saline; HPS, hematoxylin-phloxin saffron; TRIS-HC1, trls(hydroxymethyl)aminomethane-hydrochloric acid; EDTA, ethylenediaminetetra-acetic acid.
the presence of apoptotic DNA fragmentation, in situend-labeling (ISEL) of nuclei in tissue sections, and the identification of DNA "ladders" in agarose gels. ISEL is strongly suggestive of apoptotic DNA fragmentation 13 and is presently used to identify myocardial apoptosis in settings where the total n u m b e r of apoptotic cells is low. 3614-' ' However, at present the ISEL test alone is not yet generally considered to be a definitive marker. In contrast, the presence of both positive ISEL and DNA ladders is presently generally accepted as a strong indicator of a P o P totic DNA fra ~cmentation in tissues v'8 including the myocardium. 2'5' <1o Prolonged periods of myocardial ischemia can cause tissue injury and cell death. Early reperfusion of ischemic myocardium, although critical for tissue salvage, paradoxically, may also cause increased cell mortality, partly as a result of the inflammatory response and the associated neutrophil accumulation and oxidant production. 16 Although a large portion of the cell loss observed after cardiac ischemia and reperfusion occurs through necrosis, 17 there is presently increasing interest in the poss~lbility that cell death may also occur through apoptosis. Cardiomyocyte apoptosis has been d o c u m e n t e d recently in ischemic rat hearts, m'2° and in reperfused rabbit and rat myocardium. 15'2~It now seems likely that apoptosis can occur early (within 2 to 3 hours) after the onset of myocardial ischemia, 19'9° or reperfuslon," and that it is by far the major cause of cell death in the initial few hours of ischemia. 19 The role of neutrophils in this process is less well defined at the present time. Although one study has suggested that neutrophils do not potentiate apoptosis in rabbit heart, 15 another has shown that the accumulation of neutrophils in reperfused rat myocardium is associated with increased apoptosis. 21 A recent study has shown for the first time the presence of apoptosis in ischemic h u m a n myocard i u m ] ° However, the temporal and inflammatory correlates of apoptosis have not yet been d e t e r m i n e d in •
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HUMAN PATHOLOGY Volume28, No. 4 (April 1997) TABLE 1.
Clinical Features of Autopsied Patients
Case Age (yr)/ Paraffin/ No. Gender Frozen 1
39/F
P
2
47/F
P
3
65/M
P/F
4
66/F
P
5
69/M
P/F
6
75/M
P
7
79/M
P
8
85/F
P
9 10 11
47/M 42/M 63/F
P/F P/F P/F
Clinical AMI Streptokinase AMI No lytics AM1 No lytics AMI No lytics AMI Streptokinase reperfusion AMI No lytics AMI No lytics AMI tPA reperfusion Old MI TTP Pulmonary emboli
ISEL
10% neutral buffered formalin and were then processed for paraffin embedding for routine microscopic examination. In 6 cases, 4 acute infarcts and 2 negative controls, the immediately adjacent myocardium was snap frozen in OCT embedding medium (Miles Scientific, Naperville, IL) using a mixture of isopentane and dry ice, and was subsequently stored at -80°C. Five-micron sections of the frozen or paraffin block tissue were cut and were stained with hematoxylin-phloxinsaffron (HPS) or hematoxylin-eosin for routine light microscopic examination as well as with the ISEL method for apoptosis determinations. Samples of the frozen myocardium were also used for DNA extraction.
DNA Ladder
Positive
ND
Positive
ND
Positive
Positive
Positive
ND
Positive Positive
Explanted Myocardium Tissue Samples Positive
ND
Positive
ND
Positive
ND
Twelve explanted hearts from cardiac transplant recipients who had recent myocardial infarcts were examined. Nine were from men, with a mean age of 54 years (range, 42 to 64), and three were from women, with a mean age of 50 years (range, 43 to 54). Eight had undergone coronary bypass (four remote and four recent), three had mechanical support, and one had a remote percutaneous transluminal coronary angioplasty. All were given enteric-coated acetylsalicylic acid. Thrombolytic history was not available. The hearts were immersed in 10% neutral buffered formalin at the time of excision, thus minimizing autolytic changes. A healing infarct varying in age from a few days to a few weeks was noted in all tile explanted hearts. Representative areas were selected to show the infarct edge and center and normal myocardium, and were fixed in 10% neutral buffered formalin in preparation for paraffin-embedding and routine microscopic examination. Five-micron sections were cut from the paraffin blocks and stained with HPS or the ISEL method.
Negative Negative Negative Negative N e g a t i v e Negative
Abbreviations: AMI, acute myocardial infarct; tPA, tissue plasminogen activator; P, paraffin-embedded tissue available; F, frozen tissue available; ND, not done, frozen tissue not available; TTP, thrombotic thrombocytopenia purpura; ISEL, in sire end labeling.
this tissue. T h e p r i n c i p a l objectives o f the p r e s e n t study were, t h e r e f o r e , to investigate the a p p e a r a n c e o f apoptosis i n early h u m a n infarcts u s i n g b o t h the ISEL a n d D N A gel t e c h n i q u e s , a n d to d e t e r m i n e the c h a n g e s i n apoptosis that o c c u r i n r e l a t i o n s h i p to the type of cellular i n f i l t r a t i o n p r e s e n t . This study shows extensive apoptosis i n early infarcts, b e f o r e n e u t r o p h i l i c infiltration, a n d i n d e p e n d e n t of w h e t h e r r e p e r f u s i o n has occ u r r e d . Apoptosis d e c r e a s e d over the first few days of i n f a r c t e v o l u t i o n a n d , after 7 to 10 days, was o b s e r v e d p r i m a r i l y i n the i n f i l t r a t i n g m o n o n u c l e a r i n f l a m m a t o r y cells.
Effects of Postmortem Delay on Apoptosis in Ischemic Rat Myocardium
MATERIALS AND METHODS
Autopsy Tissue Samples A total of 11 postmortem cases were examined within 24 hours after the patients' death (Table 1). Six were men, with a mean age of 62.8 years (range = 42 to 79), and 5 were women, with a mean age of 59.8 years (range = 39 to 84). Of the 11 cases, 8 were found to have acute myocardial infarcts, and 1 had a remote myocardial infarct (death was caused by an arrhythmia). The remaining two consisted of one patient with pulmonary hypertension (death occurred because of a massive acute pulmonary thromboembolus), and 1 with thrombotic thrombocytopenia purpura. Neither of the two showed evidence of myocardial infarction, and they were, therefore, used as negative controls. At autopsy, the heart was cut in transverse slices at 3 to 5-ram intervals (short axis echocardiographic plane) from the apex to the midventricular level at the midpapillary muscle level, allowing maximal visualization of any ischemic injury present. Samples of fullthickness myocardium (epicardium to endocardium) were taken from grossly normal myocardium as well as from the edge and center of the visible infarct. They were fixed in
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We have shown previonsly that extensive cardiomyocyte apoptosis occurs in ischemic rat myocardium as early as 2.45 hours after occlusion of the coronary artery. 2° We therefore, used this model to examine the effects of a simulated postmortem delay on the intensity and regional distribution of apoptosis. Coronary artery occlusion was performed in rats using a modification of a previously published protocol, 22and 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/minute) with 1.5% halothane/ 100% oxygen, using a rodent respirator (model no. 683; Harvard Instruments, South Natick, MA). 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 then made through the fourth intercostal space, the ribs were gently spread to expose the heart, and a 6-0 silk ligature was tied around left main coronary artery. 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 closed using surgical clips, and the animals were ventilated with room air and regained consciousness within 5 to 10 minutes. After 4.5 hours, the rats were anesthetized with sodium pentobarbital (65 mg/kg, intraperitoneally), 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.2~ Each heart was rapidly excised and was bisected transversely midway
APOPTOSIS IN HUMAN MYOCARDIAL INFARCTS(Veinot et al) through the unstained myocardium. One half was immediately frozen over dry ice and was sectioned using a cryostat (10 #m), The other half was exposed to a simulated postmortem delay. It was placed in a small plastic bag that was susp e n d e d in a beaker containing 500 mL of water at 37°C. The temperature of the water bath was lowered to 25°C over a period of 6 hours, and the tissue was then stored at 4°C for a further 15 hours. The heart tissue was then frozen over dry ice and sectioned with a cryostat as described previously.
ISEL The protocol was based on previously published procedures. -9¢26Unless otherwise specified, all reagents were products of Sigma Chemical (St. Louis, M e ) , or BDH (Toronto, Canada). Paraffin sections were deparaffinized, were permeabilized with m e t h a n o l / a c e t o n e (1:1) for 10 min at room temperature, and were washed twice with phosphate-buffered saline (PBS). Frozen cryostat sections of rat or human myocardium were thawed and fixed in 1% glutaraldehyde for 15 minutes at room temperature and were then washed twice (5 minutes each) with PBS before permeabilization with methanol/acetone. Sections were then incubated with 20 > g / m L proteinase K in 25 m m o l / L tris (hydroxymethyl) aminometha n e - h y d r o c h l o r i c acid (TRIS-HC1) (1 mL/section), p H 6.6, for 15 minutes at room temperature, were washed twice (15 minutes each) with water, were stained with Hoechst 33258 (0.05 > g / m L ) for 30 minutes at room temperature, protected from light, and were washed 3 times (1 minute each) with PBS. The sections were then incubated in 75 #L of a buffer solution containing 200 m m o l / L potassium cacodylate, 2 m m o l / L cobalt chloride, 0.25 m g / m L bovine serum albumin, 25 m m o l / L TRIS-HC1, p H 6.6, 10 > m o l / L biotin-16-deoxyuridine triphosphate (Boehringer Mannheim Canada, Laval, Quebec), and 25 units of terminal transferase (Boehringer), for 1 hour at 37°C in a humidified chamber. The reaction was terminated by washing the sections 3 times (1 minute each) with PBS at room temperature. The sections were then incubated with 1 mL of a staining solution containing 2.5 # g / m L fluorescein isothiocyanate-avidin 4× saline-sodium citrate buffer, 0.1% Triton X-100, and 5% powdered milk, for 30 minutes at room temperature, protected from light. The sections were washed 3 times with PBS, were coverslipped in "antifade" solution containing 1 m g / m L pphenylenediamine, 90% glycerol, in PBS, and histofluorescence was monitored with a Zeiss Axiophot microscope (Carl Zeiss, Canada, Don Mills, Ontario). Positive control samples were prepared by incubating sections with 10 u n i t s / m L DNAse I for 20 minutes at 37°C before treatment with terminal transferase.
50°C. Ethanol (50% final concentration) and sodium chloride (0.5 m o l / L final concentration) were added, and the DNA was precipitated for 1 hour, or overnight, at -20°C. The DNA was collected by centrifugation at 13,000 × g for 15 minutes at 4°C, was dissolved in 500 #L of TE buffer (10 m m o l / L TRIS-HC1, 1 m m o l / L EDTA, pH 8.0), and was extracted once with p h e n o l / c h l o r o f o r m saturated with TE buffer. The DNA solution was washed once with chloroform and was precipitated in 50% ethanol, 0.5 m o l / L sodium chloride, at -20°C for 1 hour. The DNA was collected by centrifngation and was dried. It was then dissolved in 50 >L of TAE buffer (40 m m o l / L TRIS-HC1, 30 m m o l / L acetic acid, 2 m m o l / L EDTA, p H 8.0), and was subjected to electrophoresis on agarose gels (1.5% in TAE buffer).
Agarose Gel Electrophoresis of DNA The protocol was based on previously published procedures. 27'28 Frozen human myocardium (200 mg) was minced in an equal volume of Dulbecco's-PBS at 0°C and was homogenized for 30 seconds in a homogenizer (Kinematica, Brinkmann Instruments, Westbury, NY) at 10,000 revolutions per minute. A 100-#L aliquot of the homogenate was mixed with 1.25 mL of lysis buffer containing 10 m m o l / L TRIS-HC1, pH 8.0, 10 m m o l / L ethylenediaminetetra-acetic acid (EDTA), 75 m m o l / L sodium chloride, and 0.5% sodium dodecyl sulfate, and the suspension was incubated for 15 minutes at room temperature. The suspension was then centrifuged at 13,000 × g for 15 minutes at room temperature, the supernatant containing the fragmented DNA was collected, and was treated first with RNAse (100 # g / m L ) for 30 minutes at 37°C, and then with proteinase K (100 # g / m L ) for 30 minutes at
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RESULTS Light Microscopic a n d iSEL Findings All o f t h e 21 cases o f clinical m y o c a r d i a l i n f a r c t i o n (9 a u t o p s y a n d 12 e x p l a n t cases) h a d i s c h e m i a - r e l a t e d m o r p h o l o g i c a l c h a n g e s d e t e c t a b l e by r o u t i n e l i g h t mic r o s c o p i c e x a m i n a t i o n . S o m e p a t i e n t s h a d o n g o i n g rec e n t i s c h e m i a s u p e r i m p o s e d o n l a r g e p r i o r infarcts o f a few weeks age. T h e s e were p a t i e n t s w h o i n f a r c t e d a n d w e r e s u b s e q u e n t l y t r a n s p l a n t e d w h e n a d o n o r h e a r t bec a m e available. Two a u t o p s y a n d two e x p l a n t cases showed varying amounts of edema and extravasated red b l o o d cells w i t h i n t h e infarct. ISEL o f f r o z e n o r p a r a f f i n s e c t i o n s f r o m t h e s a m e r e g i o n o f t h e m y o c a r d i u m gave i d e n t i c a l results, i n d i c a t i n g t h a t fixation d i d n o t affect DNA content or fragmentation (data not shown). The h i s t o l o g i c a l age o f t h e infarcts was d e t e r m i n e d u s i n g t h e c r i t e r i a o f L o d g e - P a t c h , 29 r e c o g n i z i n g t h a t in s o m e cases e i t h e r n a t u r a l o r d r u g - r e l a t e d ( t h r o m b o l y t i c ) rep e r f u s i o n m a y have o c c u r r e d . R e p e r f u s i o n is well k n o w n to p o t e n t i a l l y a c c e l e r a t e t h e h i s t o l o g i c a l age o f i n f a r c t h e a l i n g . 3° E v i d e n c e o f m y o c a r d i a l r e p e r f u s i o n was a s s u m e d to b e p r e s e n t w h e n t h e r e was i n t r a m y o c a r dial h e m o r r h a g e with a s s o c i a t e d p r o m i n e n t c o n t r a c t i o n b a n d necrosis. T h e infarcts r a n g e d in age f r o m a few h o u r s to a few weeks a n d were d i v i d e d i n t o t h e f o l l o w i n g g r o u p s b a s e d o n t e m p o r a l c h a n g e s in m o r p h o l o g y : Twelve to 24 hours. Clinically, t h e e a r l i e s t i n f a r c t o c c u r r e d in a p a t i e n t with massive a n t e r o s e p t a l left ventricular infarction resulting from coronary artery embolization. D e a t h o c c u r r e d within 12 h o u r s o f initial c h e s t p a i n . This p a t i e n t ' s m y o c a r d i u m s h o w e d c o n t r a c t i o n b a n d s with n o e v i d e n c e o f c o a g u l a t i v e n e c r o s i s (Fig 1A). ISEL was positive in myocytes in t h e areas o f c o n t r a c t i o n b a n d s a n d n e g a t i v e in t h e n o r m a l m y o c a r d i u m (Fig 1B). I n this p a t i e n t a n d o t h e r s with early infarcts, n o s i g n i f i c a n t n e u t r o p h i l i c e x t r a v a s a t i o n was o b s e r v e d in t h e r e g i o n s o f c o n t r a c t i o n b a n d s . ISEL in this g r o u p o f p a t i e n t s s h o w e d i n t e n s e f l u o r e s c e n c e in t h e i n f a r c t e d m y o c a r d i u m , p r i m a r i l y in t h e n u c l e i o f myocytes in t h e r e g i o n o f c o n t r a c t i o n b a n d s . N o ISEL was d e t e c t e d in n o r m a l m y o c a r d i u m . I n cases in this t i m e g r o u p with slightly o l d e r infarcts, t h e i n f a r c t e d m y o c a r d i u m showed, in a d d i t i o n to c o n t r a c t i o n b a n d s , s i g n i f i c a n t i s c h e m i c m y o c y t e cell d e a t h with typical c o a g u l a t i v e necrosis, c y t o p l a s m i c e o s i n o p h i l i a , a n d wavy fibers, s i m i l a r to t h a t o b s e r v e d in t h e 1- to 5-day g r o u p (see later).
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The myocyte nuclei in the region of coagulative necrosis were either pyknotic or were absent by HPS stain. In these cases, ISEL was present in myocyte nuclei in the regions of coagulative necrosis, as illustrated in the 1- to 5-day group (see later) as well as in myocyte nuclei in the regions of contraction bands. Interestingly, in some cases of coagulative necrosis, nuclei that were no longer visible with the HPS stain nevertheless showed intense ISEL, confirming earlier observations. ~° One to 5 days. In this group, the infarcted myocardium contained areas of neutrophilic infiltration of variable a m o u n t occurring on a background of preexisting coagulative necrosis (Fig 2A). In no case were the neutrophils ISEL positive, confirming earlier observations. ~° Myocyte nuclei in the infarcted myocardium were either pyknotic or were absent. In the early cases of infarcts in this period, ISEL in the infarcted myocardium was strongly positive in the myocyte nuclei (Fig 2B), with nuclear staining often visible even when nuclei were not detectable by HPS stain. With infarcts in the latter part of this period, the myocytes in the infarcted myocardium were predominantly necrotic with accumulation of basophilic extracellular debris. As myo-
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FIGURE 2. One- to 3-day acute infarct: (A) HPS stain showing neutrophil infiltration (arrow) in a region of coagulative necrosis, Arrowhead points to lipofuscin granules at presumed location of a nucleus not stained with HPS. (B) ISELof same region showing strong fluorescence in nuclei of myocytes undergoing coagulative necrosis, (Original magnification ×400.)
FIGURE I. Early (< 12 hours) acute infarct. (A) HPS stain showing normal myocytes (n) adjacent to the blood vessel, and contraction bands (*) in myocytes fuffher removed from the vessel. (B) ISELof same region showing no fluorescence in normal myocardium next to blood vessel, and intense nuclear staining in myocytes in region of contraction bands. (Original magnification x200.) 488
cytes degenerated, the ISEL became blurred and assumed a diffuse extracellular smeared pattern, similar to that shown in the 5-day to 2-week group subsequently. No significant ISEL was observed in normal myocardium in this group. Five days to 2 weeks. In this period, macrophages were observed to infiltrate the infarcts from the edges inward. Often the center of the infarct retained eosinophilic necrotic myocardium, and the periphery reabsorbed with the presence of macrophages, hemosiderin, lymphocytes, fibroblasts, and capillaries (Fig 3A). Some residual areas of contraction bands were noted with myocytolysis and macrophage infiltration, without intervening coagulative necrosis. ISEL showed a diffuse nonspecific extracellular pattern in the necrotic cores of infarcted myocardium (Fig 3B), and positive staining of the m o n o n u c l e a r inflammatory cells at the periphery. In the areas of the contraction bands, no nuclear staining was noted other than in isolated m o n o n u c l e a r inflammatory cells (not shown). No significant ISEL was observed in the normal myocardium. Remote
These old infarcts showed well-established myocardial replacement by fibrous collagen and m o n o n u c l e a r
APOPTOSISIN HUMAN MYOCARDIALINFARCTS(Veinot et al) myocyte nuclei in the ischemic myocardinm (data not shown)• No increase in the number or fluorescence intensity of the nuclei was observed in ~he ischemic myocardium of the complementary heart fragments that were subjected to a simulated postmortem delay before freezing. In neither case were apoptotic nuclei found in the normal myocardium (not shown).
J
DISCUSSION This study confirms a recent report of myocyte apoptosis in h u m a n acute myocardial infarcts] ° However, we show for the first time that apoptosis is the first and predominant form of myocyte cell death in recently infarcted h u m a n myocardium. We observed evidence of widespread myocyte apoptosis, in the form of intense nuclear ISEL and ladder formation on agarose DNA gels, within hours of the onset of ischemia. In contrast, little morphological evidence of necrotic cell death was detected in these early infarcts. The intensity of these characteristic markers of apoptosis remained elevated in the myocytes during the next 1 to 2 days. However,
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FIGURE 3. Five-day to 2-week acute infarct. (A) HPS stain showing eosinophilic necrotic myocardium with absent nuclei and with infiltrating mononuclear interstitial cells (arrowhead), and a healing edge (*). (B) ISEL of same region showing a diffuse extracellular staining pattern in the necrotic region.
(Original magnification ×400,)
inflammatory cells, chiefly lymphocytes (Fig 4A). The adjacent myocardium had myocyte and nuclear hypertrophy and myocytolysis with dropout of myofibrils, without acute ischemic changes. Myocytes did not stain positively with ISEL, but nuclear staining of lymphocytes, endothelial cells, and smooth muscle cells was seen (Fig 4B). No ISEL was observed in the normal myocardium. Agarose Gel Electrophoresis of DNA DNA "ladders," indicative of apoptotic internucleosomal DNA fragmentation, were clearly visible in agarose gels of DNA from the middle and edge regions of recently infarcted myocardium, but not the corresponding normal myocardium (Fig 5). Ladders decreased in intensity with increasing infarct age, and were not observed with late or remote infarcts, or with the two negative controls (not shown). Effects of Postmortem Delay on Apoptosis in Ischemic Rat Myocardium ISEL of freshly frozen rat myocardium showed numerous, evenly distributed, intensely fluorescent cardio-
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FIGURE 4. Remote acute infarct. (A) HPS stain showing wellestablished myocardial replacement by fibrous collagen and mononuclear inflammatory cells, chiefly lymphocytes, (Original magnification × 100.) (B) ISELstain of similar region showing lack of myocytes staining, but strong staining of infiltrating lymphocytes, endothelial cells, and smooth muscle cells. (Original magnification ×200.)
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4
FIGURE 5. Agarose gel eiectrophoresis of myocardial DNA from an early (12 to 24 hours) acute infarct. Soluble DNA was extracted from frozen myocardium, and aliquots (7 #g) were subjected to electrophoresis on 1.5% agarose gels as described in Materials and Methods. Lane 1, normal myocardium. Lane 2, midinfarct showing typical ladders. Lane 3, edge of infarct with ladders. Lane 4, DNA molecular weight standards, containing Hindll fragments of X phage DNA (1 #g, Sigma), with arrowhead indicating molecular weight of 564.
in the subsequent several days, the n u m b e r of ISELpositive myocytes, as well as ISEL intensity, gradually decreased in the infarcts. DNA laddering also decreased in intensity over the same span of time. These data are, therefore, in agreement with recent rat studies that showed that apoptosis is the p r e d o m i n a n t form of myocyte cell death within the first several hours of myocardial ischemia. 19We did not detect apoptosis in morphologically normal myocardium t h r o u g h o u t this period. The postmortem period is unlikely to have affected
these data significantly. These data show that rat myocardium subjected to ischemia and reperfusion displays similar intensity and distribution of ISEL before and after a significant simulated postmortem delay. Previous studies have shown a similar lack of effect on ISEL by postmortem delay in other tissues. ~1 A significant finding of this study was that apoptotic cell death was detected in myocardium that was subj e c t e d to either prolonged ischemia or ischemia followed by reperfusion. Intense ISEL was observed in the nuclei of myocytes in areas of contraction bands as well as in the nuclei of myocytes in regions of early coagulative necrosis. This observation has also been made elsewhere recently. 1° It is presently generally accepted that contraction bands located within infarcts are p r o d u c e d primarily in myocytes that are reperfused after a brief, but probably irreversibly injurious, ischemic episode, whereas coagulative necrosis occurs in myocytes that are sub'ected~ to prolonged ischemia without reperfusion. 32-3 It, therefore, appears likely that the cellular apoptotic mechanism is induced in irreversibly injured myocytes, and that subsequent reperfusion does not reverse this process. In fact, our data suggest that reperfusion may" actually accelerate apoptosis. During the first few hours of myocardial infarction, we observed ISEL predominantly in myocytes in the regions of contraction bands. In contrast, the appearance of ISEL in regions of coagulative necrosis was significantly delayed and occurred concomitantly with the morphological appearance of myocytes undergoing coagulative necrosis. These data appear to support our recent rat studies that have shown that apoptosis is induced significantly earlier in reperfused ischemic myocardium than in continuously ischemic myocardium. 2° An intriguing possible interpretation of these data is that ischemia-induced myocyte cell death may begin as apoptosis and ultimately take on the morphological and biochemical characteristics of necrosis. Although such an interpretation is in apparent conflict with the assumption that apoptosis is designed to avoid the injurious inflammatory sequelae of necrosis, 9 recent evidence appears to support such a conclusion. In the rat, ischemic myocytes that show clear evidence of apoptosis within the first few hours of ischemia can ultimately assume characteristics of necrotic cell death. 19 Moreover, the only previous study of apoptosis in infarcted h u m a n myocardium also showed evidence of apoptosis in regions of coagulative necrosis) ° One possible reason for a progression from apoptosis to necrosis may be the exhaustion of the energy supply required to complete the process of apoptosis. Thus, ischemic myocytes that have initiated an apoptotic mechanism of cell death may be forced to abandon this energy-consuming process prematurely as a consequence of continuous ischemia. The present data appear to support such a hypothesis. The observed acceleration of apoptosis in the reperfused myocardium suggests that an increase in energy supply may potentiate this form of cell death. In addition, the nuclear dissolution we observed in the ISEl~positive myocytes appeared to follow a pattern normally observed in necrotic cell death, rather than apoptotic nuclear fragmentation, showing disruption of
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APOPTOSIS IN HUMAN MYOCARDIAL INFARCTS (Veinot et al)
the nuclear membrane, followed by the redistribution of the ISEL-positive nuclear contents to the entire cell and, ultimately, to the extracellular space. Clinically, a gradual progression from apoptosis to necrosis in ischemic myocardium suggests that there may be a significant window of opportunity for myocyte salvage. Recent studies have shown that a variety of treatments may rescue apoptotic cells from ultimate cell death, s5'36 In addition, we have shown recently that antioxidants or inhibitors of protein synthesis can attenuate apoptosis in ischemic rat myocardium. 37 These data indicate that early treatment of ischemic myocardium with apoptosis-inhibiting agents may increase tissue salvage. Conversely, treatments that have b e e n shown previously to salvage myocardium in the area at risk may do so by decreasing the incidence of apoptosis. For example, because this study shows that early apoptosis appeared predominantly in the regions of contraction bands, treatments that have successfully decreased the area of contraction bands 3s'39may also inhibit apoptosis. The role of neutrophils in myocardial apoptosis remains uncertain and will require further study. Although we occasionally observed significant neutrophil extravasation in our study, this infiltration was restricted to regions of coagulative necrosis and was not detected in areas of contraction bands. Because no neutrophil infiltration was observed in the regions of intense early apoptosis, it appears unlikely that neutrophils contributed significantly to this form of cell death. A recent rabbit study has suggested that neutrophils do not potentiate apoptosis in ischemic m y o c a r d i u m ] 5 However, a potentially conflicting study has shown that increased neutrophil content in reperfused rat myocardium is associated with increased apoptosis. 2~ Our recent studies, which have shown apoptosis in isolated buffer-perfused rat hearts, suggest that the induction of apoptosis can occur in the absence of blood-borne agents. 37 It is, therefore, possible that the well-documented margination of neutrophils in the microvasculature during reperfusion may temporally accompany the accelerated reperfusion-associated apoptosis but may not directly contribute to it. Recent data that suggest that the production of neutrophil-specific adhesion molecules only occurs in viable ischemic myocardium 4° indicate that immediate neutrophil extravasation is unlikely to occur in the regions containing irreversibly injured myocytes, such as the regions of contraction bands, and is, therefore, unlikely to contribute to early apoptosis. In conclusion, this study shows tbr the first time that, in early h u m a n myocardial infarcts, significant apoptosis can be observed in myocytes subjected to either p r o l o n g e d ischemia or ischemia followed by reperfusion, and apoptosis appears to constitute the predominant early form of cell death in this tissue. In older infarcts, the apoptotic mechanism appears to give way to necrosis. Although the precise biochemical mechanisms of ischemic or reperfusion injury remain to be elucidated, it is clear that cell death remains the most important clinical consequence of both types of injury. The success of any therapeutic intervention will, therefore, d e p e n d heavily on a clear understanding of the mechanisms of this cell loss. Knowledge that early cell
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death in ischemic myocardium may consist primarily of apoptosis may, therefore, prove instrumental in designing therapeutic treatment to attenuate and, potentially r e v e r s e , t i s s u e loss.
Acknowledgment. We gratefully acknowledge the technical contributions of Ute Davis and W. A. Stinson. REFERENCES 1. Tanaka M, Ito H, Adachi S, et al: Hypoxia induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat cardiomyocytes. Circ Res 75:426-433, 1994 2. Cheng W, Li BS, Kajstura J, et ah Stretch-induced programmed myocyte cell death. J Clin Invest 96:2247-2259, 1995 3. KajsturaJ, Mansukhani M, Cheng W, et al: Programmed cell death and expression of the protooncogene bcl-2 in myocytes during postnatal maturation of the heart. Exp Cell Res 219:110-121, 1995 4. Hamet P, Richard L, Dam TV, et al: Apoptosis in target organs of hypertension. Hypertension 26:642-648, 1995 5. Liu Y, Cigola E, Cheng W, et al: Myocyte nuclear mitotic dixdsion and programmed myocyte cell death characterize tile cardiac myopathy induced by rapid ventricular pacing in dogs. Lab Invest 73:771-787, 1995 6. Sharov VG, Sabbah HN, Shimoyama H, et al: Evidence of cardiocyte apoptosis in myocardium of dogs with chronic heart failure. A m J Pathol 148:141-149, 1996 7. Majno G, Joris I: Apoptosis, oncosis, and necrosis: Au overview of cell death. A m J Pathol 146:3-15, 1995 8. Hockenbery D: Defining apoptosis. Am J Pathol 146:16-19, 1995 9. Martin SJ, Green DR, Cotter TG: Dicing with death: Dissecting the components of the apoptosis machinery. Trends Biochem Sci 19:26-30, 1994 10. Itoh G, Tamura J, Suzuki M, et al: DNA fragmentation of human infarcted myocardial cells demonstrated by the nick end labeling method and DNA agarose gel electrophoresis. Am J Pathol 146:1325-1331, 1995 11. Gottlieb RA, Giesing HA, Engler RL, et al: The acid deoxyribonuclease of neutrophils: A possible participant in apoptosis-associated genome destruction. Blood 86:2414-2418, 1995 12. Yao M, Keogh A, Spratt P, et al: Elevated DNase I levels in human idiopathic dilated cardiomyopathy: An indicator of apoptosis. J Mol Cell Cardiol 28:95-101, 1996 13. Gold R, Schmied M, Giegerich G, et al: Differentiation between cellular apoptosis and necrosis by the combined use of in situ tailing and nick translation techniques. Lab Invest 71:219-225, 1994 14. Takeda K, Yu ZX, Nishikawa T, et al: Apoptosis and DNA fragmentation in the bulbus cordis of the developing rat heart. J Mol Cell Cardiol 28:209-215, 1996 15. Gottlieb RA, Burleson KO, Kloner RA, et ah Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest 94:1621-1628, 1994 16. Entman ML, Smith CW: Postreperfusion inflammation: A model for reaction to injury in cardiovascular disease. Cardiovasc Res 28:1301-1311, 1994 17. Buja LM, Eigenbrodt ML, Eigenbrodt EH: Apoptosis and necrosis: Basic types and mechanisms of cell death. Arch Pathol Lab Med 117:1208-1214, 1993 18. James TN: Normal and abnormal consequences of apoptosis in the human heart: From postnatal morphogenesis to paroxysmal arrhythmias. Circulation 90:556-573, 1994 19. Kajstura J, Cheng W, Reiss K, et ah Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats. Lab Invest 74:86-107, 1996 20. Fliss H, Gattinger D: Apoptosis in ischemic and repeffused rat myocardium. Circ Res 79:949-956, 1996 21. Buerke M, Murohara T, Skurk C, et ah Cardioprotective effect of insulin-like growth factor I in myocardial ischemia followed by reperfusion. Proc Natl Acad Sci U S A 92:8031-8035, 1995 22. Smith EF, Egan JW, Bugelski PJ, et al: Temporal relation between neutrophil accumulation and myocardial reperfusion injury. A m J Physiol 255:H1060-H1068, 1988
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23. Campo GM, Squadrito F, Ioculano M, et al: Protective effects of IRFI-016, a new antioxidant agent, in myocardial damage, following coronary artery occlusion and reperfusion in the rat. Pharmacology 48:157-166, 1994 24. Gavrieli Y, Sherman ¥, Ben-Sasson SA: Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493-501, 1992 25. Gorczyca W, Gong J, Darzynkiewicz Z: Detection of DNA strand breaks in individual apoptotic cells by the in sim terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res 53:1945-1951, 1993 26. Sei Y, Von Lubitz DKJE, Basile AS, et ah Internucleosomal DNA fragmentation in gerbil hippocampus following forebrain ischemia. Neurosci Lett 171:179-182, 1994 27. Ramakrishnan N, Catravas GN: N-(2-mercaptoethyl)-l,3-propanediamine (WR-1065) protects thymocytes from programmed cell death. J Immunol 148:1817-1821, 1992 28. Prigent P, Blanpied C, AtenJ, et al: A safe and rapid method for analyzing apoptosis-induced fragmentation of DNA extracted from tissues or cultured cells. J Immunol Methods 160:139-140, 1993 29. Lodge-Patch I: The ageing of cardiac infarcts and its influence on cardiac rupture. Br H e a r t J 13:37-42, 1951 30. Cowan MJ, Reichenbach D, Turner P, et al: Cellular response of the evolving myocardial infarction after therapeutic coronary artery reperfusion. HuH PATHOL 22:154-163, 1991 31. Petito CK, Roberts B: Effect of postmortem interval on in situ end-labeling of DNA oligonucleosomes. J Neuropathol Exp Neurol 54:761-765, 1995 32. Reimer KA, Jennings RB: Myocardial ischemia, hypoxia, and
infarction, in Fozzard HA, Jennings RB, Haber E, et al (eds): The Heart and Cardiovascular System (ed 2). NewYork, NY, Raven, 1992, pp 1875-1972 33. Miyazaki S, Fujiwara H, Onodera T, et ah Quantitative analysis of contraction band and coagulation necrosis after ischemia and reperfusion in the porcine heart. Circulation 75:1074-1082, 1987 34. Matsuda M, Fujiwara H, Onodera T, et al: Quantitative analysis of infarct size, contraction band necrosis, and coagulation necrosis in human autopsied hearts with acute myocardial infarction after treatment with selective intracoronary thrombolysis. Circulation 76:981-989, 1987 35. Oppenheim RW, Houenou LJ,JohnsonJE, et al: Developing motor neurons rescued from programmed and axotomy-induced cell death by GDNF. Nature 373:344-346, 1995 36. Thompson CB: Apoptosis in the pathogenesis and treatment of disease. Science 267:1456-1462, 1995 37. Dean DC, Fliss H: Apoptosis in an isolated reperfused rat heart model: Protection with dithiothreitol. J Mol Cell Cardiol 1996 (abstr) 38. Garcia-Dorado D, Thdroux P, DuranJM, et al: Selective inhibition of the contractile apparatus: A new approach to modification of infarct size, infarct composition, and infarct geometry during coronary artery occlusion and reperfusion. Circulation 85:1160-1174, 1992 39. Wakida Y, Nordlander R, Kobayashi S, et al: Short-term synchronized retroperfusion before reperfusion reduces infarct size after prolonged ischemia in dogs. Circulation 88:2370-2380, 1993 40. Youker KA, Hawkins HK, Kukielka GL, et al: Molecular evidence for induction of intracellular adhesion molecule-1 in the viable border zone associated with ischemia-reperfusion injury of the dog heart. Circulation 89:2736-2746, 1994
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Myocardial apoptosis, or p r o g r a m m e d cell death, has been shown recently in several injurious settings. It has been observed in isolated rat cardiomyocytes subjected to hypoxia 1 and in isolated rat heart papillary muscles exposed to sustained sn'etching. 2 In vivo studies have shown cardiomyocyte apoptosis during postnatal maturation, 3 spontaneous hypertension in rats, 4 as well as after rapid ventricular pacing, ° or microembolization-induced cardiac failure in dogs. 6 Unlike necrosis, apoptosis proceeds through a genetically p r o g r a m m e d series of biochemical and motphological steps 78 ' designed to a void t he indiscriminate release of cytosolic contents and the ensuing inflammatory response. 9 In the heart, as in other organs, the salient morphological features of apoptosis appear to be chromatin condensation, cytoplasm shrinkage, nuclear fragmentation, sarcolemmal invaginations without loss of m e m b r a n e integrity, and ultimate engulfment by macrophages. 6 However, these defining morphological features can be difficult to observe in the heart. 1° In contrast, a biochemical marker of apoptosis, fragmentation of the internucleosomal DNA in apoptotic cells to multiples of 180 to 200 base-pair fragments, seems to be more reproducible. Recent data show that cardiomyocytes contain significant amounts of endonucleases capable of internucleosomal DNA fragmentation.11'~-~ Two tests are presently commonly used to establish From the Department of Laboratory Medicine, Ottawa Civic Hospital, University of Ottawa Heart Institute; and the Department of Physiology', Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada. Accepted for publication September 7, 1996. Supported by Heart and Stroke Foundation of Ontario, and the Medical Research Council of Canada (HF). Address correspondence and reprint requests to Henry Fliss, PhD, Department of Physiology, Faculty of Medicine, University of Oltawa, 451 Smyth Rd, Ottawa, Ontario K1H 8M5, Canada. Copyright © 1997 by W.B. Saunders Company 0046-8177/97/2804-001255.00/0
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of noureperfused myoeardittm, ha older infarcts, the incidence of apoptosis declined in myocytes, but increased in invading inflammatory cells. These data suggest that apoptosis is the early and predominant form of cell death in infarcted httman myocardium, and that its appearance is accelerated in reperfused myocardium. Therapies directed at early rescue of apoptotic myocytes may, therefore, prove valuable. HUM PATHOL 28:485--492. Copyright © 1997 byW.B. Satmders Company Key words: apoptosis, human myocardium, infarct, necrosis, DNA fragmentation. Abbreviations: ISEL, in siva end labeling; PBS, phosphate-buffered saline; HPS, hematoxylin-phloxin saffron; TRIS-HC1, trls(hydroxymethyl)aminomethane-hydrochloric acid; EDTA, ethylenediaminetetra-acetic acid.
the presence of apoptotic DNA fragmentation, in situend-labeling (ISEL) of nuclei in tissue sections, and the identification of DNA "ladders" in agarose gels. ISEL is strongly suggestive of apoptotic DNA fragmentation 13 and is presently used to identify myocardial apoptosis in settings where the total n u m b e r of apoptotic cells is low. 3614-' ' However, at present the ISEL test alone is not yet generally considered to be a definitive marker. In contrast, the presence of both positive ISEL and DNA ladders is presently generally accepted as a strong indicator of a P o P totic DNA fra ~cmentation in tissues v'8 including the myocardium. 2'5' <1o Prolonged periods of myocardial ischemia can cause tissue injury and cell death. Early reperfusion of ischemic myocardium, although critical for tissue salvage, paradoxically, may also cause increased cell mortality, partly as a result of the inflammatory response and the associated neutrophil accumulation and oxidant production. 16 Although a large portion of the cell loss observed after cardiac ischemia and reperfusion occurs through necrosis, 17 there is presently increasing interest in the poss~lbility that cell death may also occur through apoptosis. Cardiomyocyte apoptosis has been d o c u m e n t e d recently in ischemic rat hearts, m'2° and in reperfused rabbit and rat myocardium. 15'2~It now seems likely that apoptosis can occur early (within 2 to 3 hours) after the onset of myocardial ischemia, 19'9° or reperfuslon," and that it is by far the major cause of cell death in the initial few hours of ischemia. 19 The role of neutrophils in this process is less well defined at the present time. Although one study has suggested that neutrophils do not potentiate apoptosis in rabbit heart, 15 another has shown that the accumulation of neutrophils in reperfused rat myocardium is associated with increased apoptosis. 21 A recent study has shown for the first time the presence of apoptosis in ischemic h u m a n myocard i u m ] ° However, the temporal and inflammatory correlates of apoptosis have not yet been d e t e r m i n e d in •
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HUMAN PATHOLOGY Volume28, No. 4 (April 1997) TABLE 1.
Clinical Features of Autopsied Patients
Case Age (yr)/ Paraffin/ No. Gender Frozen 1
39/F
P
2
47/F
P
3
65/M
P/F
4
66/F
P
5
69/M
P/F
6
75/M
P
7
79/M
P
8
85/F
P
9 10 11
47/M 42/M 63/F
P/F P/F P/F
Clinical AMI Streptokinase AMI No lytics AM1 No lytics AMI No lytics AMI Streptokinase reperfusion AMI No lytics AMI No lytics AMI tPA reperfusion Old MI TTP Pulmonary emboli
ISEL
10% neutral buffered formalin and were then processed for paraffin embedding for routine microscopic examination. In 6 cases, 4 acute infarcts and 2 negative controls, the immediately adjacent myocardium was snap frozen in OCT embedding medium (Miles Scientific, Naperville, IL) using a mixture of isopentane and dry ice, and was subsequently stored at -80°C. Five-micron sections of the frozen or paraffin block tissue were cut and were stained with hematoxylin-phloxinsaffron (HPS) or hematoxylin-eosin for routine light microscopic examination as well as with the ISEL method for apoptosis determinations. Samples of the frozen myocardium were also used for DNA extraction.
DNA Ladder
Positive
ND
Positive
ND
Positive
Positive
Positive
ND
Positive Positive
Explanted Myocardium Tissue Samples Positive
ND
Positive
ND
Positive
ND
Twelve explanted hearts from cardiac transplant recipients who had recent myocardial infarcts were examined. Nine were from men, with a mean age of 54 years (range, 42 to 64), and three were from women, with a mean age of 50 years (range, 43 to 54). Eight had undergone coronary bypass (four remote and four recent), three had mechanical support, and one had a remote percutaneous transluminal coronary angioplasty. All were given enteric-coated acetylsalicylic acid. Thrombolytic history was not available. The hearts were immersed in 10% neutral buffered formalin at the time of excision, thus minimizing autolytic changes. A healing infarct varying in age from a few days to a few weeks was noted in all tile explanted hearts. Representative areas were selected to show the infarct edge and center and normal myocardium, and were fixed in 10% neutral buffered formalin in preparation for paraffin-embedding and routine microscopic examination. Five-micron sections were cut from the paraffin blocks and stained with HPS or the ISEL method.
Negative Negative Negative Negative N e g a t i v e Negative
Abbreviations: AMI, acute myocardial infarct; tPA, tissue plasminogen activator; P, paraffin-embedded tissue available; F, frozen tissue available; ND, not done, frozen tissue not available; TTP, thrombotic thrombocytopenia purpura; ISEL, in sire end labeling.
this tissue. T h e p r i n c i p a l objectives o f the p r e s e n t study were, t h e r e f o r e , to investigate the a p p e a r a n c e o f apoptosis i n early h u m a n infarcts u s i n g b o t h the ISEL a n d D N A gel t e c h n i q u e s , a n d to d e t e r m i n e the c h a n g e s i n apoptosis that o c c u r i n r e l a t i o n s h i p to the type of cellular i n f i l t r a t i o n p r e s e n t . This study shows extensive apoptosis i n early infarcts, b e f o r e n e u t r o p h i l i c infiltration, a n d i n d e p e n d e n t of w h e t h e r r e p e r f u s i o n has occ u r r e d . Apoptosis d e c r e a s e d over the first few days of i n f a r c t e v o l u t i o n a n d , after 7 to 10 days, was o b s e r v e d p r i m a r i l y i n the i n f i l t r a t i n g m o n o n u c l e a r i n f l a m m a t o r y cells.
Effects of Postmortem Delay on Apoptosis in Ischemic Rat Myocardium
MATERIALS AND METHODS
Autopsy Tissue Samples A total of 11 postmortem cases were examined within 24 hours after the patients' death (Table 1). Six were men, with a mean age of 62.8 years (range = 42 to 79), and 5 were women, with a mean age of 59.8 years (range = 39 to 84). Of the 11 cases, 8 were found to have acute myocardial infarcts, and 1 had a remote myocardial infarct (death was caused by an arrhythmia). The remaining two consisted of one patient with pulmonary hypertension (death occurred because of a massive acute pulmonary thromboembolus), and 1 with thrombotic thrombocytopenia purpura. Neither of the two showed evidence of myocardial infarction, and they were, therefore, used as negative controls. At autopsy, the heart was cut in transverse slices at 3 to 5-ram intervals (short axis echocardiographic plane) from the apex to the midventricular level at the midpapillary muscle level, allowing maximal visualization of any ischemic injury present. Samples of fullthickness myocardium (epicardium to endocardium) were taken from grossly normal myocardium as well as from the edge and center of the visible infarct. They were fixed in
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We have shown previonsly that extensive cardiomyocyte apoptosis occurs in ischemic rat myocardium as early as 2.45 hours after occlusion of the coronary artery. 2° We therefore, used this model to examine the effects of a simulated postmortem delay on the intensity and regional distribution of apoptosis. Coronary artery occlusion was performed in rats using a modification of a previously published protocol, 22and 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/minute) with 1.5% halothane/ 100% oxygen, using a rodent respirator (model no. 683; Harvard Instruments, South Natick, MA). 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 then made through the fourth intercostal space, the ribs were gently spread to expose the heart, and a 6-0 silk ligature was tied around left main coronary artery. 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 closed using surgical clips, and the animals were ventilated with room air and regained consciousness within 5 to 10 minutes. After 4.5 hours, the rats were anesthetized with sodium pentobarbital (65 mg/kg, intraperitoneally), 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.2~ Each heart was rapidly excised and was bisected transversely midway
APOPTOSIS IN HUMAN MYOCARDIAL INFARCTS(Veinot et al) through the unstained myocardium. One half was immediately frozen over dry ice and was sectioned using a cryostat (10 #m), The other half was exposed to a simulated postmortem delay. It was placed in a small plastic bag that was susp e n d e d in a beaker containing 500 mL of water at 37°C. The temperature of the water bath was lowered to 25°C over a period of 6 hours, and the tissue was then stored at 4°C for a further 15 hours. The heart tissue was then frozen over dry ice and sectioned with a cryostat as described previously.
ISEL The protocol was based on previously published procedures. -9¢26Unless otherwise specified, all reagents were products of Sigma Chemical (St. Louis, M e ) , or BDH (Toronto, Canada). Paraffin sections were deparaffinized, were permeabilized with m e t h a n o l / a c e t o n e (1:1) for 10 min at room temperature, and were washed twice with phosphate-buffered saline (PBS). Frozen cryostat sections of rat or human myocardium were thawed and fixed in 1% glutaraldehyde for 15 minutes at room temperature and were then washed twice (5 minutes each) with PBS before permeabilization with methanol/acetone. Sections were then incubated with 20 > g / m L proteinase K in 25 m m o l / L tris (hydroxymethyl) aminometha n e - h y d r o c h l o r i c acid (TRIS-HC1) (1 mL/section), p H 6.6, for 15 minutes at room temperature, were washed twice (15 minutes each) with water, were stained with Hoechst 33258 (0.05 > g / m L ) for 30 minutes at room temperature, protected from light, and were washed 3 times (1 minute each) with PBS. The sections were then incubated in 75 #L of a buffer solution containing 200 m m o l / L potassium cacodylate, 2 m m o l / L cobalt chloride, 0.25 m g / m L bovine serum albumin, 25 m m o l / L TRIS-HC1, p H 6.6, 10 > m o l / L biotin-16-deoxyuridine triphosphate (Boehringer Mannheim Canada, Laval, Quebec), and 25 units of terminal transferase (Boehringer), for 1 hour at 37°C in a humidified chamber. The reaction was terminated by washing the sections 3 times (1 minute each) with PBS at room temperature. The sections were then incubated with 1 mL of a staining solution containing 2.5 # g / m L fluorescein isothiocyanate-avidin 4× saline-sodium citrate buffer, 0.1% Triton X-100, and 5% powdered milk, for 30 minutes at room temperature, protected from light. The sections were washed 3 times with PBS, were coverslipped in "antifade" solution containing 1 m g / m L pphenylenediamine, 90% glycerol, in PBS, and histofluorescence was monitored with a Zeiss Axiophot microscope (Carl Zeiss, Canada, Don Mills, Ontario). Positive control samples were prepared by incubating sections with 10 u n i t s / m L DNAse I for 20 minutes at 37°C before treatment with terminal transferase.
50°C. Ethanol (50% final concentration) and sodium chloride (0.5 m o l / L final concentration) were added, and the DNA was precipitated for 1 hour, or overnight, at -20°C. The DNA was collected by centrifugation at 13,000 × g for 15 minutes at 4°C, was dissolved in 500 #L of TE buffer (10 m m o l / L TRIS-HC1, 1 m m o l / L EDTA, pH 8.0), and was extracted once with p h e n o l / c h l o r o f o r m saturated with TE buffer. The DNA solution was washed once with chloroform and was precipitated in 50% ethanol, 0.5 m o l / L sodium chloride, at -20°C for 1 hour. The DNA was collected by centrifngation and was dried. It was then dissolved in 50 >L of TAE buffer (40 m m o l / L TRIS-HC1, 30 m m o l / L acetic acid, 2 m m o l / L EDTA, p H 8.0), and was subjected to electrophoresis on agarose gels (1.5% in TAE buffer).
Agarose Gel Electrophoresis of DNA The protocol was based on previously published procedures. 27'28 Frozen human myocardium (200 mg) was minced in an equal volume of Dulbecco's-PBS at 0°C and was homogenized for 30 seconds in a homogenizer (Kinematica, Brinkmann Instruments, Westbury, NY) at 10,000 revolutions per minute. A 100-#L aliquot of the homogenate was mixed with 1.25 mL of lysis buffer containing 10 m m o l / L TRIS-HC1, pH 8.0, 10 m m o l / L ethylenediaminetetra-acetic acid (EDTA), 75 m m o l / L sodium chloride, and 0.5% sodium dodecyl sulfate, and the suspension was incubated for 15 minutes at room temperature. The suspension was then centrifuged at 13,000 × g for 15 minutes at room temperature, the supernatant containing the fragmented DNA was collected, and was treated first with RNAse (100 # g / m L ) for 30 minutes at 37°C, and then with proteinase K (100 # g / m L ) for 30 minutes at
487
RESULTS Light Microscopic a n d iSEL Findings All o f t h e 21 cases o f clinical m y o c a r d i a l i n f a r c t i o n (9 a u t o p s y a n d 12 e x p l a n t cases) h a d i s c h e m i a - r e l a t e d m o r p h o l o g i c a l c h a n g e s d e t e c t a b l e by r o u t i n e l i g h t mic r o s c o p i c e x a m i n a t i o n . S o m e p a t i e n t s h a d o n g o i n g rec e n t i s c h e m i a s u p e r i m p o s e d o n l a r g e p r i o r infarcts o f a few weeks age. T h e s e were p a t i e n t s w h o i n f a r c t e d a n d w e r e s u b s e q u e n t l y t r a n s p l a n t e d w h e n a d o n o r h e a r t bec a m e available. Two a u t o p s y a n d two e x p l a n t cases showed varying amounts of edema and extravasated red b l o o d cells w i t h i n t h e infarct. ISEL o f f r o z e n o r p a r a f f i n s e c t i o n s f r o m t h e s a m e r e g i o n o f t h e m y o c a r d i u m gave i d e n t i c a l results, i n d i c a t i n g t h a t fixation d i d n o t affect DNA content or fragmentation (data not shown). The h i s t o l o g i c a l age o f t h e infarcts was d e t e r m i n e d u s i n g t h e c r i t e r i a o f L o d g e - P a t c h , 29 r e c o g n i z i n g t h a t in s o m e cases e i t h e r n a t u r a l o r d r u g - r e l a t e d ( t h r o m b o l y t i c ) rep e r f u s i o n m a y have o c c u r r e d . R e p e r f u s i o n is well k n o w n to p o t e n t i a l l y a c c e l e r a t e t h e h i s t o l o g i c a l age o f i n f a r c t h e a l i n g . 3° E v i d e n c e o f m y o c a r d i a l r e p e r f u s i o n was a s s u m e d to b e p r e s e n t w h e n t h e r e was i n t r a m y o c a r dial h e m o r r h a g e with a s s o c i a t e d p r o m i n e n t c o n t r a c t i o n b a n d necrosis. T h e infarcts r a n g e d in age f r o m a few h o u r s to a few weeks a n d were d i v i d e d i n t o t h e f o l l o w i n g g r o u p s b a s e d o n t e m p o r a l c h a n g e s in m o r p h o l o g y : Twelve to 24 hours. Clinically, t h e e a r l i e s t i n f a r c t o c c u r r e d in a p a t i e n t with massive a n t e r o s e p t a l left ventricular infarction resulting from coronary artery embolization. D e a t h o c c u r r e d within 12 h o u r s o f initial c h e s t p a i n . This p a t i e n t ' s m y o c a r d i u m s h o w e d c o n t r a c t i o n b a n d s with n o e v i d e n c e o f c o a g u l a t i v e n e c r o s i s (Fig 1A). ISEL was positive in myocytes in t h e areas o f c o n t r a c t i o n b a n d s a n d n e g a t i v e in t h e n o r m a l m y o c a r d i u m (Fig 1B). I n this p a t i e n t a n d o t h e r s with early infarcts, n o s i g n i f i c a n t n e u t r o p h i l i c e x t r a v a s a t i o n was o b s e r v e d in t h e r e g i o n s o f c o n t r a c t i o n b a n d s . ISEL in this g r o u p o f p a t i e n t s s h o w e d i n t e n s e f l u o r e s c e n c e in t h e i n f a r c t e d m y o c a r d i u m , p r i m a r i l y in t h e n u c l e i o f myocytes in t h e r e g i o n o f c o n t r a c t i o n b a n d s . N o ISEL was d e t e c t e d in n o r m a l m y o c a r d i u m . I n cases in this t i m e g r o u p with slightly o l d e r infarcts, t h e i n f a r c t e d m y o c a r d i u m showed, in a d d i t i o n to c o n t r a c t i o n b a n d s , s i g n i f i c a n t i s c h e m i c m y o c y t e cell d e a t h with typical c o a g u l a t i v e necrosis, c y t o p l a s m i c e o s i n o p h i l i a , a n d wavy fibers, s i m i l a r to t h a t o b s e r v e d in t h e 1- to 5-day g r o u p (see later).
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The myocyte nuclei in the region of coagulative necrosis were either pyknotic or were absent by HPS stain. In these cases, ISEL was present in myocyte nuclei in the regions of coagulative necrosis, as illustrated in the 1- to 5-day group (see later) as well as in myocyte nuclei in the regions of contraction bands. Interestingly, in some cases of coagulative necrosis, nuclei that were no longer visible with the HPS stain nevertheless showed intense ISEL, confirming earlier observations. ~° One to 5 days. In this group, the infarcted myocardium contained areas of neutrophilic infiltration of variable a m o u n t occurring on a background of preexisting coagulative necrosis (Fig 2A). In no case were the neutrophils ISEL positive, confirming earlier observations. ~° Myocyte nuclei in the infarcted myocardium were either pyknotic or were absent. In the early cases of infarcts in this period, ISEL in the infarcted myocardium was strongly positive in the myocyte nuclei (Fig 2B), with nuclear staining often visible even when nuclei were not detectable by HPS stain. With infarcts in the latter part of this period, the myocytes in the infarcted myocardium were predominantly necrotic with accumulation of basophilic extracellular debris. As myo-
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FIGURE 2. One- to 3-day acute infarct: (A) HPS stain showing neutrophil infiltration (arrow) in a region of coagulative necrosis, Arrowhead points to lipofuscin granules at presumed location of a nucleus not stained with HPS. (B) ISELof same region showing strong fluorescence in nuclei of myocytes undergoing coagulative necrosis, (Original magnification ×400.)
FIGURE I. Early (< 12 hours) acute infarct. (A) HPS stain showing normal myocytes (n) adjacent to the blood vessel, and contraction bands (*) in myocytes fuffher removed from the vessel. (B) ISELof same region showing no fluorescence in normal myocardium next to blood vessel, and intense nuclear staining in myocytes in region of contraction bands. (Original magnification x200.) 488
cytes degenerated, the ISEL became blurred and assumed a diffuse extracellular smeared pattern, similar to that shown in the 5-day to 2-week group subsequently. No significant ISEL was observed in normal myocardium in this group. Five days to 2 weeks. In this period, macrophages were observed to infiltrate the infarcts from the edges inward. Often the center of the infarct retained eosinophilic necrotic myocardium, and the periphery reabsorbed with the presence of macrophages, hemosiderin, lymphocytes, fibroblasts, and capillaries (Fig 3A). Some residual areas of contraction bands were noted with myocytolysis and macrophage infiltration, without intervening coagulative necrosis. ISEL showed a diffuse nonspecific extracellular pattern in the necrotic cores of infarcted myocardium (Fig 3B), and positive staining of the m o n o n u c l e a r inflammatory cells at the periphery. In the areas of the contraction bands, no nuclear staining was noted other than in isolated m o n o n u c l e a r inflammatory cells (not shown). No significant ISEL was observed in the normal myocardium. Remote
These old infarcts showed well-established myocardial replacement by fibrous collagen and m o n o n u c l e a r
APOPTOSISIN HUMAN MYOCARDIALINFARCTS(Veinot et al) myocyte nuclei in the ischemic myocardinm (data not shown)• No increase in the number or fluorescence intensity of the nuclei was observed in ~he ischemic myocardium of the complementary heart fragments that were subjected to a simulated postmortem delay before freezing. In neither case were apoptotic nuclei found in the normal myocardium (not shown).
J
DISCUSSION This study confirms a recent report of myocyte apoptosis in h u m a n acute myocardial infarcts] ° However, we show for the first time that apoptosis is the first and predominant form of myocyte cell death in recently infarcted h u m a n myocardium. We observed evidence of widespread myocyte apoptosis, in the form of intense nuclear ISEL and ladder formation on agarose DNA gels, within hours of the onset of ischemia. In contrast, little morphological evidence of necrotic cell death was detected in these early infarcts. The intensity of these characteristic markers of apoptosis remained elevated in the myocytes during the next 1 to 2 days. However,
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FIGURE 3. Five-day to 2-week acute infarct. (A) HPS stain showing eosinophilic necrotic myocardium with absent nuclei and with infiltrating mononuclear interstitial cells (arrowhead), and a healing edge (*). (B) ISEL of same region showing a diffuse extracellular staining pattern in the necrotic region.
(Original magnification ×400,)
inflammatory cells, chiefly lymphocytes (Fig 4A). The adjacent myocardium had myocyte and nuclear hypertrophy and myocytolysis with dropout of myofibrils, without acute ischemic changes. Myocytes did not stain positively with ISEL, but nuclear staining of lymphocytes, endothelial cells, and smooth muscle cells was seen (Fig 4B). No ISEL was observed in the normal myocardium. Agarose Gel Electrophoresis of DNA DNA "ladders," indicative of apoptotic internucleosomal DNA fragmentation, were clearly visible in agarose gels of DNA from the middle and edge regions of recently infarcted myocardium, but not the corresponding normal myocardium (Fig 5). Ladders decreased in intensity with increasing infarct age, and were not observed with late or remote infarcts, or with the two negative controls (not shown). Effects of Postmortem Delay on Apoptosis in Ischemic Rat Myocardium ISEL of freshly frozen rat myocardium showed numerous, evenly distributed, intensely fluorescent cardio-
489
FIGURE 4. Remote acute infarct. (A) HPS stain showing wellestablished myocardial replacement by fibrous collagen and mononuclear inflammatory cells, chiefly lymphocytes, (Original magnification × 100.) (B) ISELstain of similar region showing lack of myocytes staining, but strong staining of infiltrating lymphocytes, endothelial cells, and smooth muscle cells. (Original magnification ×200.)
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4
FIGURE 5. Agarose gel eiectrophoresis of myocardial DNA from an early (12 to 24 hours) acute infarct. Soluble DNA was extracted from frozen myocardium, and aliquots (7 #g) were subjected to electrophoresis on 1.5% agarose gels as described in Materials and Methods. Lane 1, normal myocardium. Lane 2, midinfarct showing typical ladders. Lane 3, edge of infarct with ladders. Lane 4, DNA molecular weight standards, containing Hindll fragments of X phage DNA (1 #g, Sigma), with arrowhead indicating molecular weight of 564.
in the subsequent several days, the n u m b e r of ISELpositive myocytes, as well as ISEL intensity, gradually decreased in the infarcts. DNA laddering also decreased in intensity over the same span of time. These data are, therefore, in agreement with recent rat studies that showed that apoptosis is the p r e d o m i n a n t form of myocyte cell death within the first several hours of myocardial ischemia. 19We did not detect apoptosis in morphologically normal myocardium t h r o u g h o u t this period. The postmortem period is unlikely to have affected
these data significantly. These data show that rat myocardium subjected to ischemia and reperfusion displays similar intensity and distribution of ISEL before and after a significant simulated postmortem delay. Previous studies have shown a similar lack of effect on ISEL by postmortem delay in other tissues. ~1 A significant finding of this study was that apoptotic cell death was detected in myocardium that was subj e c t e d to either prolonged ischemia or ischemia followed by reperfusion. Intense ISEL was observed in the nuclei of myocytes in areas of contraction bands as well as in the nuclei of myocytes in regions of early coagulative necrosis. This observation has also been made elsewhere recently. 1° It is presently generally accepted that contraction bands located within infarcts are p r o d u c e d primarily in myocytes that are reperfused after a brief, but probably irreversibly injurious, ischemic episode, whereas coagulative necrosis occurs in myocytes that are sub'ected~ to prolonged ischemia without reperfusion. 32-3 It, therefore, appears likely that the cellular apoptotic mechanism is induced in irreversibly injured myocytes, and that subsequent reperfusion does not reverse this process. In fact, our data suggest that reperfusion may" actually accelerate apoptosis. During the first few hours of myocardial infarction, we observed ISEL predominantly in myocytes in the regions of contraction bands. In contrast, the appearance of ISEL in regions of coagulative necrosis was significantly delayed and occurred concomitantly with the morphological appearance of myocytes undergoing coagulative necrosis. These data appear to support our recent rat studies that have shown that apoptosis is induced significantly earlier in reperfused ischemic myocardium than in continuously ischemic myocardium. 2° An intriguing possible interpretation of these data is that ischemia-induced myocyte cell death may begin as apoptosis and ultimately take on the morphological and biochemical characteristics of necrosis. Although such an interpretation is in apparent conflict with the assumption that apoptosis is designed to avoid the injurious inflammatory sequelae of necrosis, 9 recent evidence appears to support such a conclusion. In the rat, ischemic myocytes that show clear evidence of apoptosis within the first few hours of ischemia can ultimately assume characteristics of necrotic cell death. 19 Moreover, the only previous study of apoptosis in infarcted h u m a n myocardium also showed evidence of apoptosis in regions of coagulative necrosis) ° One possible reason for a progression from apoptosis to necrosis may be the exhaustion of the energy supply required to complete the process of apoptosis. Thus, ischemic myocytes that have initiated an apoptotic mechanism of cell death may be forced to abandon this energy-consuming process prematurely as a consequence of continuous ischemia. The present data appear to support such a hypothesis. The observed acceleration of apoptosis in the reperfused myocardium suggests that an increase in energy supply may potentiate this form of cell death. In addition, the nuclear dissolution we observed in the ISEl~positive myocytes appeared to follow a pattern normally observed in necrotic cell death, rather than apoptotic nuclear fragmentation, showing disruption of
490
APOPTOSIS IN HUMAN MYOCARDIAL INFARCTS (Veinot et al)
the nuclear membrane, followed by the redistribution of the ISEL-positive nuclear contents to the entire cell and, ultimately, to the extracellular space. Clinically, a gradual progression from apoptosis to necrosis in ischemic myocardium suggests that there may be a significant window of opportunity for myocyte salvage. Recent studies have shown that a variety of treatments may rescue apoptotic cells from ultimate cell death, s5'36 In addition, we have shown recently that antioxidants or inhibitors of protein synthesis can attenuate apoptosis in ischemic rat myocardium. 37 These data indicate that early treatment of ischemic myocardium with apoptosis-inhibiting agents may increase tissue salvage. Conversely, treatments that have b e e n shown previously to salvage myocardium in the area at risk may do so by decreasing the incidence of apoptosis. For example, because this study shows that early apoptosis appeared predominantly in the regions of contraction bands, treatments that have successfully decreased the area of contraction bands 3s'39may also inhibit apoptosis. The role of neutrophils in myocardial apoptosis remains uncertain and will require further study. Although we occasionally observed significant neutrophil extravasation in our study, this infiltration was restricted to regions of coagulative necrosis and was not detected in areas of contraction bands. Because no neutrophil infiltration was observed in the regions of intense early apoptosis, it appears unlikely that neutrophils contributed significantly to this form of cell death. A recent rabbit study has suggested that neutrophils do not potentiate apoptosis in ischemic m y o c a r d i u m ] 5 However, a potentially conflicting study has shown that increased neutrophil content in reperfused rat myocardium is associated with increased apoptosis. 2~ Our recent studies, which have shown apoptosis in isolated buffer-perfused rat hearts, suggest that the induction of apoptosis can occur in the absence of blood-borne agents. 37 It is, therefore, possible that the well-documented margination of neutrophils in the microvasculature during reperfusion may temporally accompany the accelerated reperfusion-associated apoptosis but may not directly contribute to it. Recent data that suggest that the production of neutrophil-specific adhesion molecules only occurs in viable ischemic myocardium 4° indicate that immediate neutrophil extravasation is unlikely to occur in the regions containing irreversibly injured myocytes, such as the regions of contraction bands, and is, therefore, unlikely to contribute to early apoptosis. In conclusion, this study shows tbr the first time that, in early h u m a n myocardial infarcts, significant apoptosis can be observed in myocytes subjected to either p r o l o n g e d ischemia or ischemia followed by reperfusion, and apoptosis appears to constitute the predominant early form of cell death in this tissue. In older infarcts, the apoptotic mechanism appears to give way to necrosis. Although the precise biochemical mechanisms of ischemic or reperfusion injury remain to be elucidated, it is clear that cell death remains the most important clinical consequence of both types of injury. The success of any therapeutic intervention will, therefore, d e p e n d heavily on a clear understanding of the mechanisms of this cell loss. Knowledge that early cell
491
death in ischemic myocardium may consist primarily of apoptosis may, therefore, prove instrumental in designing therapeutic treatment to attenuate and, potentially r e v e r s e , t i s s u e loss.
Acknowledgment. We gratefully acknowledge the technical contributions of Ute Davis and W. A. Stinson. REFERENCES 1. Tanaka M, Ito H, Adachi S, et al: Hypoxia induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat cardiomyocytes. Circ Res 75:426-433, 1994 2. Cheng W, Li BS, Kajstura J, et ah Stretch-induced programmed myocyte cell death. J Clin Invest 96:2247-2259, 1995 3. KajsturaJ, Mansukhani M, Cheng W, et al: Programmed cell death and expression of the protooncogene bcl-2 in myocytes during postnatal maturation of the heart. Exp Cell Res 219:110-121, 1995 4. Hamet P, Richard L, Dam TV, et al: Apoptosis in target organs of hypertension. Hypertension 26:642-648, 1995 5. Liu Y, Cigola E, Cheng W, et al: Myocyte nuclear mitotic dixdsion and programmed myocyte cell death characterize tile cardiac myopathy induced by rapid ventricular pacing in dogs. Lab Invest 73:771-787, 1995 6. Sharov VG, Sabbah HN, Shimoyama H, et al: Evidence of cardiocyte apoptosis in myocardium of dogs with chronic heart failure. A m J Pathol 148:141-149, 1996 7. Majno G, Joris I: Apoptosis, oncosis, and necrosis: Au overview of cell death. A m J Pathol 146:3-15, 1995 8. Hockenbery D: Defining apoptosis. Am J Pathol 146:16-19, 1995 9. Martin SJ, Green DR, Cotter TG: Dicing with death: Dissecting the components of the apoptosis machinery. Trends Biochem Sci 19:26-30, 1994 10. Itoh G, Tamura J, Suzuki M, et al: DNA fragmentation of human infarcted myocardial cells demonstrated by the nick end labeling method and DNA agarose gel electrophoresis. Am J Pathol 146:1325-1331, 1995 11. Gottlieb RA, Giesing HA, Engler RL, et al: The acid deoxyribonuclease of neutrophils: A possible participant in apoptosis-associated genome destruction. Blood 86:2414-2418, 1995 12. Yao M, Keogh A, Spratt P, et al: Elevated DNase I levels in human idiopathic dilated cardiomyopathy: An indicator of apoptosis. J Mol Cell Cardiol 28:95-101, 1996 13. Gold R, Schmied M, Giegerich G, et al: Differentiation between cellular apoptosis and necrosis by the combined use of in situ tailing and nick translation techniques. Lab Invest 71:219-225, 1994 14. Takeda K, Yu ZX, Nishikawa T, et al: Apoptosis and DNA fragmentation in the bulbus cordis of the developing rat heart. J Mol Cell Cardiol 28:209-215, 1996 15. Gottlieb RA, Burleson KO, Kloner RA, et ah Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest 94:1621-1628, 1994 16. Entman ML, Smith CW: Postreperfusion inflammation: A model for reaction to injury in cardiovascular disease. Cardiovasc Res 28:1301-1311, 1994 17. Buja LM, Eigenbrodt ML, Eigenbrodt EH: Apoptosis and necrosis: Basic types and mechanisms of cell death. Arch Pathol Lab Med 117:1208-1214, 1993 18. James TN: Normal and abnormal consequences of apoptosis in the human heart: From postnatal morphogenesis to paroxysmal arrhythmias. Circulation 90:556-573, 1994 19. Kajstura J, Cheng W, Reiss K, et ah Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats. Lab Invest 74:86-107, 1996 20. Fliss H, Gattinger D: Apoptosis in ischemic and repeffused rat myocardium. Circ Res 79:949-956, 1996 21. Buerke M, Murohara T, Skurk C, et ah Cardioprotective effect of insulin-like growth factor I in myocardial ischemia followed by reperfusion. Proc Natl Acad Sci U S A 92:8031-8035, 1995 22. Smith EF, Egan JW, Bugelski PJ, et al: Temporal relation between neutrophil accumulation and myocardial reperfusion injury. A m J Physiol 255:H1060-H1068, 1988
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23. Campo GM, Squadrito F, Ioculano M, et al: Protective effects of IRFI-016, a new antioxidant agent, in myocardial damage, following coronary artery occlusion and reperfusion in the rat. Pharmacology 48:157-166, 1994 24. Gavrieli Y, Sherman ¥, Ben-Sasson SA: Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493-501, 1992 25. Gorczyca W, Gong J, Darzynkiewicz Z: Detection of DNA strand breaks in individual apoptotic cells by the in sim terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res 53:1945-1951, 1993 26. Sei Y, Von Lubitz DKJE, Basile AS, et ah Internucleosomal DNA fragmentation in gerbil hippocampus following forebrain ischemia. Neurosci Lett 171:179-182, 1994 27. Ramakrishnan N, Catravas GN: N-(2-mercaptoethyl)-l,3-propanediamine (WR-1065) protects thymocytes from programmed cell death. J Immunol 148:1817-1821, 1992 28. Prigent P, Blanpied C, AtenJ, et al: A safe and rapid method for analyzing apoptosis-induced fragmentation of DNA extracted from tissues or cultured cells. J Immunol Methods 160:139-140, 1993 29. Lodge-Patch I: The ageing of cardiac infarcts and its influence on cardiac rupture. Br H e a r t J 13:37-42, 1951 30. Cowan MJ, Reichenbach D, Turner P, et al: Cellular response of the evolving myocardial infarction after therapeutic coronary artery reperfusion. HuH PATHOL 22:154-163, 1991 31. Petito CK, Roberts B: Effect of postmortem interval on in situ end-labeling of DNA oligonucleosomes. J Neuropathol Exp Neurol 54:761-765, 1995 32. Reimer KA, Jennings RB: Myocardial ischemia, hypoxia, and
infarction, in Fozzard HA, Jennings RB, Haber E, et al (eds): The Heart and Cardiovascular System (ed 2). NewYork, NY, Raven, 1992, pp 1875-1972 33. Miyazaki S, Fujiwara H, Onodera T, et ah Quantitative analysis of contraction band and coagulation necrosis after ischemia and reperfusion in the porcine heart. Circulation 75:1074-1082, 1987 34. Matsuda M, Fujiwara H, Onodera T, et al: Quantitative analysis of infarct size, contraction band necrosis, and coagulation necrosis in human autopsied hearts with acute myocardial infarction after treatment with selective intracoronary thrombolysis. Circulation 76:981-989, 1987 35. Oppenheim RW, Houenou LJ,JohnsonJE, et al: Developing motor neurons rescued from programmed and axotomy-induced cell death by GDNF. Nature 373:344-346, 1995 36. Thompson CB: Apoptosis in the pathogenesis and treatment of disease. Science 267:1456-1462, 1995 37. Dean DC, Fliss H: Apoptosis in an isolated reperfused rat heart model: Protection with dithiothreitol. J Mol Cell Cardiol 1996 (abstr) 38. Garcia-Dorado D, Thdroux P, DuranJM, et al: Selective inhibition of the contractile apparatus: A new approach to modification of infarct size, infarct composition, and infarct geometry during coronary artery occlusion and reperfusion. Circulation 85:1160-1174, 1992 39. Wakida Y, Nordlander R, Kobayashi S, et al: Short-term synchronized retroperfusion before reperfusion reduces infarct size after prolonged ischemia in dogs. Circulation 88:2370-2380, 1993 40. Youker KA, Hawkins HK, Kukielka GL, et al: Molecular evidence for induction of intracellular adhesion molecule-1 in the viable border zone associated with ischemia-reperfusion injury of the dog heart. Circulation 89:2736-2746, 1994
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