(Circulation. 1997;96:1598-1604.)
© 1997 American Heart Association, Inc.
Articles |
Ischemic Preconditioning Decreases Apoptosis in Rat Hearts In Vivo
From the Cardiovascular Research Institute, Department of Medicine (Cardiology Division) (C.A.P., D.P., R.E.S., C.L.W.) and Department of Pathology (P.C.U.), University of California, San Francisco.
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Abstract |
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Background Previous studies have demonstrated that ischemic preconditioning prevents lethal cell injury and, as a consequence, limits infarct size in rat heart. Although both apoptosis and necrosis have been shown to contribute to myocardial cell death after myocardial ischemia and reperfusion, the ability of ischemic preconditioning to prevent programmed cell death remains unknown.
Methods and Results To test the hypothesis that ischemic preconditioning reduces irreversible ischemic injury in part by decreasing apoptosis, rats that underwent ischemic preconditioning and controls were subjected to 30 minutes of left coronary artery occlusion followed by 180 minutes of reperfusion. Ischemic preconditioning was achieved by five 5-minute cycles of ischemia, each followed by 5 minutes of reperfusion. Infarct size, determined by dual staining with triphenyltetrazolium chloride and phthalocyanine blue dye, was significantly reduced in preconditioned compared with nonpreconditioned rats (11.4±1.4% versus 58.7±1.4%; n=20 in each group; P<.001; infarct size/risk area). Genomic DNA from preconditioned hearts showed little or no oligonucleosome-sized fragments (200-bp multiples), whereas genomic DNA from nonpreconditioned hearts showed a typical nucleosome fragmentation. The TUNEL assay localized fewer and sparsely stained nuclei within the infarct zone of ischemic preconditioned hearts compared with nonpreconditioned hearts. Consistent with these findings, the number of cytosolic histone–associated low-molecular-weight DNA fragments was significantly decreased in preconditioned hearts compared with controls (0.17±0.02 versus 1.07±0.09 U; n=10 in each group; P<.001; absorbance 405 nm/490 nm).
Conclusions This study suggests that ischemic preconditioning reduces irreversible ischemic injury in part by decreasing apoptosis after prolonged ischemia and reperfusion.
Key Words: apoptosis • ischemia • myocardial infarction • reperfusion
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Introduction |
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The cardioprotective effect of ischemic preconditioning was first described in 1986 by Murry et al.1 Since then, numerous studies have shown that brief periods of acute myocardial ischemia protect the heart against detrimental consequences of ischemia-reperfusion injuries in different animal species,2 3 4 5 6 7 8 9 10 including humans.11 12 13 14 In addition to reducing ventricular arrhythmias2 3 and postischemic contractile dysfunction (myocardial stunning),4 5 ischemic preconditioning causes a significant reduction in infarct size (ie, myocyte cell death).1 5 6 7 8 9 10 14 The mechanism by which preconditioning prevents irreversible cell injury, however, remains unclear.
Recently, cardiomyocyte apoptosis (a mechanism of programmed cell death) has been linked to heart failure15 16 17 18 as well as to myocardial ischemia-reperfusion injury in vitro19 and in vivo.20 Apoptotic cell death is characterized morphologically by chromatin condensation and biochemically by degradation of DNA into a specific pattern of fragments.21 It has been suggested that apoptosis and necrosis, two distinct mechanisms of cell death, may contribute independently to infarct size in rat hearts.22 In contrast to necrosis, which is considered to be a catastrophic metabolic failure resulting in loss of cell membrane integrity, apoptosis is the result of an active cellular response ("cell suicide") involving a specific cascade of molecular events and possibly can be prevented.23 24 Thus, several specific gene families, such as bcl-2, that modulate apoptosis25 have been shown to be inversely related to programmed cell death in myocytes.26
Because apoptosis appears to be a much more cell-regulated biological phenomenon than is necrosis, it is possible that ischemic preconditioning prevents myocyte cell death in part by preventing programmed cell death. Recently, Gottlieb et al27 found that myocyte apoptosis was reduced by preconditioning in vitro. To test the hypothesis that ischemic preconditioning reduces irreversible ischemic injury in part by decreasing apoptosis in vivo, both specific DNA fragmentation and infarct size were assessed in preconditioned and in nonpreconditioned rat hearts.
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Methods |
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Animal Model of Acute Myocardial Ischemia and Reperfusion
A rat model of reversible myocardial ischemia and reperfusion was used as previously described.9 10 28 29 Briefly, female Sprague-Dawley rats weighing 225 to 250 g were anesthetized with intraperitoneal pentobarbital (40 mg/kg) and ventilated via a tracheostomy on a Harvard rodent respirator (tidal volume, 0.5 to 1.5 mL; respiratory rate, 95 to 105 breaths per minute). A midline sternotomy was performed, and a reversible coronary artery snare occluder was placed around the proximal LCA. After a 1- to 2-second test occlusion, too short to induce preconditioning, the presence of myocardial ischemia was confirmed by regional cyanosis, and reperfusion was verified by hyperemia after the snare was released. The rats were then allowed to stabilize for 20 minutes and subjected to one of the experimental protocols described below. Throughout the experiments, core body temperature of the rats was monitored by a rectal thermometer and was maintained by heating pads. All experiments in this study were performed in accordance with the guidelines for animal research from the National Institutes of Health (NIH publication 85-23, revised 1985) and were approved by the Committee on Animal Research at the University of California, San Francisco.
Experimental Protocols
Rats were randomly assigned to one of five groups subjected to
different protocols: control animals with no ischemia;
nonpreconditioned animals with 10, 20, or 30 minutes of
LCA occlusion followed by 180 minutes of reperfusion (10/180, 20/180,
and 30/180, respectively); and IP animals. The preconditioning protocol
consisted of five consecutive 5-minute episodes of LCA occlusion, each
followed by a 5-minute period of reperfusion.
Nonpreconditioned animals had a comparable 50-minute
nonischemic period with the snare occluder open. Preconditioned
rats were then subjected to an immediate 30-minute period of LCA
occlusion followed by 180 minutes of reperfusion. All rats were finally
assessed for infarct size and/or DNA cleavage as described below. The
body temperature was carefully maintained constant (between 36.8°C
and 37°C) throughout the protocol.
Infarct Sizing
Infarct size and ischemic risk area were determined as
described previously.9 10 28 29 After the prolonged
ischemia and reperfusion, the LCA was reoccluded, and 1 mL
phthalocyanine blue dye was injected into the LV cavity in vivo and
allowed to perfuse the nonischemic portions of the heart. The
entire heart was excised, rinsed of excess blue dye, trimmed of right
ventricular and atrial tissue, and sliced transversely into
sections 2 mm thick. These slices were incubated in a 1% solution
of TTC for 12 minutes to stain the viable myocardium brick
red. The samples were then fixed in a 10% formalin solution for 24
hours and weighed, and both sides of each slice were photographed with
an Olympus OM2 camera using a 90-mm macrolens and a 2x teleconverter.
The ischemic risk area (unstained by phthalocyanine blue dye)
and the infarcted area (unstained by TTC) were outlined on each
photograph and measured by planimetry. The area from each region was
averaged from the photographs of each side for each slice and
multiplied by the weight of that tissue section. Infarct size was
expressed both as a percentage of total LV mass and as a percentage of
the ischemic risk area.
Genomic DNA Analysis
For determination of genomic DNA fragmentation, rat hearts were
rapidly removed after the prolonged ischemia and reperfusion,
and LV samples from completely normal (nonischemic) and
ischemic areas were isolated with the phthalocyanine blue dye
perfusion as a guide, washed of blood, frozen in liquid nitrogen, and
ground to powder. The powdered tissue, transferred to a 50-mL
centrifuge tube with 
10 vol extraction buffer (10
mmol/L Tris-HCl [pH 8.0], 0.1 mol/L EDTA [pH 8.0], 0.5% SDS, and
20 µg/mL pancreatic RNAase), was first incubated for 1 hour at room
temperature and then digested in the same buffer with 200 µg/mL
proteinase K (Sigma) at 50°C overnight. An equal volume of phenol
equilibrated with 1 mol/L Tris buffer (pH 8.0) was then added, and the
tube was placed on a roller apparatus for 1 hour. After the
two phases were separated by centrifugation at
5000g for 30 minutes at room temperature, the viscous
aqueous phase was transferred to a clean 50-mL tube, and the extraction
was repeated with an equal volume of phenol/chloroform. After the
second extraction, the aqueous phase was transferred to a new 50-mL
tube and the DNA precipitated by addition of 0.1 vol 3 mol/L sodium
acetate and 2 vol 100% ethanol. DNA precipitate was collected by
centrifugation at 5000g for 20 minutes at
room temperature, rinsed with 70% ethanol, and finally resuspended in
0.5 mL extraction buffer in a 1.5-mL microcentrifuge tube until
dissolved. The concentration of DNA in each sample was measured by
spectrophotometry (260 nm). To detect DNA internucleosomal cleavage, 10
µg of each DNA was electrophoretically fractionated on 1.5% agarose
gel with 0.5 µg/mL ethidium bromide. HaeIII digest was run
in parallel as molecular size standard. The DNA in the gel was
visualized and photographed under UV light. A qualitative
analysis of DNA fragmentation was performed by analyzing the
pattern of low-molecular-weight DNA (180-bp multiples).
TUNEL Staining
To localize and assess cells undergoing DNA fragmentation, TdT
was used for the incorporation of biotin-16-dUTP to free 3'-OH ends to
DNA strand breaks in situ.30 After perfusion in vivo with
the phthalocyanine blue dye, rat hearts were rapidly removed, washed of
blood, sliced transversely into sections 2 mm thick, incubated in
TTC for 12 minutes, and fixed in phosphate-buffered 4% formaldehyde at
4°C overnight. The slices of heart were photographed, dehydrated in
graded alcohols and xylene, and embedded in paraffin according to
standard methods. Sections 5 µm thick were cut and mounted on
Fisher SuperFrost Plus glass slides. The sections were then rehydrated.
Endogenous peroxide activity was quenched by a 30-minute
incubation in 3% hydrogen peroxide in methanol (Sigma) at room
temperature, and the sections were washed several times in PBS. The
sections were incubated in 0.1% saponin and 1 mmol/L EGTA (Sigma)
in PBS for 30 minutes at room temperature and then washed several times
in PBS. The TUNEL procedure was carried out as follows: the sections
were incubated for 60 minutes at 37°C in a humid chamber in a
solution containing 5 U TdT, 3 µL cobalt chloride (1.5 mmol/L
final), 0.5 µL biotin-16-dUTP (0.5 nmol/L final), 10 µL TdT buffer,
and distilled water to bring the volume up to 50 µL (all reagents are
available from Boehringer-Mannheim). The sections were then
washed several times in PBS. The reaction was stopped in 4x saline
citrate buffer (Fisher) and 5% powdered milk for 30 minutes at room
temperature. The slides were washed in PBS before processing by
standard immunoperoxidase techniques using diaminobenzidine as
chromogen. The sections were then dehydrated and cleared before
coverslipping. Slides incubated without TdT and thymus sections were
used as negative and positive controls, respectively.
Sandwich Enzyme Immunoassay
For quantification of DNA fragmentation, specific determination
of cytosolic mononucleosomes and oligonucleosomes was performed with an
ELISA kit (Boehringer Mannheim) designed to quantify cytosolic
oligonucleosome–bound DNA. This assay is based on a quantitative
sandwich enzyme immunoassay principle using mouse monoclonal antibodies
directed against DNA and histones, respectively.31
Transmural samples from 25 mg of completely normal areas (central
portion of the septum) and ischemic areas (central portion of
the LV free wall, halfway from the edge of the septum) were isolated
with the phthalocyanine blue dye perfusion as a guide, washed of blood,
disintegrated in tissue grinder, and incubated for 30 minutes at room
temperature in 400 µL lysis buffer supplied with the kit. The
homogenate was centrifuged at 13 000g
for 20 minutes. The supernatant (ie, cytosolic fraction) was further
diluted 50-fold in PBS buffer (in mmol/L: NaCl 137, KCl 2.7,
Na2HPO4·7H2O 4.3, and
KH2PO4 1.4; pH 7.4) and used directly as
antigen source in the sandwich ELISA. Incubation buffer instead of the
sample solution and DNA-histone complex included in the kit were used
as background control and positive control, respectively. Three values
from the double absorbance measurements (405 nm/490 nm) of the samples
were averaged, and the background value of the immunoassay was
subtracted from each of these averages. The positive control was used
as internal control for daily variability.
Statistical Analysis
All values are expressed as mean±SEM. Comparisons between
groups were assessed by one-way ANOVA with post hoc analysis
with the Student-Newman-Keuls test. Statistical significance was
defined as a value of P<.05.
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Results |
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Anatomy and Sizing of Myocardial Infarcts
After incubation in TTC, slices from nonpreconditioned rat hearts (20 and 30/180) showed a large homogeneous myocardial infarct in the anterior and lateral free wall of the left ventricle. Within the larger ischemic area (defined by absence of phthalocyanine blue), there was usually a subepicardial and/or subendocardial rim of surviving myocardium adjacent to the infarct. The IP rat hearts had smaller infarcts; some were more patchy but mostly distributed in the central portion of the LV free wall.
The infarct sizes of preconditioned and
nonpreconditioned rats are summarized in Table 1
and Fig 1. As shown, myocardial infarct
size continuously increased as ischemia time increased in
nonpreconditioned rats subjected to 10, 20, and 30
minutes of LCA occlusion and 180 minutes of reperfusion, respectively.
In contrast, infarct size was significantly reduced in the
preconditioned group compared with the
nonpreconditioned group subjected to 30 minutes of LCA
occlusion and 180 minutes of reperfusion. There was no difference in
the risk area between these different groups.
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Detection of Genomic DNA Fragmentation
For detection and qualitative evaluation of DNA fragmentation, we
examined whether genomic DNA isolated from ischemic hearts
produced a typical "ladder" pattern (180-bp multiples) when
analyzed on an agarose gel. As illustrated (Fig 2, lanes 2 and 3), all ischemic LV regions
obtained from rats subjected to 20 (n=6) or 30 (n=6) minutes of LCA
occlusion and 180 minutes of reperfusion showed a typical DNA
electrophoretic pattern characterized by mononucleosomal and
oligonucleosomal DNA fragmentation. Specific nucleosomal cleavage was
associated with a diffuse pattern of DNA damage (random digestion of
DNA), indicating that apoptosis and necrosis were present
simultaneously. In contrast, neither nucleosome ladders nor
smears of DNA could be seen in nonischemic LV areas. No
internucleosomal DNA fragmentation could be detected in hearts obtained
from either control rats (ie, no ischemia) (n=4) or rats
subjected to 10 minutes of LCA occlusion and 180 minutes of reperfusion
(n=6) (Fig 2, lane 1).
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Similarly, we examined whether genomic DNA isolated from IP rat hearts
showed a typical internucleosomal DNA fragmentation when
analyzed on an agarose gel. As illustrated in Fig 3, DNA from preconditioned hearts (n=12) showed little
or no internucleosomal cleavage (lane 3), whereas DNA from
nonpreconditioned hearts (n=12) showed typical
nucleosome fragmentation (lane 1). No nucleosome ladders could be
detected in nonischemic LV areas (lane 2 and 4).
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Localization and Assessment of In Situ DNA Fragmentation by
TUNEL Staining
By hematoxylin-eosin staining, the sections of rat hearts showed
normal myocardium in the infarct zone determined by TTC
after 30 minutes of LCA occlusion and 180 minutes of reperfusion; there
were no histological features of myocardial infarction
and no inflammatory reaction at this stage (Fig 4A). In
contrast, the TUNEL assay clearly localized nuclei in apoptotic
myocardial cells. The reaction product was dark brown, and there
was minimal background. In the nonischemic area, there were no
stained nuclei for either preconditioned or
nonpreconditioned rat hearts (Fig 4B). In the infarct
zone of nonpreconditioned hearts (30/180), numerous
ovoid centrally oriented nuclei within myofibers contained reaction
product (Fig 4C); there were no other stained nuclei within the
myocardium (n=3). The TUNEL method localized scattered
nuclei with precipitate within the infarct zone of IP hearts, but the
nuclei were sparse compared with the nonpreconditioned
hearts (Fig 4D) (n=3).
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Quantification of Cytosolic DNA Fragmentation by Sandwich
Enzyme Immunoassay
Quantitative determination of fragmented DNA into mononucleosomes
and oligonucleosomes was determined by an ELISA specific for cytosolic
histone–bound DNA. As noted in Table 2 and Fig 5, DNA fragmentation determined by ELISA was very low
and not significantly different in nonischemic LV regions
obtained from rats subjected to 10 (n=5), 20 (n=5), and 30 minutes
(n=10) of LCA occlusion and 180 minutes of reperfusion compared with
control animals (no ischemia) (n=5). In contrast, according to
agarose gel analysis, fragmented DNA from ischemic LV
regions obtained from rats subjected to 20 or 30 minutes of LCA
occlusion and 180 minutes of reperfusion was significantly increased
compared with nonischemic regions (Table 2 and Fig 5).
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Similarly, quantitative analysis of cytoplasmic fragmented DNA
was performed in IP animals (n=10). As illustrated in Table 2
and Fig 5, cytoplasmic mononucleosomes and oligonucleosomes were significantly
reduced in preconditioned compared with
nonpreconditioned rat hearts.
Relationship Between Infarct Size and DNA Fragmentation
To establish the relationship between infarct size and fragmented
DNA, we assessed infarct size determined by TTC and internucleosomal
DNA cleavage determined by sandwich enzyme immunoassay in the same
animals. As shown in Fig 6, there was a direct
correlation between myocardial infarct size and specific DNA
fragmentation in this animal model in vivo. Moreover, the correlation
between infarct size and DNA fragmentation was maintained after
ischemic preconditioning (r=.96,
P<.001).
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Discussion |
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The results of the present study indicate that both internucleosomal DNA fragmentation and infarct size were significantly reduced in preconditioned rat hearts compared with nonpreconditioned rat hearts after ischemia and reperfusion in vivo. Because specific DNA degradation by activation of an endogenous endonuclease is a hallmark of apoptosis, these data suggest that ischemic preconditioning reduces irreversible ischemic injury (ie, myocyte cell death) in part by preventing programmed cell death after prolonged ischemia and reperfusion.
Ischemic Preconditioning in Rat Hearts
A number of investigators have demonstrated that ischemic
preconditioning limits infarct size in different animal
species,1 5 6 including rats.7 8 9 10 The
mechanism by which preconditioning prevents myocyte cell death in rat
hearts appears to be transient7 but is not well
understood. Although adenosine A1 receptor
activation and ATP-sensitive potassium channels have been proposed as a
mechanism for preconditioning in some animal species, neither
adenosine32 33 nor ATP-sensitive potassium
current34 appears to mediate the protective effects of
ischemic preconditioning in the rat heart. Recently, Hu and
Nattel35 have suggested that ischemic
preconditioning against ventricular
tachyarrhythmias and myocardial stunning in the rat
heart was due to the stimulation of 
1B-adrenergic
receptors by the release of endogenous
catecholamines, resulting in the activation of a pertussis
toxin–sensitive G protein that enhances PKC activity. Whether the
antiarrhythmic action of preconditioning shares a common mechanism with
the effect on myocardial infarction or not, there is abundant evidence
supporting the role of PKC as a final common pathway in the rat heart.
Li and Kloner36 found that blockade of PKC with calphostin
C, a novel and specific inhibitor of PKC, completely
aborted the protective effect of ischemic preconditioning on
infarct size. Speechly-Dick et al37 showed that
1,2-dioctanoyl-sn-glycerol, a diacylglycerol analogue and
specific agonist of PKC, reduced infarct size to an extent similar to
that of preconditioning in rat hearts. Moreover, any receptor
stimulation that can activate PKC may mediate the protective
effects of ischemic preconditioning and explain differences
observed across various species.38 39 Activation by
preconditioning of PKC through a cellular signaling pathway involving G
protein–stimulated phospholipase C activity may theoretically induce
phosphorylation of many proteins in myocytes, causing a
reduction in cell death.
In addition, reduction in intracellular acidosis after ischemic preconditioning has been extensively described.9 40 41 42 A previous study from our laboratory suggested that the reduction in the fall of intracellular pH was associated with endogenous glycogen depletion before prolonged ischemia (ie, reduced proton production from anaerobic glycolysis).9 Furthermore, Barbosa et al10 showed that infarct size reduction by preconditioning correlated with the degree of glycogen depletion in rat hearts in vivo. Thus, regulation of pH homeostasis by ischemic preconditioning may contribute to a reduction in lethal ischemic injury in rat hearts.
Postischemic Apoptotic Cell Death in
Myocytes
Despite important clinical implications, relatively little is
known about the nature of cardiomyocyte death after
ischemic injury. Necrosis and apoptosis are two
distinct mechanisms of lethal cell injury and may potentially be
implicated in myocyte cell death. Necrotic cell death, which is
considered to be a catastrophic metabolic failure resulting
directly from severe damage, is characterized by early loss of membrane
integrity and late random digestion of DNA. In contrast,
apoptotic cell death is an active, regulated, energy-requiring
cellular response that appears to be under genetic
control.23 24 Programmed cell death occurs in the absence
of membrane rupture and is characterized primarily by early
fragmentation of nuclear DNA into internucleosomal
fragments.21 Recently, Tanaka et al43 showed
that hypoxia does not merely cause necrosis but also may
activate the suicide program of neonatal rat
cardiomyocytes in culture. Moreover, Kajstura et
al22 reported that apoptosis was the major initial
form of myocardial damage produced by permanent occlusion (no reflow)
of the left main coronary artery in rat hearts. They concluded
that apoptotic and necrotic myocyte cell deaths were
independent contributing variables of infarct size in this in vivo
animal model.
In the present study, we found that apoptotic and necrotic cell death (ie, internucleosomal and random DNA fragmentation, respectively) were simultaneously present in rat hearts subjected to 20 and 30 minutes of occlusion followed by 180 minutes of reperfusion. In the large, homogeneous infarct zone defined by TTC, the TUNEL method localized numerous nuclei of apoptotic cells. Consistent with these findings, the amount of cytosolic mononucleosomes and oligonucleosomes quantified by photometric enzyme immunoassay in the ischemic area in which the dUTP-stained myocyte nuclei were distributed was significantly increased compared with the nonischemic area without dUTP-stained nuclei in rat hearts subjected to 20 and 30 minutes of occlusion and 180 minutes of reperfusion. We also found a correlation between the amount of cytoplasmic histone–associated low-molecular-weight DNA fragments and infarct size, suggesting that the magnitude of infarct size in rat hearts in vivo is associated with the degree of specific DNA fragmentation after ischemia and reperfusion.
Previous studies have shown that reperfusion may trigger (or accelerate) programmed cell death after a prolonged period of ischemia. Data obtained in both in vitro19 and in vivo20 44 models of ischemia and reperfusion have demonstrated that internucleosomal DNA fragmentation was significantly greater after ischemia followed by reperfusion than after ischemia without reperfusion. Reactive oxygen species release and/or modifications in pH homeostasis during reperfusion may potentially mediate this response.45
Regulation of apoptosis involves a large number of gene products. Some of them, such as Fas antigen and bcl-2 protein, appear to be upregulated by hypoxia and/or reperfusion. Fas antigen, a cell surface protein involved in the negative selection of autoreactive T cells, has been shown to be increased both in cultured neonatal rat cardiomyocytes in vitro43 and in adult rat myocytes in vivo after prolonged ischemia.22 Similarly, bcl-2 protein, the mammalian proto-oncogene product analogous to the cell death inhibitor Ced-9 in Caenorhabditis elegans, was overexpressed in rat cardiomyocytes in vivo after coronary artery occlusion.22 Moreover, Misao et al46 reported that bcl-2 protein was expressed in the salvaged myocytes of human hearts with acute infarctions, suggesting that some cardiac cells may be salvaged by the expression of bcl-2 in the early stage of infarction. However, the pathophysiological role of these proteins in the apoptosis of cardiomyocytes after ischemia and/or reperfusion needs further study.
Prevention of Apoptotic Cell Death by Ischemic
Preconditioning
Although ischemic preconditioning has been extensively
shown to reduce infarct size in different animal species, little is
known about its effect on programmed cell death. We report here that
ischemic preconditioning reduces both infarct size and
internucleosomal DNA fragmentation in rat hearts in vivo after
prolonged ischemia and reperfusion. We found that genomic DNA
obtained from preconditioned rat hearts showed little or no
internucleosomal cleavage when analyzed on an agarose gel. The
TUNEL method localized few and sparsely stained nuclei within the small
infarct zone of these preconditioned hearts. In accord with these
findings, the amount of cytoplasmic mononucleosomes and
oligonucleosomes quantified by ELISA was significantly decreased in
preconditioned rat hearts compared with
nonpreconditioned rat hearts. The results of the
present study are consistent with those of a previous
investigation showing that preconditioning may prevent programmed
myocyte cell death in vitro.27
The basic mechanism by which ischemic preconditioning could prevent apoptosis remains unknown. Recent experiments have demonstrated that apoptosis could be delayed in neutrophils47 and in hematopoietic stem cells48 by preservation of intracellular pH homeostasis. On the basis of these findings, Gottlieb et al27 suggested that activation of the vacuolar proton ATPase by PKC during preconditioning may attenuate intracellular acidification during metabolic inhibition and thereby protect myocytes from apoptosis in vitro. Consistent with this hypothesis, previous studies from our laboratory9 and elsewhere40 41 42 have demonstrated that ischemic preconditioning was accompanied by a reduction in intracellular acidosis in rat hearts. In addition to increased proton export through vacuolar proton ATPase by PKC activation, reduced proton production from anaerobic glycolysis may also contribute to prevent myocyte apoptosis in rat hearts in vivo.
We observed a close correlation between infarct size and the amount of apoptosis in rat hearts subjected to prolonged ischemia and reperfusion. Moreover, the correlation was maintained after ischemic preconditioning. Although apoptosis and necrosis are considered to be distinct mechanisms of cell death, our data suggest the possibility that these pathways of cell death may be interrelated in this in vivo model.
Conclusions
This study shows that ischemic preconditioning reduces
both infarct size and internucleosomal DNA fragmentation after
prolonged ischemia and reperfusion in rat hearts in vivo. These
data suggest the possibility that preconditioning reduces irreversible
ischemic injury in part by decreasing apoptosis.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by the California Affiliate of the American Heart Association (grant 95-225 to Dr Wolfe). Dr Piot is the recipient of an overseas fellowship grant from the French Federation of Cardiology. We wish to thank Hovsep Melkonyan for his advice on the DNA analysis procedure and Margaret Mayes for her technical assistance with TUNEL staining. We acknowledge Chandra Adivi for his technical assistance.
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Footnotes |
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Reprint requests to Christopher L. Wolfe, MD, University of California, San Francisco, Moffitt Hospital, M-1186, 505 Parnassus Ave, San Francisco, CA 94143-0124.
Received December 18, 1996; revision received March 3, 1997; accepted March 7, 1997.
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