# Apoptosis in Human Acute Myocardial Infarction: The Rationale for

Authors: Simon W. Rabkin
Journal: Scholarly Research Exchange
 (2009)

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## Abstract

The objective of the present review was to examine apoptosis in patients with acute myocardial infarction (MI) and to address  (i) the prevalence of apoptosis in acute MI, (ii) techniques to determine apoptosis, (iii) time period from the onset of acute MI to the detection of apoptosis, (iii) criticisms about apoptosis in acute MI.  A systematic literature search identified over 20 publications comprising over 400 patients. The prevalence of apoptosis varied from over 90%  in nuclear imaging studies using annexin binding to 0.25%  in an autopsy study using monoclonal antibody to single-stranded DNA. Apoptosis was present in 50–60%  of infarcted hearts within 24 hours of MI (detected by Bax and activated caspase-3), 26%  of myocytes in patients who died within 11 days of MI (pooled mean from 5 studies using only TUNEL staining), and 12%  of the myocytes of patients who died, on average, 20 days after onset of MI (pooled mean from eight studies using dual staining with caspase-3 plus TUNEL). Criticisms of the TUNEL assay appear unjustified as TUNEL is at least 85%  specific using caspase-3 activation as a marker of apoptosis. Taken together, DNA fragmentation on agarose gel electrophoresis, TUNEL staining of nuclei, caspase-3 activation, bcl-2 and Bax expression, and annexin V binding overwhelming support apoptotic cell death as an important component of MI. The amount of cardiac apoptosis correlates with the presence of heart failure and fatal arrhythmias. Heart failure as a complication of MI carries a high mortality and indicates the amount of myocardium lost during the infarct. Taken together, these findings suggest the need for clinical trials in acute MI to confirm whether inhibition of apoptosis can reduce patient morbidity and mortality.


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## Body

## 1. Introduction

Apoptosis, one of the main forms of
cell death, is a highly regulated process which leads to the destruction of a
cell utilizing a unique set of biochemical processes and leading to a
characteristic set of morphologic changes [1]. Oncosis, the other major
form of cell death, has been traditionally considered to be the dominant form
of cell death in acute myocardial infarction (MI) [2]. There was a long delay from the
initial description of apoptosis [1, 3] until the recognition of its
occurrence in the heart [4]. Experimental models of
ischemia followed by reperfusion, or hypoxia followed by reoxygenation induce
apoptosis in the heart of different species including mice, rabbit, rat, dog,
and pig [4, 5]. There is abundant
experimental evidence that isolated cardiomyocytes undergo apoptotic cell death
from activation of the extrinsic pathway through the activation of cell surface
receptors or through the intrinsic pathway mainly involving mitochondria. Stimuli
such as high concentrations of the saturated fatty acids, nitric oxide, some catecholamines,
or cytokines induce apoptosis in isolated cardiomyocytes [6–9]. Despite the experimental
evidence, there is recurrent concern that apoptosis does not occur in the human
heart because it has not been demonstrated to occur in human heart to any
meaningful extent. It is universally accepted, however, that the death of
cardiomyocytes is the fundamental basis of myocardial infarction and its
subsequent complications. Therefore, it is of paramount importance to clearly
understand the extent to which apoptosis maybe the primary mode of cell death
in patients with acute MI. The purpose of this paper is to critically review
the evidence supporting the hypothesis that apoptosis occurs in patients with
acute MI and to evaluate whether apoptosis, in the setting of acute MI, is a
worthwhile therapeutic target.


## 2. Methods

The Medline database was searched
from its inception to August 2008. The search terms were “apoptosis” and “myocardial
infarction.” The search was restricted to humans and further restricted to studies
that examined the hearts of humans after the onset of an acute myocardial
infarction.This
review was performed to specifically address the following issues: (i) prevalence
of apoptosis in acute myocardial infarction, (ii) techniques that were used to determine whether apoptosis
was present, (iii) time period from the onset of the acute myocardial
infarction to the detection of apoptosis and its potential for producing variations
in the prevalence of apoptosis in acute myocardial infarction, and (iv) criticisms
in the literature about cardiac apoptosis in acute MI.


## 3. Results

A review of the human
pathologic studies and their methodology for the detection of apoptosis shows
that there were 20 autopsy studies over a ten-year period from 1995 to 2007
(Table1). One study examined the myocardium in a patient with an infarcted
intraventricular septum that was partially resected at surgery [10]. Studies excluded were those contained cases
with profound ischemia without documented myocardial infarction and those that
were explanted at the time of a cardiac transplant with ischemia but without a
recent infarction or examined only the right ventricle. The present review,
therefore, encompasses 437 acute MI patients that were included in studies that
evaluated cases with documented myocardial infarction. As some of the authors published
several of these papers and since the respective methodology sections did not
specifically indicate which cases, if any, were included in other publications,
it is assumed that no overlap exists in the cases reported across these studies.Table 1
The number of studies that examined apoptosis in
patients with acute MI, the number of cases, the time from the onset of
symptoms to death, and the time from death to autopsy. (Studies present the times as either mean or median.)
AuthorYearNumber of MITime after onset MITime death to autopsyItoh et
al.  [11]199519“12 hours
to several days”UncertainOlivetti et
al.  [12]199620~4 (<10)
days<24 hoursBardales et al.  [13]199632UncertainUncertainVeinot et al.  [14]19978Uncertain<24 hoursSaraste [15]1997838 (6 to
120) hours17.3 (4
to 48) hoursToyoda et
al. [10]199817 days0aJames [16]199912UncertainUncertainOttaviani et al.  [17]199910<2.5<24 hoursPiro et
al.  [18]2000160.4 days (<14 hours)UncertainRodríguez-Calvo et al. [19]2001142.9 (0.5 to 11) daysUncertainAbbate et
al.  [20]200224~27 (15
to 40) daysUncertainNakatome et
al.  [21]20026<2 hours<24 hoursNakatome et
al.  [21]200260.5 to 5
hours24 to 63
hoursBaldi et al.  [22]20021623
(12–62) days<30 hoursEdston et al.  [23]200210Uncertain
(Sudden deaths)3.6 daysBussani et
al.  [24]200312~20 (14
to 44) daysUncertainAbbate et al.  [25]20033023 (14 to
40) daysUncertainAbbate et
al.  [26]200314~16 (12
to 34) daysUncertainBiondi-Zoccai et al.  [27]200421~25
(11–80) days<30 hoursBiondi-Zoccai et al.  [28, 29]200521~22
(12–50) daysUncertainAbbate et
al.  [30]20053017 (9 to
60) days<30Akasaka et
al.  [31]200543Uncertain<6 hoursZidar et
al.  [32]200630<24 hoursUncertainZidar et
al.  [32]200620>24 hoursUncertainSinagra et al.  [33]20071418.8 (10
to 62) daysUncertainAcross these respective
studies, there was found to be a wide range of allowable time intervals for
patient inclusion from the initial onset of acute MI symptoms to eventual
patient death. Some studies only focused on cases where death had recently occurred
within 2 hours while other studies included cases 80 days after the acute MI. The
time from the onset of symptoms to death is a determinant of the presence and
thus the prevalence of apoptosis [14] as well as the assessment of comparability
between studies. Two studies were subdivided as reported by the authors into
different time periods from the onset of symptoms. Another issue is that 5 of 22
of the published studies did not report the time from the onset of symptoms to
death. Most autopsies were performed within 24 to 30 hours of patient death because
excessively long time periods would have limited the accurate detection of
apoptosis [21]. Unfortunately, in half of
the studies, the time from the death to autopsy is uncertain.Different methodologies were utilized
to assess the heart for the presence of apoptosis (Table2). These methods
encompassed direct detection of the specific DNA fragmentation characteristic of apoptosis seen on agarose gel electrophoresis, as
well as immunohistochemical changes of DNA fragmentation or immunohistochemical evidence of activation of apoptotic pathways.
Not all of these techniques can provide quantitative information. The results should
be considered for their consistency between studies using the same methodology.Table 2
Methodologies used to determine the presence of apoptosis in the heart.
AuthorYearNumber MIDNA gel electrophoresisISEL or TUNELCaspase-3ssDNABaxItoh et
al.  [11]199519YesYesNoOlivetti et
al.  [12]199620YesYesNoBardales
et al. [13]199632NoYesNoVeinot et
al. [14]19978YesYesNoSaraste et
al.  [34]19978YesYesNoToyoda et
al. [10]19981NoYesNoJames [16]199812NoYesNoOttaviani et al.  [17]199910NoYesNoPiro et
al.  [18]200016YesYesNoRodríguez-Calvo
et al. [19]200114YesYesNoAbbate et
al.  [20]200224NoYesYesNakatome et
al.  [21]200212YesYesNoBaldi et
al.  [22]200216NoYesYesYesEdston et
al. [23]200210NoYesNoAbbate et al. [25]200330NoYesYesBussani et
al.  [24]200312NoYesYesAbbate et
al. [26]200314NoYesYesBiondi-Zoccai et al.  [27]200421NoYesYesYesBiondi-Zoccai et al. [28, 29]200521NoYesYesYesAbbate et
al. [30]200530NoYesYesAkasaka et
al.  [31]200543NoNoNoYesZidar et
al.  [32]200650NoNoYesSinagra et
al.  [33]200714NoYesYesApoptosis
was originally defined based on distinct morphological features due to internucleosomal
DNA degradation resulting from
activation of specific endonucleases or DNAses [1]. The morphologic features of apoptosis include chromatic margination,
nuclear condensation and fragmentation, as well as condensation of the cell
with preservation of organelles [1]. The cellular enzymes active
in the process of apoptosis also produce fragmentation of the cell into
membrane-bound apoptotic bodies, which undergo phagocytosis by nearby cells
without associated inflammation [3, 35].


### 3.1. Histologic Evidence of DNA Damage Consistent with Apoptosis

Histologic staining of the
myocardium to show nuclei with specific DNA fragmentation has been accomplished by staining for in situ end-labeling (ISEL), TUNEL, or ssDNA. The early studies
used ISEL or TUNEL and reported either the presence or absence of apoptosis
with the conclusion that apoptosis was present in most cases of acute MI [10, 14, 16, 21] (Table 3). These studies, therefore,
demonstrated that widespread apoptosis occurs
in acute MI since apoptosis was evident after only a few hours after the onset
of MI and prior to the appearance of necrosis [14]. In addition, apoptosis was not detected in normal
myocardium [14]. In older infarcts, the
incidence of apoptosis declined in
myocytes, but increased in invading inflammatory cells [14].Table 3
The percentage of confirmed apoptosis using
different assays. (Data are presented in two ways: the data in parenthesis
is the percentage of infarcted hearts with apoptosis; otherwise it is the
percentage of cardiomyocytes that show evidence of apoptosis.)
TUNELTUNEL + CaspaseTUNEL ± CaspaseCasp-3NMI (%)MI (%)Remote (%)% HeartsControl (%)NControlRemoteOlivetti et al. [12]2012 B0.74P<.05Bardales et
al. [13]32(All)813Saraste et
al. [34]80.8 B0.0050.0076Veinot et
al. [14]8(All)03James
[16]12(All)Ottaviani et al. [17]10AA3P<.05Piro et
al. [18]1643.638.100Not donensRodríguez-Calvo
et al. [19]143605Nakatome et
al.  [21]6(All)0Abbate et
al. [20]2419.50.5P<.001Baldi et
al. [22]1625.40.7P<.001Edston
et al. [23]102608Abbate et
al. [25]30114Abbate et
al. [26]1419Bussani
et al. [24]1216.7Biondi-Zoccai et al. [27]210.710.014Biondi-Zoccai et al. [28, 29]218.10.41+0.014Abbate et
al. [30]306.90.90.015P<.01P<.01Zidar et
al. [32]30(60)05Zidar et
al. [32]20(15)05Sinagra et
al. [33]145.9A: data expressed as arcosineAll: present in all hearts and there is no quantitative dataB: border zone+: the data are extracted from the graph.Quantitation of the number of
myocytes showing evidence of ISEL- or TUNEL-stained nuclei, as a proportion of
the total myocytes nuclei visualized microscopically has been referred to as
the “apoptotic index.” The earliest study found that 12% of myocytes in the
border zone of the myocardial infarct showed DNA strand breaks, whereas 1% of cells were undergoing apoptosis in the remote myocardium; indicating
a significant increase in apoptosis in acute MI [12]. Subsequent studies reported
apoptotic indexes using this detection methodology range from 0.8 to 43.6%
(Table 3). Although the 0.8% value is the outlier, it is from a study in which
the occluded artery had been reopened, potentially, minimizing the amount of cell death.
This value is lower than the level of apoptosis in a similar study [30]. Some of the studies
report
data as the mean and others as median.  If one assumes that the two indexes of
central tendency are similar and calculate a weighted average, then 25% of myocyte
nuclei show evidence of apoptosis in acute MI (Table 3). Apoptosis index varies
dramatically across studies, in part because some studies did not indicate from
which  region of the infarct the
apoptotic index was derived [18, 19]. This is unfortunate because
there is little or no TUNEL staining in the core of the infarct where cellular
nuclei are either absent or severely disintegrated [23], considerable TUNEL-stained
nuclei in the border zone and much less at a site remote form the infarct. The
increase in apoptotic cells in the border zone around the infarct area was consistently
many fold greater than the area of myocardium remote from the infarct [12, 34].The presence of TUNEL staining in
nuclei of myocardial cells is a function of the time from death to autopsy because
postmortem autolysis damages the DNA and limits detection of the characteristic apoptosis. Nakatome et al. reported that  all 6 cases with autopsies between 5 to 30 hours
after death showed TUNEL-positive cells in the infarct while none of 6 cases
who had autopsies 24 to 63 after death showed TUNEL-positive cells [21].Monoclonal antibody to
single-stranded DNA (ssDNA) is more
specific and sensitive cellular marker of apoptosis than TUNEL. Akasaka et al.
studied 43 persons with acute MI at autopsy and related the detection of ssDNA to histologic estimation of the stage of MI
from earliest, to coagulation necrosis, macrophage infiltration,
granulation tissue, and scar tissue [31]. This study is difficult to
relate to the others because it reported more stages of MI than hearts
examined. The maximum prevalence of apoptosis was only 0.25% [31]. As the time from death to
autopsy is uncertain in that study, it is possible that this variable
influenced their results.  It is difficult
to reconcile this assessment of apoptosis in MI with the other studies of
apoptosis in the heart (Table 3).


### 3.2. DNA Fragmentation on Agarose Gel Electrophoresis

DNA fragmentation into fragments of 180 to 200 base pairs detected on agarose gel
electrophoresis is the hallmark of apoptosis because of the activation of
specific endonucleases during on the apoptosis process [1]. Itoh et al. were the first
to report  DNA fragmentation in the infarcted myocardium but not in normal myocardium [11]. It has been examined in
seven studies and the characteristic DNA fragmentation has been consistently  demonstrated (Table 2) in the myocardium of
patents following MI [11, 12, 14, 18, 19, 21, 34].
In contrast, there is no DNA fragmentation in hearts from individuals who did not die of MI or in normal
myocardium remote from the MI [12, 14, 19]. The DNA ladder pattern is found in cases of MI less than 72 hours old and more often
with high prevalence of apoptotic DNA [19]. DNA fragmentation was seen in the early stages of AMI (12 to 24 hours) and was not seen in older infarcts [14]. With the caveat that most
studies evaluated only a few cases from their study population, the consistency
of the findings across all studies in which it was examined is noteworthy.


### 3.3. Caspase-3

Caspases are a family of proteases
that cleave cellular polypeptides with the subsequent production of the
majority of cellular and morphological events that occur during apoptotic cell
death [3]. Caspase activation is unique
to apoptosis as it does not occur in other forms of cell death and provides strong
evidence for the presence of apoptosis [35]. Caspase-3 activation has
been found in each of the 9 studies of acute MI in which it was examined [20, 22, 24–27, 30, 32, 33]. Immunohistochemical
examination of MI demonstrated caspase-3 activation in myocardial cells in 60%
of patients who died within 24 hours of the onset of MI, 30% of those dying 1
to 7 days after the infarct, and in none of the  cases dying 1 to 4 weeks after the onset of
infarction [32].Because caspase activation is a
relatively specific marker of apoptosis, investigators have used double staining
of cardiomyocytes with TUNEL plus caspase-3 as the criteria to indicate
apoptosis. Using this set of criteria, the apoptosis index varies from 5.9 to
25.4%. On average, 13.2% of cardiomyocytes in the heart of patients dying of
acute MI manifested apoptosis (Table3). The dual labeling (TUNEL plus
caspase-3) approach has been utilized mainly by a common group of
investigators. Unfortunately, most of the studies using the more specific TUNEL
plus caspase-3 criteria examined heart of patients who had died 20 days after
the onset of MI (Table 1). Considering that caspase-3 activation is maximum within
24 hours after acute MI and declines thereafter [32], the percentage of apoptotic
cells using this criteria is dependent of the time after acute MI that the
heart was examined.Caspase-3 activation was
significantly greater in patients who received reperfusion treatment compared
to those that did not receive this form of therapy [32]. The site of the infarct that
expresses the greatest amount of caspase-3 activation, that is, center or
border zone is unresolved between studies [22, 32].


### 3.4. Bax and Bcl-2 Expression

The Bcl-2 family of proteins regulate the various protein-protein
and protein-membrane interactions affecting the permeability of the outer
mitochondrial membrane which maybe viewed as the point of no return in apoptotic
cell deaths [36]. Two important Bcl-2 signaling
molecules in apoptosis related to the intrinsic or mitochondrial pathway in
apoptosis are Bax and bcl-2 [36] both of which regulate
caspase activation.Myocytes with positive bcl-2
immunoreactivity were seen in 60% of hearts with acute MI while there were no
myocytes with positive bcl-2 immunoreactivity in the controls or in hearts with
old MI [37]. There is a predominance of bcl-2
localized to the border areas surrounding the infarcted tissues [37].  Piro et al. did not find immunoreactivity for
bcl-2 but attributed this failure to the autolysis process as most of their
specimens for analysis of bcl-2 were taken 24 hours after death [18]. Bax expression is increased
in acute MI. Bax immuno-reactivity is evident in 52% of myocytes at the site of
the infarction which is significantly greater than in areas remote from the MI [22]; Bax protein expression
colocalizes with TUNEL [22]. Myocardial Bax expression
was increased in patients with multivessel disease [27].


### 3.5. Demonstration of Apoptosis in Patients with MI by Cardiac Imaging

The studies reviewed above have been mainly autopsy
studies with the attendant concerns about postmortem changes in tissue and
technical issues surrounding TUNEL or caspase-3 immunohistochemistry. Annexin V
is a naturally occurring human protein that binds avidly to membrane-associated
phosphatidylserine  which is actively
transported to the outer layer as an early event in apoptosis, and indicated
the presence of apoptosis in isolated cardiomyocytes
[38]. In six of seven patients,
increased uptake of Tc-m99-labelled annexin-V was seen in the
infarct area of the heart on nuclear (SPECT) imaging [39]. No increased uptake was seen in the
heart outside the infarct area. All patients with increased Tc-m99-labelled
annexin-V uptake in the infarct area showed a matching perfusion defect [39]. In a control individual, no
increased uptake in the heart was seen [39]. In another series of 9 patients, all
patients showed accumulation of Tc-m99-labelled annexin A5 at the
infarct site [40]. These data suggest that 94% of patients with acute MI manifest
apoptosis [39, 40]. The major critique of this
methodology is the inability to distinguish between apoptosis in cardiomyocytes
from apoptosis in the other cell types in the heart, and annexin V labeling of platelets
in the coronary thrombus producing the infarct [41, 42]. Although labeling of
platelets may not be that relevant of an issue [43], the proportion of the total
labeling that can be confirmed to be only due to myocytes remains a challenge.
This technique, however, has validity [44] and provides supporting
evidence for the existence of apoptosis in the process of acute MI.


### 3.6. Relationship of Apoptosis to Reperfusion Injury or Heart Failure

While experimental data support a relationship between
apoptosis and reperfusion injury, it is difficult to obtain this kind of data in
patients after acute MI in large part because autopsy studies do not come from
randomized trials comparing reperfusion and control therapy. However, apoptosis
was associated with histologic features of myocardium that is reperfused after
brief ischemia whereas necrosis occurs more often in areas of prolonged
ischemia without reperfusion [14]. DNA fragmentation (ISEL) was visible in recent infarcts  primarily in myocytes containing contraction
bands, which occur predominantly in regions of reperfused myocardium [14]. The myocardium supplied by
an occluded infarct-related artery (IRA) was associated with significantly
higher amount of apoptosis compared to the myocardium supplied by a patent IRA [30, 45]. The investigators suggested
that the benefits observed with a patent IRA may in part be due to reduced
myocardial apoptosis [30, 45].The amount of apoptosis present in the heart at autopsy is
a significant indicator of the presence of heart failure. Considering that heart
failure was a complication of MI that carried a high mortality and acts as a major indicator of the
amount of myocardium lost during the infarct, individuals who experienced early
occurrence of heart failure had a four-fold increase in the
prevalence of apoptosis compared to individuals without heart failure [26]. Multivariate analysis
including variables such as the amount of apoptosis at autopsy, the use of fibrinolytic
agents, as well as the
history of a previous remote MI showed that only heart failure
remained significantly associated with increased apoptosis [26]. Individuals with poor
prognosis at the onset of acute MI, assessed by the Norris coronary prognostic
index (age, presence of pulmonary congestion, heart size, and history of
previous MI), had a higher prevalence or greater proportion of apoptosis both in
the infarct as well as the area remote from the infarct compared to low-risk
individuals [25]. Patients with biventricular
enlargement had extremely high cardiomyocyte apoptosis at necropsy compared to
individuals who had no cardiac dilatation after MI  [24]. Patients with severe postinfarction
LV diastolic dysfunction had significantly higher rates of apoptosis,
determined by colocalization of TUNEL and
caspase-3 [33]. Thus, the amount of
apoptosis is an important determinant of the amount of left ventricular
dysfunction which in turn is a major determinant of an unfavorable outcome.


### 3.7. Potential Relevance of Apoptosis in the Border Zone of Acute MI

Several studies have shown that apoptosis is greater in
the border zone of MI or the area at the border of the infarct compared to
normal myocardium [31, 34]. The number of apoptotic
nuclei is consistently greater in the peri-infarcted region than in that away
from infarction [12].
Potentially fatal arrhythmias such as ventricular tachycardia can originate in pathways
in the heart at the border zone of an acute MI [46, 47] perhaps due to the mixture of
living and death cells  at the border
zone [12, 31]. Thus, apoptosis may be an
important component in the generation of potentially fatal cardiac arrhythmias.


## 3.1. Histologic Evidence of DNA Damage Consistent with Apoptosis

Histologic staining of the
myocardium to show nuclei with specific DNA fragmentation has been accomplished by staining for in situ end-labeling (ISEL), TUNEL, or ssDNA. The early studies
used ISEL or TUNEL and reported either the presence or absence of apoptosis
with the conclusion that apoptosis was present in most cases of acute MI [10, 14, 16, 21] (Table 3). These studies, therefore,
demonstrated that widespread apoptosis occurs
in acute MI since apoptosis was evident after only a few hours after the onset
of MI and prior to the appearance of necrosis [14]. In addition, apoptosis was not detected in normal
myocardium [14]. In older infarcts, the
incidence of apoptosis declined in
myocytes, but increased in invading inflammatory cells [14].Table 3
The percentage of confirmed apoptosis using
different assays. (Data are presented in two ways: the data in parenthesis
is the percentage of infarcted hearts with apoptosis; otherwise it is the
percentage of cardiomyocytes that show evidence of apoptosis.)
TUNELTUNEL + CaspaseTUNEL ± CaspaseCasp-3NMI (%)MI (%)Remote (%)% HeartsControl (%)NControlRemoteOlivetti et al. [12]2012 B0.74P<.05Bardales et
al. [13]32(All)813Saraste et
al. [34]80.8 B0.0050.0076Veinot et
al. [14]8(All)03James
[16]12(All)Ottaviani et al. [17]10AA3P<.05Piro et
al. [18]1643.638.100Not donensRodríguez-Calvo
et al. [19]143605Nakatome et
al.  [21]6(All)0Abbate et
al. [20]2419.50.5P<.001Baldi et
al. [22]1625.40.7P<.001Edston
et al. [23]102608Abbate et
al. [25]30114Abbate et
al. [26]1419Bussani
et al. [24]1216.7Biondi-Zoccai et al. [27]210.710.014Biondi-Zoccai et al. [28, 29]218.10.41+0.014Abbate et
al. [30]306.90.90.015P<.01P<.01Zidar et
al. [32]30(60)05Zidar et
al. [32]20(15)05Sinagra et
al. [33]145.9A: data expressed as arcosineAll: present in all hearts and there is no quantitative dataB: border zone+: the data are extracted from the graph.Quantitation of the number of
myocytes showing evidence of ISEL- or TUNEL-stained nuclei, as a proportion of
the total myocytes nuclei visualized microscopically has been referred to as
the “apoptotic index.” The earliest study found that 12% of myocytes in the
border zone of the myocardial infarct showed DNA strand breaks, whereas 1% of cells were undergoing apoptosis in the remote myocardium; indicating
a significant increase in apoptosis in acute MI [12]. Subsequent studies reported
apoptotic indexes using this detection methodology range from 0.8 to 43.6%
(Table 3). Although the 0.8% value is the outlier, it is from a study in which
the occluded artery had been reopened, potentially, minimizing the amount of cell death.
This value is lower than the level of apoptosis in a similar study [30]. Some of the studies
report
data as the mean and others as median.  If one assumes that the two indexes of
central tendency are similar and calculate a weighted average, then 25% of myocyte
nuclei show evidence of apoptosis in acute MI (Table 3). Apoptosis index varies
dramatically across studies, in part because some studies did not indicate from
which  region of the infarct the
apoptotic index was derived [18, 19]. This is unfortunate because
there is little or no TUNEL staining in the core of the infarct where cellular
nuclei are either absent or severely disintegrated [23], considerable TUNEL-stained
nuclei in the border zone and much less at a site remote form the infarct. The
increase in apoptotic cells in the border zone around the infarct area was consistently
many fold greater than the area of myocardium remote from the infarct [12, 34].The presence of TUNEL staining in
nuclei of myocardial cells is a function of the time from death to autopsy because
postmortem autolysis damages the DNA and limits detection of the characteristic apoptosis. Nakatome et al. reported that  all 6 cases with autopsies between 5 to 30 hours
after death showed TUNEL-positive cells in the infarct while none of 6 cases
who had autopsies 24 to 63 after death showed TUNEL-positive cells [21].Monoclonal antibody to
single-stranded DNA (ssDNA) is more
specific and sensitive cellular marker of apoptosis than TUNEL. Akasaka et al.
studied 43 persons with acute MI at autopsy and related the detection of ssDNA to histologic estimation of the stage of MI
from earliest, to coagulation necrosis, macrophage infiltration,
granulation tissue, and scar tissue [31]. This study is difficult to
relate to the others because it reported more stages of MI than hearts
examined. The maximum prevalence of apoptosis was only 0.25% [31]. As the time from death to
autopsy is uncertain in that study, it is possible that this variable
influenced their results.  It is difficult
to reconcile this assessment of apoptosis in MI with the other studies of
apoptosis in the heart (Table 3).


## 3.2. DNA Fragmentation on Agarose Gel Electrophoresis

DNA fragmentation into fragments of 180 to 200 base pairs detected on agarose gel
electrophoresis is the hallmark of apoptosis because of the activation of
specific endonucleases during on the apoptosis process [1]. Itoh et al. were the first
to report  DNA fragmentation in the infarcted myocardium but not in normal myocardium [11]. It has been examined in
seven studies and the characteristic DNA fragmentation has been consistently  demonstrated (Table 2) in the myocardium of
patents following MI [11, 12, 14, 18, 19, 21, 34].
In contrast, there is no DNA fragmentation in hearts from individuals who did not die of MI or in normal
myocardium remote from the MI [12, 14, 19]. The DNA ladder pattern is found in cases of MI less than 72 hours old and more often
with high prevalence of apoptotic DNA [19]. DNA fragmentation was seen in the early stages of AMI (12 to 24 hours) and was not seen in older infarcts [14]. With the caveat that most
studies evaluated only a few cases from their study population, the consistency
of the findings across all studies in which it was examined is noteworthy.


## 3.3. Caspase-3

Caspases are a family of proteases
that cleave cellular polypeptides with the subsequent production of the
majority of cellular and morphological events that occur during apoptotic cell
death [3]. Caspase activation is unique
to apoptosis as it does not occur in other forms of cell death and provides strong
evidence for the presence of apoptosis [35]. Caspase-3 activation has
been found in each of the 9 studies of acute MI in which it was examined [20, 22, 24–27, 30, 32, 33]. Immunohistochemical
examination of MI demonstrated caspase-3 activation in myocardial cells in 60%
of patients who died within 24 hours of the onset of MI, 30% of those dying 1
to 7 days after the infarct, and in none of the  cases dying 1 to 4 weeks after the onset of
infarction [32].Because caspase activation is a
relatively specific marker of apoptosis, investigators have used double staining
of cardiomyocytes with TUNEL plus caspase-3 as the criteria to indicate
apoptosis. Using this set of criteria, the apoptosis index varies from 5.9 to
25.4%. On average, 13.2% of cardiomyocytes in the heart of patients dying of
acute MI manifested apoptosis (Table3). The dual labeling (TUNEL plus
caspase-3) approach has been utilized mainly by a common group of
investigators. Unfortunately, most of the studies using the more specific TUNEL
plus caspase-3 criteria examined heart of patients who had died 20 days after
the onset of MI (Table 1). Considering that caspase-3 activation is maximum within
24 hours after acute MI and declines thereafter [32], the percentage of apoptotic
cells using this criteria is dependent of the time after acute MI that the
heart was examined.Caspase-3 activation was
significantly greater in patients who received reperfusion treatment compared
to those that did not receive this form of therapy [32]. The site of the infarct that
expresses the greatest amount of caspase-3 activation, that is, center or
border zone is unresolved between studies [22, 32].


## 3.4. Bax and Bcl-2 Expression

The Bcl-2 family of proteins regulate the various protein-protein
and protein-membrane interactions affecting the permeability of the outer
mitochondrial membrane which maybe viewed as the point of no return in apoptotic
cell deaths [36]. Two important Bcl-2 signaling
molecules in apoptosis related to the intrinsic or mitochondrial pathway in
apoptosis are Bax and bcl-2 [36] both of which regulate
caspase activation.Myocytes with positive bcl-2
immunoreactivity were seen in 60% of hearts with acute MI while there were no
myocytes with positive bcl-2 immunoreactivity in the controls or in hearts with
old MI [37]. There is a predominance of bcl-2
localized to the border areas surrounding the infarcted tissues [37].  Piro et al. did not find immunoreactivity for
bcl-2 but attributed this failure to the autolysis process as most of their
specimens for analysis of bcl-2 were taken 24 hours after death [18]. Bax expression is increased
in acute MI. Bax immuno-reactivity is evident in 52% of myocytes at the site of
the infarction which is significantly greater than in areas remote from the MI [22]; Bax protein expression
colocalizes with TUNEL [22]. Myocardial Bax expression
was increased in patients with multivessel disease [27].


## 3.5. Demonstration of Apoptosis in Patients with MI by Cardiac Imaging

The studies reviewed above have been mainly autopsy
studies with the attendant concerns about postmortem changes in tissue and
technical issues surrounding TUNEL or caspase-3 immunohistochemistry. Annexin V
is a naturally occurring human protein that binds avidly to membrane-associated
phosphatidylserine  which is actively
transported to the outer layer as an early event in apoptosis, and indicated
the presence of apoptosis in isolated cardiomyocytes
[38]. In six of seven patients,
increased uptake of Tc-m99-labelled annexin-V was seen in the
infarct area of the heart on nuclear (SPECT) imaging [39]. No increased uptake was seen in the
heart outside the infarct area. All patients with increased Tc-m99-labelled
annexin-V uptake in the infarct area showed a matching perfusion defect [39]. In a control individual, no
increased uptake in the heart was seen [39]. In another series of 9 patients, all
patients showed accumulation of Tc-m99-labelled annexin A5 at the
infarct site [40]. These data suggest that 94% of patients with acute MI manifest
apoptosis [39, 40]. The major critique of this
methodology is the inability to distinguish between apoptosis in cardiomyocytes
from apoptosis in the other cell types in the heart, and annexin V labeling of platelets
in the coronary thrombus producing the infarct [41, 42]. Although labeling of
platelets may not be that relevant of an issue [43], the proportion of the total
labeling that can be confirmed to be only due to myocytes remains a challenge.
This technique, however, has validity [44] and provides supporting
evidence for the existence of apoptosis in the process of acute MI.


## 3.6. Relationship of Apoptosis to Reperfusion Injury or Heart Failure

While experimental data support a relationship between
apoptosis and reperfusion injury, it is difficult to obtain this kind of data in
patients after acute MI in large part because autopsy studies do not come from
randomized trials comparing reperfusion and control therapy. However, apoptosis
was associated with histologic features of myocardium that is reperfused after
brief ischemia whereas necrosis occurs more often in areas of prolonged
ischemia without reperfusion [14]. DNA fragmentation (ISEL) was visible in recent infarcts  primarily in myocytes containing contraction
bands, which occur predominantly in regions of reperfused myocardium [14]. The myocardium supplied by
an occluded infarct-related artery (IRA) was associated with significantly
higher amount of apoptosis compared to the myocardium supplied by a patent IRA [30, 45]. The investigators suggested
that the benefits observed with a patent IRA may in part be due to reduced
myocardial apoptosis [30, 45].The amount of apoptosis present in the heart at autopsy is
a significant indicator of the presence of heart failure. Considering that heart
failure was a complication of MI that carried a high mortality and acts as a major indicator of the
amount of myocardium lost during the infarct, individuals who experienced early
occurrence of heart failure had a four-fold increase in the
prevalence of apoptosis compared to individuals without heart failure [26]. Multivariate analysis
including variables such as the amount of apoptosis at autopsy, the use of fibrinolytic
agents, as well as the
history of a previous remote MI showed that only heart failure
remained significantly associated with increased apoptosis [26]. Individuals with poor
prognosis at the onset of acute MI, assessed by the Norris coronary prognostic
index (age, presence of pulmonary congestion, heart size, and history of
previous MI), had a higher prevalence or greater proportion of apoptosis both in
the infarct as well as the area remote from the infarct compared to low-risk
individuals [25]. Patients with biventricular
enlargement had extremely high cardiomyocyte apoptosis at necropsy compared to
individuals who had no cardiac dilatation after MI  [24]. Patients with severe postinfarction
LV diastolic dysfunction had significantly higher rates of apoptosis,
determined by colocalization of TUNEL and
caspase-3 [33]. Thus, the amount of
apoptosis is an important determinant of the amount of left ventricular
dysfunction which in turn is a major determinant of an unfavorable outcome.


## 3.7. Potential Relevance of Apoptosis in the Border Zone of Acute MI

Several studies have shown that apoptosis is greater in
the border zone of MI or the area at the border of the infarct compared to
normal myocardium [31, 34]. The number of apoptotic
nuclei is consistently greater in the peri-infarcted region than in that away
from infarction [12].
Potentially fatal arrhythmias such as ventricular tachycardia can originate in pathways
in the heart at the border zone of an acute MI [46, 47] perhaps due to the mixture of
living and death cells  at the border
zone [12, 31]. Thus, apoptosis may be an
important component in the generation of potentially fatal cardiac arrhythmias.


## 4. Discussion

The major contribution of this paper
is the synthesis of data involving over 400 MI patients whose myocardium was
examined for the presence of apoptosis. The demonstration of DNA fragmentation, caspase-3 activation, Bax
expression, and annexin V binding provides overwhelming support for the
contention that apoptotic cell death is a component of myocardial infarction in
acute MI patients. The major question is whether the extent of apoptosis in
acute MI was evaluated. The differences in the technique that is used to quantitate
apoptosis makes it difficult to compare the studies but it is estimated that the
amount of apoptosis in the initial (24 hours) of acute MI may be approximately 60%
of cases based on myocytes that demonstrate the presence of Bax, Bcl-2, or
caspase-3 activation [22, 32, 37]. After 20 days, 12% of
myocytes show evidence of DNA fragmentation (TUNEL) and caspase-3 activation in the absence of signs of DNA repair [20, 22, 24–27, 30, 32, 33].Most of the studies have relied,
all or in part, on TUNEL staining of nuclei to conclude that apoptosis was
evident in MI.  A number of the earlier studies
presented dichotomous results namely that TUNEL-positive cells were either
present or not present. The existence of apoptotic nuclei suggested that apoptosis
was present in all cells in acute MI and clashed with prevailing thinking of
the dominance of oncosis as the main form of cell death in acute MI [2]. Criticism of the TUNEL methodology,
upon which opinions of the prevalence of apoptosis in MI were based, was
powerful. Some investigators contend that there is no direct evidence of
apoptosis in the heart and rather “all of the evidence for apoptosis in
cardiomyocytes is indirect, based on detection of DNA fragmentation and/or so-called apoptosis-related factors” [48]. This contention is based on
the following arguments: (i) electron microscopy is the gold standard for the
diagnosis of apoptosis and no human studies have utilized this methodology to
establish apoptosis in acute MI [48], (ii) TUNEL methodology does
not have a high specificity for apoptosis because it detects both apoptotic as
well as oncotic cell death [49], (iii) TUNEL can also
indicate DNA activity associated
with DNA repair or increased
proteins synthesis and not only apoptosis [50, 51], (iv) DNA fragmentation in the nucleus is not necessarily related to apoptotic morphology
in the nucleus or other indicators of apoptosis such as caspase activation [49, 52], (iv) DNA ladders, as performed, are nonspecific for cardiomyocytes as they are from
extracts of heart that may also contain the nonmyocytes elements of the heart [48]. Another criticism of the occurrence
of apoptosis in the heart questions whether cells initially undergoing
apoptosis eventually die by oncotic mechanisms so that apoptosis is not a major
concern for the heart. Stated in another way, the triggers for both kinds of
cell death can be one and the same, and the cell may switch from one type of
cell death (apoptosis)  to the other (oncosis)
mode of cell death [53]. Secondly, DNA fragmentation as indicated by TUNEL or caspase
activation does not inevitably mean cell death [54]. These criticisms can be
examined by applying the patient data examined in this study.


### 4.1. Analysis and Rebuttal to the Critique of Apoptosis in Acute Myocardial Infarction

TUNEL Methodology Does Not Have a High Specificity for Apoptosis Because It Detects both Apoptotic As Well As Oncotic Cell Death
This criticism was initially
advanced by Ohno et al. based
on the poor correlation of electron microscopic (EM) evaluation for signs of
apoptosis and TUNEL staining of cardiomyocytes in rabbit heart subjected to 30
minutes of ischemia followed by reperfusion [49]. There are two camps on this
issue. Takemura and Fujiwara contend that apoptosis can only be conclusively
identified by EM which does not identify enough of the characteristic
morphologic features to substantiate the existence of apoptosis in
cardiomyocytes [48].  In contrast, the other position has been well
formulated by Anversa who contend that EM evaluates too small a sample of the
myocardium or cardiac nucleus to be able to identify cardiac apoptosis so that
immunohistochemical techniques are preferred [55]. Nevertheless, there are EM data
to substantiate that apoptosis does exist in human heart [56]. Studies in human heart with
classic indicator of apoptosis, namely, caspase-3 activation found a high
correlation of TUNEL-positive cells with caspase-3, as 85% of TUNEL-positive
cells also manifest caspase-3 activation [22]. Considering that the
processes of DNA fragmentation and
caspase-3 activation should not necessarily be expected to occur simultaneously
as the process of apoptosis takes time to complete, a 100% correlation should
not be anticipated. Furthermore, the classic marker of apoptosis, DNA ladder pattern in cases of MI, correlated strongly
with a high prevalence of TUNEL-stained nuclei in cardiac myocytes [19]. The high correlation between
TUNEL and caspase-3 activation and DNA laddering assures that TUNEL is at least 85% specific for the presence of
apoptosis in the heart and supports
the contention that TUNEL-positive cells are primarily apoptotic cells.TUNEL Can Also Indicate DNA Activity Associated with DNA Repair or Increased Protein Synthesis and Not Only Apoptosis
False-positive TUNEL staining
may be more of a problem in heart failure or cardiomyopathy [51]. In patients with MI,
however, TUNEL-positive-stained cardiomyocytes are overwhelmingly negative for
immunohistochemical evidence of DNA repair. Specifically, none of the myocytes (0%) which coexpressed TUNEL and
caspase-3 were positive for PCNA or SC35 which, respectively, indicates an increased DNA and RNA synthesis [22]. Thus, during the early part
of the time frame and sequence of cellular events that occur during an acute MI,
TUNEL-positive cells do not represent cells undergoing DNA repair or increased protein synthesis.DNA Fragmentation on Agarose Gel Electrophoresis Is Not Specific for Cardiomyocytes
Although DNA fragmentation of agarose gel electrophoresis is the classic hallmark of
apoptosis, criticism of this data centers on the uncertainty of how much
cardiomyocytes rather than the other elements of the heart contributed to the
generation of the DNA fragmentation and the inability of this technology to provide data on what percentage
of cardiomyocytes underwent apoptosis. However, DNA fragmentation on agarose gel was observed mainly in the areas of the myocardial
infarction that had the majority of apoptotic cells identified by intense ISEL-
or TUNEL-stained nuclei of cardiomyocytes [34]. Similarly, there was no
relationship between areas of the myocardium that manifested DNA fragmentation and the distribution of ISEL-positive
nonmyocyte or inflammatory cells [34].TUNEL-Positive Cells May Include Apoptosis in Other Elements of the Heart and in Inflammatory Cells
Elements of the heart other than
cardiomyocytes undergo apoptosis after acute MI. Inflammatory cells that invade
the infarct also undergo apoptosis. This event is a later stage in acute MI
while the greatest amount of apoptosis occurs before the inflammatory response [14]. While these non-cardiomyocytes
can stain TUNEL, autopsy studies comment specifically on their evaluation only of
the myocyte element of the heart either directly or using special stains for
myocytes [14, 22, 25, 27, 45]. Thus, there is only a low
probability that TUNEL-positive cells represent apoptosis in non-cardiomyocyte
elements of the heart.Cardiomyocytes Undergoing Apoptosis Eventually Die by Oncotic Mechanisms So That Apoptosis Is Not a Major Concern for the Heart
Currently available data shows  a strong relationship
between evidence for apoptosis and the severity of the myocardial infarction thus
providing a compelling argument that apoptosis is an important process in the
heart during acute MI. The amount of apoptosis present in the heart at autopsy is
a significant predictor of the presence of heart failure—a complication of
myocardial infarction that carried a high mortality. Individuals who
experienced early occurrence of clinical manifestations of heart failure had a four-fold greater prevalence of apoptosis compared to individuals who
did not have heart failure [25, 26]. Patients with biventricular
enlargement had extremely high rates of cardiomyocyte apoptosis, compared to
individuals who had no cardiac dilatation after MI [24]. Patients who died after an
acute MI with severe postinfarction left ventricular (LV) diastolic dysfunction
had significantly higher rates of apoptosis than those without LV dysfunction [33]. Thus, the amount of
apoptosis is an important determinant of the amount of LV dysfunction which in
turn is a major determinant of an unfavorable outcome pertaining to patient
morbidity and mortality.DNA Fragmentation As Indicated by TUNEL or Caspase Activation Does Not Inevitably Mean Cell Death
Clinical trial data on the effect of interruption of
apoptosis on clinical outcomes in patients after acute MI would address this
issue but are absent. This necessitates the reliance on experimental data. These
studies can be grouped into two kinds. Those studies using genetic overexpression
or knockout models have been uniformly supportive of a role for apoptosis in acute
MI while studies with synthetic caspase inhibitors have been controversial. Caspase-3 transgenic mice
that had cardiac tissue-specific overexpression of the proapoptotic gene caspase-3 manifested
increased infarct size and a pronounced susceptibility to die after
experimentally induced MI [57]. Adenoviral gene transfer of
the caspase inhibitor p35 leads to a significant reduction of myocardial
infarct size and improved cardiac function after induction of acute MI in the
rat [58]. Caspase-1-knockout (KO)
mice, lacking caspase-1 exhibited both improved peri-infarct survival and a
decreased rate of ventricular dilatation, possibly due in part to a reduction
in the rate of apoptosis after experimental MI [59]. Reduction in bax levels in
the bax knock out mouse was associated with reduced infarct size and improved
long-term function after MI [60].Synthetic caspase inhibitors have the risk of inherent
drug toxicity as well as their potential beneficial effect from caspase inhibition.
Several caspase inhibitors protected the heart against lethal reperfusion
injury [61–64] while others do not [65–68]. This is more difficult to
extrapolate from data on caspase inhibitors because the details of their
systemic adverse effects may vary between the different caspase inhibitors.Interestingly, some drug therapy
that improves prognosis after myocardial infarction such as ACE inhibitors, beta-adrenergic receptor antagonists, and aldosternone antagonists have antiapoptotic
properties [69–71]. Some novel therapies such as
the interleukin-1 receptor antagonist anakinra, which inhibits caspase-1 and -9
activities, limits cardiomyocyte apoptosis and ameliorates the remodeling after
experimentally induced acute MI [72]. These comments are not meant
to provide a detailed review of strategies to prevent cardiac apoptosis. Rather
data on caspase inhibition in acute MI suggest that if the apoptosis process is
not antagonized, then cell death is more likely to occur or that accentuation
of apoptosis leads to enhanced cardiac cell death. These findings refute the
criticism that caspase activation is of no consequence in myocardial
infarction.


### 4.2. Summary

The strength of the overwhelming evidence supports the contention that
apoptosis occurs in cardiomyocytes in patients following acute MI. This is
based on immunohistochemical staining of myocytes showing DNA fragmentation, caspase-3 activation, as well as
bcl-2 and Bax overexpression: the presence of the characteristic DNA fragmentation on agarose gel electrophoresis;
and  is
nuclear imaging using radio-labeled annexin V. Some authors
contend that TUNEL methodology is not accurate or specific for the recognition
of cardiac apoptosis. Review of this contention suggests that TUNEL may have
been excessively criticized and its role in detection of apoptosis has been too
readily dismissed. While each methodology has its flaws, they are all
consistent in their demonstration of the presence of apoptosis, and taken
together, the data offer a strong and compelling argument in favor of this
hypothesis.The principle question that exists is the extent of apoptosis
in acute MI. The studies that were reviewed do not provide a clear answer for
several reasons, the predominant reason is that present study designs and methodology
do not allow for serial follow-up, in the human heart, starting with the
activation of apoptotic pathways in individual cardiomyocytes as well as in the
determination of whether their eventual death is exclusively due to apoptosis. Nuclear
imaging studies in patients with an antibody specific for caspase activation or
annexin V, coupled with a probe to ensure the radiolabel is targeted to
cardiomyocytes, may prove to be the best method to identify apoptosis in
patients to obviate the need to obtain myocardial tissue. Cleary, autopsy
studies and sectioning of the heart at the time of death provide a picture at a
single time point, and the numbers of myocytes that have already died due to
apoptosis are not included in the evaluation. The prevalence of apoptosis may be
as high as 60% in the first day after acute MI and approximately 12% three
weeks later. The above is a disturbingly high number because the dynamic nature
of acute MI means the maximum cell death occurs in the early stages of MI and
the major mode of cell death may actually be apoptosis.Thus, this potentially high prevalence of apoptotic cell
death appearing to be unreasonably high has been has been discussed by Elsässer et al.[73]. In 2000, Elsässer et al.
drew attention to the unresolved issues in the role of apoptosis in myocardial
ischemia including some that are clinically relevant  today, namely, the need to determine the reason cells, under
ischemic conditions, die by either apoptosis or oncosis and the need to
determine the reversibility of apoptosis [73]. The association of high
amounts of apoptosis in myocytes in the heart in heart failure or in areas of
the heart that can generate potentially fatal arrhythmias underscores the
importance of apoptosis in MI.Clinical trials are needed in patients with acute MI to
determine the extent to which apoptosis is reversible or preventable in
patients with acute MI. The latter may lead to considerable changes to
treatment strategies that are considered best practices for MI.
Considering that our present treatment strategies still allow the
occurrence of a high morbidity and mortality from acute MI; these clinical
trials are long overdue.


## 4.1. Analysis and Rebuttal to the Critique of Apoptosis in Acute Myocardial Infarction

TUNEL Methodology Does Not Have a High Specificity for Apoptosis Because It Detects both Apoptotic As Well As Oncotic Cell Death
This criticism was initially
advanced by Ohno et al. based
on the poor correlation of electron microscopic (EM) evaluation for signs of
apoptosis and TUNEL staining of cardiomyocytes in rabbit heart subjected to 30
minutes of ischemia followed by reperfusion [49]. There are two camps on this
issue. Takemura and Fujiwara contend that apoptosis can only be conclusively
identified by EM which does not identify enough of the characteristic
morphologic features to substantiate the existence of apoptosis in
cardiomyocytes [48].  In contrast, the other position has been well
formulated by Anversa who contend that EM evaluates too small a sample of the
myocardium or cardiac nucleus to be able to identify cardiac apoptosis so that
immunohistochemical techniques are preferred [55]. Nevertheless, there are EM data
to substantiate that apoptosis does exist in human heart [56]. Studies in human heart with
classic indicator of apoptosis, namely, caspase-3 activation found a high
correlation of TUNEL-positive cells with caspase-3, as 85% of TUNEL-positive
cells also manifest caspase-3 activation [22]. Considering that the
processes of DNA fragmentation and
caspase-3 activation should not necessarily be expected to occur simultaneously
as the process of apoptosis takes time to complete, a 100% correlation should
not be anticipated. Furthermore, the classic marker of apoptosis, DNA ladder pattern in cases of MI, correlated strongly
with a high prevalence of TUNEL-stained nuclei in cardiac myocytes [19]. The high correlation between
TUNEL and caspase-3 activation and DNA laddering assures that TUNEL is at least 85% specific for the presence of
apoptosis in the heart and supports
the contention that TUNEL-positive cells are primarily apoptotic cells.TUNEL Can Also Indicate DNA Activity Associated with DNA Repair or Increased Protein Synthesis and Not Only Apoptosis
False-positive TUNEL staining
may be more of a problem in heart failure or cardiomyopathy [51]. In patients with MI,
however, TUNEL-positive-stained cardiomyocytes are overwhelmingly negative for
immunohistochemical evidence of DNA repair. Specifically, none of the myocytes (0%) which coexpressed TUNEL and
caspase-3 were positive for PCNA or SC35 which, respectively, indicates an increased DNA and RNA synthesis [22]. Thus, during the early part
of the time frame and sequence of cellular events that occur during an acute MI,
TUNEL-positive cells do not represent cells undergoing DNA repair or increased protein synthesis.DNA Fragmentation on Agarose Gel Electrophoresis Is Not Specific for Cardiomyocytes
Although DNA fragmentation of agarose gel electrophoresis is the classic hallmark of
apoptosis, criticism of this data centers on the uncertainty of how much
cardiomyocytes rather than the other elements of the heart contributed to the
generation of the DNA fragmentation and the inability of this technology to provide data on what percentage
of cardiomyocytes underwent apoptosis. However, DNA fragmentation on agarose gel was observed mainly in the areas of the myocardial
infarction that had the majority of apoptotic cells identified by intense ISEL-
or TUNEL-stained nuclei of cardiomyocytes [34]. Similarly, there was no
relationship between areas of the myocardium that manifested DNA fragmentation and the distribution of ISEL-positive
nonmyocyte or inflammatory cells [34].TUNEL-Positive Cells May Include Apoptosis in Other Elements of the Heart and in Inflammatory Cells
Elements of the heart other than
cardiomyocytes undergo apoptosis after acute MI. Inflammatory cells that invade
the infarct also undergo apoptosis. This event is a later stage in acute MI
while the greatest amount of apoptosis occurs before the inflammatory response [14]. While these non-cardiomyocytes
can stain TUNEL, autopsy studies comment specifically on their evaluation only of
the myocyte element of the heart either directly or using special stains for
myocytes [14, 22, 25, 27, 45]. Thus, there is only a low
probability that TUNEL-positive cells represent apoptosis in non-cardiomyocyte
elements of the heart.Cardiomyocytes Undergoing Apoptosis Eventually Die by Oncotic Mechanisms So That Apoptosis Is Not a Major Concern for the Heart
Currently available data shows  a strong relationship
between evidence for apoptosis and the severity of the myocardial infarction thus
providing a compelling argument that apoptosis is an important process in the
heart during acute MI. The amount of apoptosis present in the heart at autopsy is
a significant predictor of the presence of heart failure—a complication of
myocardial infarction that carried a high mortality. Individuals who
experienced early occurrence of clinical manifestations of heart failure had a four-fold greater prevalence of apoptosis compared to individuals who
did not have heart failure [25, 26]. Patients with biventricular
enlargement had extremely high rates of cardiomyocyte apoptosis, compared to
individuals who had no cardiac dilatation after MI [24]. Patients who died after an
acute MI with severe postinfarction left ventricular (LV) diastolic dysfunction
had significantly higher rates of apoptosis than those without LV dysfunction [33]. Thus, the amount of
apoptosis is an important determinant of the amount of LV dysfunction which in
turn is a major determinant of an unfavorable outcome pertaining to patient
morbidity and mortality.DNA Fragmentation As Indicated by TUNEL or Caspase Activation Does Not Inevitably Mean Cell Death
Clinical trial data on the effect of interruption of
apoptosis on clinical outcomes in patients after acute MI would address this
issue but are absent. This necessitates the reliance on experimental data. These
studies can be grouped into two kinds. Those studies using genetic overexpression
or knockout models have been uniformly supportive of a role for apoptosis in acute
MI while studies with synthetic caspase inhibitors have been controversial. Caspase-3 transgenic mice
that had cardiac tissue-specific overexpression of the proapoptotic gene caspase-3 manifested
increased infarct size and a pronounced susceptibility to die after
experimentally induced MI [57]. Adenoviral gene transfer of
the caspase inhibitor p35 leads to a significant reduction of myocardial
infarct size and improved cardiac function after induction of acute MI in the
rat [58]. Caspase-1-knockout (KO)
mice, lacking caspase-1 exhibited both improved peri-infarct survival and a
decreased rate of ventricular dilatation, possibly due in part to a reduction
in the rate of apoptosis after experimental MI [59]. Reduction in bax levels in
the bax knock out mouse was associated with reduced infarct size and improved
long-term function after MI [60].Synthetic caspase inhibitors have the risk of inherent
drug toxicity as well as their potential beneficial effect from caspase inhibition.
Several caspase inhibitors protected the heart against lethal reperfusion
injury [61–64] while others do not [65–68]. This is more difficult to
extrapolate from data on caspase inhibitors because the details of their
systemic adverse effects may vary between the different caspase inhibitors.Interestingly, some drug therapy
that improves prognosis after myocardial infarction such as ACE inhibitors, beta-adrenergic receptor antagonists, and aldosternone antagonists have antiapoptotic
properties [69–71]. Some novel therapies such as
the interleukin-1 receptor antagonist anakinra, which inhibits caspase-1 and -9
activities, limits cardiomyocyte apoptosis and ameliorates the remodeling after
experimentally induced acute MI [72]. These comments are not meant
to provide a detailed review of strategies to prevent cardiac apoptosis. Rather
data on caspase inhibition in acute MI suggest that if the apoptosis process is
not antagonized, then cell death is more likely to occur or that accentuation
of apoptosis leads to enhanced cardiac cell death. These findings refute the
criticism that caspase activation is of no consequence in myocardial
infarction.


## 4.2. Summary

The strength of the overwhelming evidence supports the contention that
apoptosis occurs in cardiomyocytes in patients following acute MI. This is
based on immunohistochemical staining of myocytes showing DNA fragmentation, caspase-3 activation, as well as
bcl-2 and Bax overexpression: the presence of the characteristic DNA fragmentation on agarose gel electrophoresis;
and  is
nuclear imaging using radio-labeled annexin V. Some authors
contend that TUNEL methodology is not accurate or specific for the recognition
of cardiac apoptosis. Review of this contention suggests that TUNEL may have
been excessively criticized and its role in detection of apoptosis has been too
readily dismissed. While each methodology has its flaws, they are all
consistent in their demonstration of the presence of apoptosis, and taken
together, the data offer a strong and compelling argument in favor of this
hypothesis.The principle question that exists is the extent of apoptosis
in acute MI. The studies that were reviewed do not provide a clear answer for
several reasons, the predominant reason is that present study designs and methodology
do not allow for serial follow-up, in the human heart, starting with the
activation of apoptotic pathways in individual cardiomyocytes as well as in the
determination of whether their eventual death is exclusively due to apoptosis. Nuclear
imaging studies in patients with an antibody specific for caspase activation or
annexin V, coupled with a probe to ensure the radiolabel is targeted to
cardiomyocytes, may prove to be the best method to identify apoptosis in
patients to obviate the need to obtain myocardial tissue. Cleary, autopsy
studies and sectioning of the heart at the time of death provide a picture at a
single time point, and the numbers of myocytes that have already died due to
apoptosis are not included in the evaluation. The prevalence of apoptosis may be
as high as 60% in the first day after acute MI and approximately 12% three
weeks later. The above is a disturbingly high number because the dynamic nature
of acute MI means the maximum cell death occurs in the early stages of MI and
the major mode of cell death may actually be apoptosis.Thus, this potentially high prevalence of apoptotic cell
death appearing to be unreasonably high has been has been discussed by Elsässer et al.[73]. In 2000, Elsässer et al.
drew attention to the unresolved issues in the role of apoptosis in myocardial
ischemia including some that are clinically relevant  today, namely, the need to determine the reason cells, under
ischemic conditions, die by either apoptosis or oncosis and the need to
determine the reversibility of apoptosis [73]. The association of high
amounts of apoptosis in myocytes in the heart in heart failure or in areas of
the heart that can generate potentially fatal arrhythmias underscores the
importance of apoptosis in MI.Clinical trials are needed in patients with acute MI to
determine the extent to which apoptosis is reversible or preventable in
patients with acute MI. The latter may lead to considerable changes to
treatment strategies that are considered best practices for MI.
Considering that our present treatment strategies still allow the
occurrence of a high morbidity and mortality from acute MI; these clinical
trials are long overdue.



---

*Source: 979318-2009-03-25.xml*