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.
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 [
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.
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
(Table
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.)
Author | Year | Number of MI | Time after onset MI | Time death to autopsy |
---|---|---|---|---|
Itoh et
al. [ | 1995 | 19 | “12 hours to several days” | Uncertain |
Olivetti et
al. [ | 1996 | 20 | ~4 ( | |
Bardales et al. [ | 1996 | 32 | Uncertain | Uncertain |
Veinot et al. [ | 1997 | 8 | Uncertain | |
Saraste [ | 1997 | 8 | 38 (6 to 120) hours | 17.3 (4 to 48) hours |
Toyoda et
al. [ | 1998 | 1 | 7 days | 0 |
James [ | 1999 | 12 | Uncertain | Uncertain |
Ottaviani et al. [ | 1999 | 10 | ||
Piro et
al. [ | 2000 | 16 | 0.4 days ( | Uncertain |
Rodríguez-Calvo et al. [ | 2001 | 14 | 2.9 (0.5 to 11) days | Uncertain |
Abbate et
al. [ | 2002 | 24 | ~27 (15 to 40) days | Uncertain |
Nakatome et
al. [ | 2002 | 6 | ||
Nakatome et
al. [ | 2002 | 6 | 0.5 to 5 hours | 24 to 63 hours |
Baldi et al. [ | 2002 | 16 | 23 (12–62) days | |
Edston et al. [ | 2002 | 10 | Uncertain (Sudden deaths) | 3.6 days |
Bussani et
al. [ | 2003 | 12 | ~20 (14 to 44) days | Uncertain |
Abbate et al. [ | 2003 | 30 | 23 (14 to 40) days | Uncertain |
Abbate et
al. [ | 2003 | 14 | ~16 (12 to 34) days | Uncertain |
Biondi-Zoccai et al. [ | 2004 | 21 | ~25 (11–80) days | |
Biondi-Zoccai et al. [ | 2005 | 21 | ~22 (12–50) days | Uncertain |
Abbate et
al. [ | 2005 | 30 | 17 (9 to 60) days | |
Akasaka et
al. [ | 2005 | 43 | Uncertain | |
Zidar et
al. [ | 2006 | 30 | Uncertain | |
Zidar et
al. [ | 2006 | 20 | >24 hours | Uncertain |
Sinagra et al. [ | 2007 | 14 | 18.8 (10 to 62) days | Uncertain |
Across 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 [
Different methodologies were utilized
to assess the heart for the presence of apoptosis (Table
Methodologies used to determine the presence of apoptosis in the heart.
Author | Year | Number MI | DNA gel electrophoresis | ISEL or TUNEL | Caspase-3 | ssDNA | Bax |
---|---|---|---|---|---|---|---|
Itoh et
al. [ | 1995 | 19 | Yes | Yes | No | ||
Olivetti et
al. [ | 1996 | 20 | Yes | Yes | No | ||
Bardales
et al. [ | 1996 | 32 | No | Yes | No | ||
Veinot et
al. [ | 1997 | 8 | Yes | Yes | No | ||
Saraste et
al. [ | 1997 | 8 | Yes | Yes | No | ||
Toyoda et
al. [ | 1998 | 1 | No | Yes | No | ||
James [ | 1998 | 12 | No | Yes | No | ||
Ottaviani et al. [ | 1999 | 10 | No | Yes | No | ||
Piro et
al. [ | 2000 | 16 | Yes | Yes | No | ||
Rodríguez-Calvo
et al. [ | 2001 | 14 | Yes | Yes | No | ||
Abbate et
al. [ | 2002 | 24 | No | Yes | Yes | ||
Nakatome et
al. [ | 2002 | 12 | Yes | Yes | No | ||
Baldi et
al. [ | 2002 | 16 | No | Yes | Yes | Yes | |
Edston et
al. [ | 2002 | 10 | No | Yes | No | ||
Abbate et al. [ | 2003 | 30 | No | Yes | Yes | ||
Bussani et
al. [ | 2003 | 12 | No | Yes | Yes | ||
Abbate et
al. [ | 2003 | 14 | No | Yes | Yes | ||
Biondi-Zoccai et al. [ | 2004 | 21 | No | Yes | Yes | Yes | |
Biondi-Zoccai et al. [ | 2005 | 21 | No | Yes | Yes | Yes | |
Abbate et
al. [ | 2005 | 30 | No | Yes | Yes | ||
Akasaka et
al. [ | 2005 | 43 | No | No | No | Yes | |
Zidar et
al. [ | 2006 | 50 | No | No | Yes | ||
Sinagra et
al. [ | 2007 | 14 | No | Yes | Yes |
Apoptosis
was originally defined based on distinct morphological features due to internucleosomal
DNA degradation resulting from
activation of specific endonucleases or DNAses [
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 [
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.)
TUNEL | TUNEL + Caspase | TUNEL ± Caspase | Casp-3 | ||||||
---|---|---|---|---|---|---|---|---|---|
MI (%) | MI (%) | Remote (%) | % Hearts | Control (%) | Control | Remote | |||
Olivetti et al. [ | 20 | 12 B | 0.74 | ||||||
Bardales et
al. [ | 32 | (All) | 8 | 13 | |||||
Saraste et
al. [ | 8 | 0.8 B | 0.005 | 0.007 | 6 | ||||
Veinot et
al. [ | 8 | (All) | 0 | 3 | |||||
James
[ | 12 | (All) | |||||||
Ottaviani et al. [ | 10 | A | A | 3 | |||||
Piro et
al. [ | 16 | 43.6 | 38.1 | 0 | 0 | Not done | ns | ||
Rodríguez-Calvo
et al. [ | 14 | 36 | 0 | 5 | |||||
Nakatome et
al. [ | 6 | (All) | 0 | ||||||
Abbate et
al. [ | 24 | 19.5 | 0.5 | ||||||
Baldi et
al. [ | 16 | 25.4 | 0.7 | ||||||
Edston
et al. [ | 10 | 26 | 0 | 8 | |||||
Abbate et
al. [ | 30 | 11 | 4 | ||||||
Abbate et
al. [ | 14 | 19 | |||||||
Bussani
et al. [ | 12 | 16.7 | |||||||
Biondi-Zoccai et al. [ | 21 | 0.71 | 0.01 | 4 | |||||
Biondi-Zoccai et al. [ | 21 | 8.1 | 0.01 | 4 | |||||
Abbate et
al. [ | 30 | 6.9 | 0.9 | 0.01 | 5 | ||||
Zidar et
al. [ | 30 | (60) | 0 | 5 | |||||
Zidar et
al. [ | 20 | (15) | 0 | 5 | |||||
Sinagra et
al. [ | 14 | 5.9 |
A: data expressed as arcosine
All: present in all hearts and there is no quantitative data
B: 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 [
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 [
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 [
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 [
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 [
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 (Table
Caspase-3 activation was
significantly greater in patients who received reperfusion treatment compared
to those that did not receive this form of therapy [
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 [
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 [
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
[
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 [
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 [
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 [
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 [
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 [
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 [
False-positive TUNEL staining
may be more of a problem in heart failure or cardiomyopathy [
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 [
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 [
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 [
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 [
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 [
Interestingly, some drug therapy
that improves prognosis after myocardial infarction such as ACE inhibitors, beta-adrenergic receptor antagonists, and aldosternone antagonists have antiapoptotic
properties [
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.[
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.