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Content uploaded by Youngil Lee
Author content
All content in this area was uploaded by Youngil Lee on Jun 30, 2015
Content may be subject to copyright.
Content uploaded by Youngil Lee
Author content
All content in this area was uploaded by Youngil Lee on Jun 30, 2015
Content may be subject to copyright.
CELL DEATH AND DISEASE
Role of apoptosis in cardiovascular disease
Youngil Lee ÆA
˚sa B. Gustafsson
Published online: 14 January 2009
ÓSpringer Science+Business Media, LLC 2009
Abstract Apoptosis plays a key role in the pathogenesis
in a variety of cardiovascular diseases due to loss of ter-
minally differentiated cardiac myocytes. Cardiac myocytes
undergoing apoptosis have been identified in tissue samples
from patients suffering from myocardial infarction, dia-
betic cardiomyopathy, and end-stage congestive heart
failure. Apoptosis is a highly regulated program of cell
death and can be mediated by death receptors in the plasma
membrane, as well as the mitochondria and the endoplas-
mic reticulum. The cell death program is activated in
cardiac myocytes by various stressors including cytokines,
increased oxidative stress and DNA damage. Many studies
have demonstrated that inhibition of apoptosis is cardio-
protective and can prevent the development of heart
failure. This review provides a current overview of the
evidence of apoptosis in cardiovascular diseases and dis-
cusses the molecular pathways involved in cardiac
myocyte apoptosis.
Keywords Apoptosis Heart failure Death receptors
Mitochondria Bcl-2
Introduction
Cardiovascular disease is the leading cause of morbidity
and mortality in the developed world. A multitude of recent
studies suggest that loss of terminally differentiated cardiac
myocytes contributes to development of heart failure.
There are three morphologically and biochemically distinct
forms of cell death that occur in the heart; necrosis,
apoptosis, and possibly autophagy. Autophagy is a cellular
process that degrades long lived proteins and dysfunctional
organelles [1]. Autophagy is characterized by sequestration
of cytoplasm in double-membrane vesicles called auto-
phagosomes. Autophagosomes subsequently fuse with
lysosomes, leading to degradation of their content [1]. This
process is important for cellular homeostasis, and is a
survival response upregulated in response to stress or
starvation. However, excess autophagy can lead to cell
death due to excessive digestion of organelles and essential
proteins [2]. Necrosis is a passive form of cell death caused
by ATP depletion and rapid disruption of cell membrane
integrity resulting in spillage of intracellular contents into
interstitial and extracellular space, which initiates inflam-
mation and induces damage to neighboring cells [3,4]. In
contrast, apoptosis is an energy requiring form of pro-
grammed cell death whereby damaged cells are removed
without provoking inflammation. Apoptosis is character-
ized by chromatin condensation, DNA fragmentation,
plasma membrane blebbing (i.e., externalization of phos-
phatidylserine), and cell shrinkage due to reduction in
cytoplasm and organelles [5–7]. Finally, membrane-bound
apoptotic bodies containing cytosol and processed organ-
elles are formed and then removed by macrophages via
phagocytosis [8].
It is now evident that apoptosis plays a key role in the
pathogenesis of a variety of cardiovascular diseases.
Studies have reported that apoptosis occurs in myocardial
tissue samples from patients suffering from myocardial
infarction, dilated cardiomyopathy and end-stage heart
failure [9–12] as well as in animal models of ischemia–
reperfusion injury [13–15]. Apoptosis is activated in car-
diac myocytes by multiple stressors that are commonly
seen in cardiovascular disease such as cytokine production
Y. Lee A
˚. B. Gustafsson (&)
BioScience Center, San Diego State University, 5500 Campanile
Drive, San Diego, CA 92182-4650, USA
e-mail: agustafs@sciences.sdsu.edu
123
Apoptosis (2009) 14:536–548
DOI 10.1007/s10495-008-0302-x
[16,17], increased oxidative stress [18], and DNA damage
[19]. Apoptosis is a highly regulated program of cell death
and inhibition of this process is cardioprotective under
many conditions. Therefore, this process represents a
potential target for therapeutic intervention to prevent heart
failure. This review provides a current overview of the
evidence and functional role of apoptosis in cardiovascular
diseases and discusses the molecular pathways involved in
cardiac myocyte apoptosis.
Evidence of apoptosis in cardiovascular diseases
Cardiac cells undergoing apoptosis have been detected in
many different diseases of the cardiovascular system
including atherosclerosis, myocardial ischemia and reper-
fusion injury, diabetic cardiomyopathy, and chronic heart
failure. Atherosclerotic vascular disease is a leading cause
of myocardial infarction and heart failure, and a major
factor in the development of acute coronary syndrome is
disruption of an atherosclerotic plaque. It has been reported
that macrophages and smooth muscle cells undergo apop-
tosis in unstable atherosclerotic plaques which can lead to
rupture of the plaque and thrombosis [20]. Recently, Clarke
et al. [21] demonstrated that vascular smooth muscle cell
apoptosis during either atherogenesis or within established
plaques of apolipoprotein (Apo) E(-/-) mice accelerated
plaque growth and was associated with features of plaque
vulnerability such as a thin fibrous cap and loss of collagen
and matrix. In addition, it has been proposed that poor
clearance of apoptotic macrophages may lead to accumu-
lation of cellular debris within the lipid-rich core of
atherosclerotic plaque, thus contributing to plaque pro-
gression and rupture. Rupture of atherosclerotic plaques
and thrombus formation cause occlusion of coronary
arteries which reduces the blood supply to the myocardium
and leads to myocardial infarction and possibly death.
Reperfusion is the treatment for acute myocardial infarc-
tion, but the reintroduction of oxygen can aggravate the
tissue damage, a process called reperfusion injury [22].
Loss of cardiac cells in response to ischemia/reperfusion
(I/R) injury was long considered to be due to necrotic cell
death, but studies over the past decade have identified
apoptosis as a significant component of cell loss during
reperfusion after a myocardial infarction [9,10,14].
Apoptosis seems to occur primarily after reperfusion fol-
lowing ischemia, whereas prolonged ischemia leads to
necrosis. There is also mounting evidence that apoptosis
plays an important role in both acute and chronic loss of
cardiac myocytes after a myocardial infarction. Studies
have reported the presence of apoptotic cells in the border
zone of the infarct and in remote myocardium in the early
phase [9], as well as months after myocardial infarction
[23], suggesting that apoptosis plays a role in remodeling
and development of heart failure after myocardial infarc-
tion. Since the regenerative capacity of the myocardium is
limited, there is intense interest in the prevention of car-
diomyocyte loss during ischemia and reperfusion.
Moreover, diabetes is a rapidly growing epidemic in the
Western world which is well known to increase the risk of
developing cardiovascular disease [24]. Increased levels of
apoptosis have been detected in the hearts of diabetic
patients and animal models of diabetes, and the loss of
myocytes has been implicated in the development of dia-
betic cardiomyopathy [25,26]. In addition, it has been
reported that the mortality of myocardial infarction in dia-
betic patients is more than double that of non-diabetic
patients [27]. It appears that cardiac myocytes in the dia-
betic myocardium are more susceptible to apoptosis [25],
suggesting that the higher sensitivity to MI is due to a higher
loss of cardiac myocytes via apoptosis in response to the
stress [28]. Also, Kuethe et al. [29] found that diabetic
patients with dilated cardiomyopathy had significantly more
apoptotic cells in the heart compared to patients without
diabetes. The pathogenesis of diabetic cardiomyopathy is a
complex process that is attributed to abnormal cellular
metabolism and defects in organelles such as mitochondria,
sarcolemma, and endoplasmic reticulum (ER) which leads
to activation of apoptosis. Hyperglycemia appears to play a
central role in the development of diabetic cardiomyopathy
by inducing apoptosis of cardiac myocytes via increased
levels of reactive oxygen and nitrogen species [30]. Using a
mouse model of diabetes, Cai et al. [25] found that
administration of insulin suppressed hyperglycemia and
inhibited diabetes-induced apoptosis in the heart. Other
studies have shown that suppression of myocardial cell
death by antioxidants or inhibition of apoptotic signaling
pathways result in a significant prevention of diabetic car-
diotoxicity, suggesting that oxidative-stress mediated
apoptosis plays an important role in the development of
diabetic cardiomyopathy [31,32].
There is also a connection between chronic heart failure
and apoptosis. It has been reported that patients with
advanced heart failure have higher rates of cardiac myocyte
apoptosis than normal subjects, (0.08–0.25 vs. 0.001–
0.002%) [9,10,33]. Genetic and pharmacological studies
from animal models suggest that apoptosis plays a crucial
role in the development and progression of heart failure.
Using transgenic mice that express a conditionally active
caspase-8 in the heart, Wencker et al. [34] found that
extremely low levels of chronic myocyte apoptosis were
sufficient to cause a lethal dilated cardiomyopathy. Inhi-
bition of cell death with a caspase inhibitor prevented left
ventricular dilation and improved ventricular function.
Thus, even if apoptosis occurs at a very low frequency after
an insult such as I/R, it is a constant process and results in
Apoptosis (2009) 14:536–548 537
123
progressive loss of myocytes, which gradually reduces the
ability of the heart to maintain contractile function. As a
result, the remaining myocytes are forced to work harder to
compensate for the loss in contractility, which, in turn, may
contribute to cardiac hypertrophy resulting in cardiomy-
opathy and heart failure.
Apoptotic signaling pathways in the heart
The major pathways involved in apoptotic signaling in the
heart involve the death receptor pathway, the mitochon-
drial and ER-stress death pathways. A schematic view of
apoptotic cascades is shown in Fig. 1.
Death receptor pathway in cardiovascular disease
Apoptosis can be initiated through activation of the death
receptor pathway (also called the extrinsic pathway) via a
complex signal transduction from the plasma membrane
leading to activation of the caspase cascade. The death
receptors belong to the tumor necrosis factor/nerve growth
factor receptor superfamily and are transmembrane pro-
teins with an extracellular ligand-interacting domain, a
transmembrane domain, and an intracellular death domain
[35]. Binding of the corresponding ligand to the death
receptor causes oligomerization and activation of the
receptor. For instance, after Fas ligand (FasL) binding, Fas
receptors undergo trimerization and recruit Fas-associated
death domain (FADD). Fas/FADD complex binds to pro-
caspase 8 leading to cleavage of the initiator pro-caspase 8
to active caspase 8. Activated caspase 8 propagates the
apoptotic signal through a direct activation of executioner
downstream caspases and via the release of cytochrome c
by mitochondria.
Several studies strongly suggest an important patho-
physiological role for the death receptor pathway in the
pathogenesis of heart failure. The best-characterized death
receptors are Fas (also called CD95 or Apo1) and tumor
necrosis factor receptor 1 (TNFR1). Both receptors are
present in cardiac myocytes and have been implicated to
contribute to cardiovascular disease. TRAIL is ligand for
Death Receptor 4 and 5 (DR4 and DR5) and has also been
reported to be released by cardiac myocytes [36], but not
much is known about this ligand and its receptors in the
heart. Patients with end-stage congestive heart failure have
elevated circulating levels of TNF-a, the ligand for TNFR1
[37–39], and studies suggest that there is a relationship
between the serum levels of TNF- aand the severity of
heart failure [39,40]. In addition, several studies have
reported that cardiomyocytes can be an abundant source of
TNF-a[41–43], and that failing human myocardium, but
not nonfailing hearts, express high levels of TNF-a[44,
45]. Following I/R, there is a sustained increase of TNF-a
both locally in the heart as well as in circulating levels in
blood [43,46]. The functional role of elevated levels of
TNF-ahas been investigated using transgenic mice over-
expressing TNF-aspecifically in the heart. For instance,
cardiac specific overexpression of TNF-ain transgenic
mice caused development of dilated cardiomyopathy and
heart failure [16,17], suggesting that increased levels of
Fig. 1 Scheme of apoptotic
signaling in cardiac myocytes.
Apoptosis can be mediated via
activation of death receptors,
permeabilization of the outer
mitochondrial membrane, or
ER-stress
538 Apoptosis (2009) 14:536–548
123
TNF-ais detrimental to the heart by activation of the death
receptor pathway.
In addition, both Fas and FasL expression are increased in
myocytes exposed to hypoxia [47] and activation of the Fas
pathway has been shown to induce apoptosis in cardiac
myocytes [48]. There is also evidence suggesting that Fas-
mediated apoptosis is associated with myocarditis [49],
myocardial reperfusion injury [50], and post-infarction
ventricular remodeling [51]. For instance, Gomez et al. [52]
recently reported that mice lacking functional Fas (lpr mice)
had no difference in infarct size and apoptosis after I/R,
suggesting that Fas plays a minor role in mediating acute I/R
injury. Similarly, Li et al. [51] found that lpr mice and mice
lacking Fas ligand (gld mice) had similar infarct sizes 2 days
after myocardial infarction. Instead, this study found
decreased apoptosis of granulation tissue cells which
reduced post-infarct remodeling. Granulation tissue cells are
removed via apoptosis to eventually make scar tissue [23].
Also, they found that infecting hearts with an adenovirus
encoding soluble Fas, a competitive inhibitor of FasL,
3 days after a myocardial infarction improved cardiac
function and survival. This suggests that the Fas pathway
contributes to cell death in the later stages of an infarction.
In contrast, two different studies found that the lpr mice
were resistant to acute I/R injury and hearts had reduced
infarct size and apoptosis compared to wild type [43,50]. In
support of this observation, vanadyl sulfate treatment caused
decreased expression of FasL which correlated with reduced
caspase-3 activation and cell death in an in vivo model of
I/R, suggesting that the Fas pathway plays a key role in
I/R-mediated apoptosis [53]. The reasons for the differences
in the findings in these studies using the same mice are not
clear and further studies are needed to clarify the role of Fas
in acute I/R injury. In addition, monocyte chemoattractant
protein-1 (MCP-1) plays a crucial role in initiating coronary
heart disease by recruiting monocytes/macrophages to the
vessel wall, and transgenic mice overexpressing MCP-1 in
the heart manifest cardiac inflammation and develop heart
failure. Interestingly, inhibition of FasL function through
cardiac-specific expression of soluble Fas rescued the MCP-1
transgenic mice from developing heart failure, suggesting
that the FasL derived from the infiltrating mononuclear cells
causes death of cardiac cells resulting in the development of
heart failure [54]. Clearly, Fas-mediated apoptosis plays a
role in contributing to heart failure but further studies are
needed to elucidate exactly how and under what conditions
(chronic or acute) Fas activates apoptosis.
Mitochondrial death pathway in heart disease
The primary function of mitochondria is to provide energy
for the cell in the form of ATP through oxidative
phosphorylation. In cardiac myocytes, mitochondria con-
stitute about 30% of cell volume and are located below the
sarcolemma as well as in intermyofibrillar spaces. This
ubiquitous presence and strategic location of mitochondria
ensures efficient ATP supply and delivery to the continu-
ously contracting myocyte. However, mitochondria can
also contribute to cell death in response to intracellular
stress such as increased oxidative stress, serum deprivation,
and DNA damage. In response to stress, mitochondria
release several pro-apoptotic factors such as cytochrome c,
apoptosis inducing factor (AIF), endonuclease G (Endo G),
second mitochondria-derived activator of caspases (Smac/
Diablo), and HtrA2/Omi, resulting in initiation of apopto-
sis. The mitochondrial death pathway appears to play a
significant role in contributing to cell death particularly in
I/R injury. Studies have reported that there is substantial
release of cytochrome cand activation of caspase-9 after
myocardial I/R injury [55–57]. Also, it has been reported
that protecting mitochondrial integrity protects against I/R
injury [56,58–60].
The mitochondrial cell death pathway is regulated by
the pro- and anti-apoptotic Bcl-2 proteins. These proteins
share up to four conserved Bcl-2 homology (BH) domains.
Anti-apoptotic Bcl-2 proteins, such as Bcl-2 and Bcl-X
L
,
contain all four subtypes of BH domains (BH1-4) and
promote cell survival. The pro-apoptotic Bcl-2 proteins
contain one or three BH domains and therefore are divided
into two structurally distinctive subfamilies: (1) multido-
main proteins such as Bax and Bak that share three BH
regions (BH1-3), and (2) BH3-only domain proteins such
as Bnip3, Nix/Bnip3L, Bad, Bid, Noxa, and Puma [61,62].
The anti-apoptotic members such as Bcl-2 and Bcl-X
L
are important in cell survival and protect cardiac myocytes
against various stressors. For instance, overexpression of
Bcl-X
L
in H9c2 cardiac cells protected against doxorubi-
cin- and hypoxia-mediated apoptosis by preserving
mitochondrial integrity [63], and Bcl-2 was shown to pre-
vent p53-mediated apoptosis in cardiac myocytes [64].
Moreover, transgenic mice showing accelerated accumu-
lation of mitochondrial DNA (mtDNA) mutations due to
expression of an error-prone mtDNA polymerase specifi-
cally in the heart activated a strong prosurvival response by
upregulation of Bcl-2 and Bcl-X
L
[65]. Transgenic mice
overexpressing Bcl-2 in the heart have reduced I/R injury
and fewer apoptotic cells compared to wild type mice,
suggesting that the cardioprotective effect of Bcl-2 is via
inhibition of the mitochondrial death pathway [60,66,67].
Bcl-2 has been reported to protect against cell death by
preventing permeabilization of the outer mitochondrial
membrane by inhibiting activation of pro-apoptotic Bax/
Bak [68]. For instance, mice null for desmin, a muscle-
specific member of the intermediate filament gene family,
develop cardiomyopathy characterized by extensive
Apoptosis (2009) 14:536–548 539
123
cardiomyocyte death, and subsequent heart failure [69].
Weisleder et al. [70] found that overexpression of Bcl-2 in
the desmin null heart attenuated the cardiomyopathy phe-
notype by preventing mitochondrial defects. However, new
protective functions for Bcl-2 have also been identified.
During ischemia, the sodium calcium exchanger (NCX)
operates in reverse to extrude Na
?
and as a result the cell
becomes loaded with Ca
2?
[71]. Interestingly, Bcl-2 was
reported to reduce NCX activity as well as increase resis-
tance to permeability transition in the mitochondria by
increasing the Ca
2?
threshold for mitochondrial perme-
ability transition pore opening in heart mitochondria [72].
Bcl-2 also provides protection by inhibiting consumption
of glycolytically generated ATP by the ATPase. During
myocardial ischemia, mitochondrial ATP generation is
inhibited and the F1F0-ATPase is running in reverse,
thereby consuming ATP generated by glycolysis [73].
Interestingly, Imahashi et al. [74] found that Bcl-2 over-
expression in the heart reduced the rate of ATP decline and
decreased acidosis during ischemia. These studies suggest
that the anti-apoptotic proteins can protect against cell
death via multiple mechanisms and have attractive thera-
peutic potential to treat or prevent heart disease.
The pro-apoptotic Bcl-2 proteins activate cell death by
permeabilizing the outer mitochondrial membrane, which
releases pro-apoptotic proteins such as cytochrome cand
AIF from the intermembrane space. The pro-apoptotic BH3-
only proteins function as cellular stress sensors and integrate
diverse cell death stimuli. For instance, Bnip3 is activated in
response to increased oxidative stress [75], whereas Bad is
activated in response to growth factor deprivation [76].
Activation of a BH3-only protein in response to a specific
stress induces activation of downstream effectors Bax and/
or Bak either by directly interacting with these proteins or
indirectly by sequestering anti-apoptotic Bcl-2/Bcl-X
L
[68].
Activation of Bax and/or Bak induce their oligomerization
in the mitochondrial membrane and formation of a pore in
the outer mitochondrial membrane which allows for release
of proteins from the intermembrane space [68]. Bax and Bak
are essential for cell death mediated via the mitochondrial
pathway, and mouse embryonic fibroblast (MEFs) isolated
from the Bax/Bak double knockout mouse are completely
resistant to cell death by staurosporine, growth factor
deprivation, and UV, as well as to overexpression of BH3-
only proteins such a tBid, Bad, and Bnip3 [75,77,79]. The
pro-apoptotic Bcl-2 proteins have been widely implicated in
cardiovascular disease. In the myocardial cells, Bax has
been reported to be activated in response to oxidative stress
[56] and simulated I/R [78,80,81]. Moreover, hearts of Bax
deficient mice have reduced mitochondrial damage and
infarcts size compared to wild type mice, suggesting that
Bax is an important contributor to I/R injury [82]. The most
studied BH3-only protein in the heart is Bnip3 which has
been reported to contribute to acute I/R injury [58] and post-
infarct remodeling [83]. Bnip3 is transcriptionally upregu-
lated by hypoxia in neonatal cardiac myocytes via HIF-1a
[84] and E2F-1 [85], and Frazier et al. [86] have reported
that the Bnip3 protein is stabilized by acidosis. Recently, we
reported that Bnip3 is activated by oxidative stress which
promotes homodimerization and activation of Bnip3 in the
myocardium [75]. Since apoptosis occurs mainly during
reperfusion, these studies suggest that oxygen deprivation
and acidosis promotes upregulation and stabilization of
Bnip3, respectively, and that increased oxidative stress
during reperfusion promotes homodimerization and acti-
vation of Bnip3 in the mitochondria. Bnip3 has been
reported to activate downstream Bax/Bak [75] as well as
induce opening of the mitochondrial permeability transition
pore [87,88]. In contrast, the Bnip3 homologue Nix/Bnip3L
has been implicated in cardiac hypertrophy and develop-
ment of cardiomyopathy [89]. Moreover, Bid is cleaved to
its truncated and active form in response to I/R [55,90], and
Bad is upregulated in response to I/R [91]. Since ischemia
and reperfusion induces multiple stress signals (oxygen
deprivation, oxidative stress, DNA damage, growth factor
deprivation etc.) in the cardiac myocyte, multiple BH3-only
proteins are activated in response to I/R ensuring activation
of the cell death program. Thus, the BH3-only proteins are
important activators of the mitochondrial cell death pathway
in response to myocardial infarction and pathological car-
diac hypertrophy.
Importantly, the relative level of the pro- and anti-
apoptotic Bcl-2 proteins determines whether a cell will
survive or die following an apoptotic stimulus. It has been
reported that there is a shift in the ratio of anti- to pro-
apoptotic Bcl-2 proteins to proteins that promote apoptosis
in the hearts of patients after a myocardial infarction, in
severe dilated cardiomyopathy and ischemic heart disease
[92–94]. In addition, there was a significant shift in the
ratio of Bax to Bcl-X
L
during the transition from com-
pensated hypertrophy to heart failure in a model of pressure
overload which correlated with increased levels of cyto-
chrome cin the cytosol and caspase-3 activation [95].
Although the ratio is shifted towards anti-apoptotic pro-
teins, it may not activate apoptosis. However, the myocytes
will have a lower threshold for additional stress and will
induce apoptosis easier than a normal cell, making these
hearts more susceptible to develop heart failure.
Permeabilization of the mitochondrial membrane
Cytochrome c
In healthy cells, the role of cytochrome cis to shuttle
electrons from complex III to IV of the respiratory chain.
540 Apoptosis (2009) 14:536–548
123
However, during apoptosis, cytochrome cis released into
the cytosol where it binds to Apaf-1 and dATP which
induces a conformational change in Apaf-1 which recruits
and subsequently activates caspases-9. The activated cas-
pase-9 cleaves pro-caspases-3 to produce active effector
caspase-3 which leads to the cleavage of cellular target
proteins [96–98]. In addition, it has been reported that
activated caspase-3 translocates to nucleus, then cleaves
the DNA repairing enzyme, poly (ADP-ribose) polymerase
(PARP) and activates endonucleases which cleave DNA.
These events culminate in apoptotic cell death [96,99,
100].
Apoptosis-inducing factor (AIF)
AIF is a flavoprotein localized in the mitochondrial inter-
membrane space and is required for oxidative
phosphorylation and for the assembly and/or stabilization
of respiratory complex I [101]. AIF is essential for the
maintenance of normal heart function, and inactivation of
AIF results in dilated cardiomyopathy [102]. In addition,
cardiac myocytes from Harlequin (Hq) mice, which has
*80% reduction in AIF protein levels due to a proviral
insertion in the first intron of the Aif gene, are sensitized to
oxidative stress-induced cell death, and Hq hearts display
more severe ischemic damage compared to wild-type
hearts after acute ischemia/reperfusion injury [103]. AIF is
anchored to the mitochondrial inner membrane via its
N-terminus. However, upon induction of apoptosis, AIF is
cleaved and released into the cytosol [104] where it then
translocates to the nucleus and mediates chromatin con-
densation and large-scale DNA fragmentation [105].
Interestingly, it was recently reported that increased levels
of mitochondrial Ca
2?
activated a mitochondrial calpain
which cleaved membrane bound AIF. Permeabilization of
the mitochondrial membrane resulted in release of the
soluble AIF [104]. It is well known that I/R causes Ca
2?
overload of mitochondria [106], and reduction of mito-
chondrial Ca
2?
uptake has beneficial effects on cardiac
function following I/R with improved functional recovery
and reduced infarct size [107,108]. Since it has been
reported that I/R induces release of AIF [109,110], it will
be interesting to investigate whether this is due to pro-
cessing and release of AIF by Ca
2?
-activated Calpain.
Moreover, considering the fact that AIF functions as a
NADH-oxidase involved in electron transport in complex I
of the electron transport chain, loss of AIF from mito-
chondria may cause further mitochondrial dysfunction due
to reduction in electron transport capacity. Thus, release of
AIF not only initiates apoptosis, but also subsequently
induces mitochondrial dysfunction which will ensure cell
death.
Omi/HtrA2, Smac/Diablo, and endonuclease G
(EndoG)
Other apoptotic effectors are released from mitochondria,
including the serine protease Omi/HtrA2, Smac/Diablo,
and Endo G. Similar to cytochrome c-mediated activation
of the caspase cascade, Smac/Diablo and Omi/HtrA2 are
indirectly involved in caspase activation. For example,
inhibitors of apoptosis proteins (IAPs) are endogenous
inhibitors of caspases and are bound to caspase-3 and -9
under normal conditions [111,112]. However, when
released to cytosol, Smac/Diablo and Omi/HtrA2 prevent
IAPs from inhibiting the caspases, resulting in activation of
caspase-9 and -3. A role for Omi/HtrA2 in the heart was
demonstrated by two studies where inhibition of Omi/
HtrA2 using ucf-101 reduced apoptosis and infarct size in
mice as well as rat after in vivo I/R [113,114]. Also,
transgenic mice overexpressing IAP2 had reduced infarct
size and fewer TUNEL-positive cells after I/R [115].
Endo G is a nuclear-encoded endonuclease and localized
in the inner membrane space of mitochondria. Under nor-
mal conditions, Endo G plays a role in maintenance of
mitochondria DNA by removing defective DNA [116].
However, similar to AIF once released, Endo G translo-
cates to the nucleus where it cleaves DNA. Although few
studies using Endo G-/-splenocytes and fibroblasts
report that Endo G is not essential for nuclear DNA frag-
mentation and apoptosis, studies using intact heart or
isolated cardiomyocytes demonstrate that Endo G plays a
role in I/R-mediated cell death. For instance, Javadov et al.
[117] reported that VAE-480, a specific Na
?
/H
?
exchan-
ger-1 (NHE-1) inhibitor, reduced release of Endo G and I/R
injury, suggesting that attenuation of Endo G release is
cardioprotective. In addition, Bahi et al. [118] demon-
strated that Endo G was released from mitochondria and
induced DNA damage, and importantly when Endo G was
downregulated using siRNA, DNA damage was signifi-
cantly reduced in adult cardiomyocytes during I/R.
Collectively, these studies suggest that Endo G is a critical
contributor to DNA degradation during I/R.
Activation of the ER stress death pathway in the heart
The ER is responsible for synthesis and folding of secreted
proteins as well as Ca
2?
storage. Under normal conditions,
there is a balance between import of newly unfolded pro-
teins into the ER and secretion of folded mature proteins.
When this balance is disturbed and there is an accumula-
tion of protein aggregates in the ER lumen, the unfolded
protein response (UPR) is activated in an attempt to restore
ER homeostasis [119]. Aberrant Ca
2?
regulation can also
activate the UPR. The UPR activates a transcriptional
Apoptosis (2009) 14:536–548 541
123
program to increase the protein folding capacity of the ER,
and the degradation of misfolded proteins, as well as sup-
press proteins synthesis in the cell [120]. Thus, the UPR is
primarily an adaptive response to restore ER homeostasis
and protect the cell from stress. However, if the stress is
prolonged and overwhelming, the UPR activates a pro-
apoptotic response instead which is independent of the
mitochondria.
Several recent studies have demonstrated a role for ER
stress in cardiovascular disease. For instance, Okada et al.
[121] found that pressure overload by aortic constriction
induced extensive ER stress during progression from car-
diac hypertrophy to heart failure. In addition, studies have
reported that ER stress occurs in response to myocardial
I/R [122,123], and that enhanced ER stress is associated
with the development of ischemic heart disease [124].
There is also evidence that ER stress-induced apoptosis is
involved in pathogenesis of diabetes and heart failure [26],
and ultrastructural analysis revealed swelling of the ER in
diabetic myocardium [125]. Consistent with these obser-
vations, defective ER quality control in transgenic mice
with mutant KDEL receptor (a receptor for ER chaperones)
caused dilated cardiomyopathy [126]. These studies sug-
gest that apoptosis mediated by ER stress may be a
significant contributor to cardiovascular disease.
Endoplasmic reticulum stress induces cell death via two
different mechanisms. Under ER stress, activated caspase-
12 activates caspase-3, leading to apoptosis [119]. The
second death-signaling pathway activated by ER stress is
activation of a transcriptional program via upregulation of
the transcription factor CHOP/GADD 153. CHOP activates
transcription of genes encoding pro-apoptotic proteins
including the BH3-only protein Puma [127]. Szegezdi et al.
[122] found that during prolonged ischemia, the UPR
switched to a pro-apoptotic response by activation of the
ER stress death pathway with upregulation of the tran-
scription factor CHOP, and activation of caspase-12 in
neonatal cardiac myocytes. Studies in cardiac myocytes
and the heart have demonstrated that increased CHOP
expression during I/R contributed towards apoptosis, and
inhibition of CHOP expression using a PKCdinhibitor
significantly attenuated ER-mediated apoptosis [128]. This
study suggests that translocation of PKCdto ER membrane
induced the UPR, which led to upregulation of CHOP and
subsequent apoptosis. In addition, Okada et al. [121]
demonstrated that pharmacological intervention inducing
ER stress increased CHOP expression and apoptosis in the
heart.
Interestingly, the BH3-only protein Puma was reported
to be upregulated in isolated cardiac myocytes exposed to
hypoxia/re-oxygensation, and genetic deletion of Puma
reduced I/R-mediated cell death and decreased infarct size
in Langendorff perfused hearts [129]. Recently, it was
demonstrated that myocytes deficient in Puma or down-
regulation via siRNA were resistant to ER stress-induced
apoptosis [130], suggesting that Puma is a critical com-
ponent of ER-stress induced apoptosis in cardiac myocytes.
The Bcl-2 proteins have been shown to localize to the ER
where they can regulate the levels of Ca
2?
stored in the ER
[131].
Cross talk between the apoptotic pathways
There is cross talk between the death receptor and mito-
chondrial cell death pathways. Activation of caspase 8 by
the death receptor pathway directly activates executioner
caspase-3, but also cleaves the BH3-only protein Bid at
aspartate 60 to generate a 15 kDa truncated form (tBid)
that facilitates release of cytochrome cfrom the mito-
chondria [132,133]. The release of cytochrome ccauses
activation of caspase-9 which in turn also activates cas-
pase-3, thus amplifying the death signal from the plasma
membrane. The cross talk between the pathways via death
receptor activation has been demonstrated in cardiac
myocytes and the heart. For instance, Date et al. [134]
found that overexpression of FasL activated both caspase-8
and -9 in neonatal cardiac myocytes. Moreover, cardiac
restricted overexpression of TNF-aresults in increased
apoptosis and development of heart failure, but when these
mice were crossed with transgenic mice overexpressing
Bcl-2 in the heart cardiac myocytes apoptosis and LV
remodeling was attenuated [135]. In addition, caspase-8
was activated, and Bid was cleaved to t-Bid, suggesting
concurrent activation of both the death receptor and
mitochondrial death pathways in the heart by TNF-a.In
support of this, Bcl-2 overexpression only partially atten-
uated cardiomyocyte apoptosis and had no effect on
extrinsic signaling.
There have also been reports of cross-talk between the
death receptor pathway and the ER-stress pathway. For
instance, treatment of L929 cells with FasL induced pro-
cessing of caspase-12 as well as activation of caspases-3,
-7, and -9 [136]. In addition, Bajaj and Sharma [137]
recently reported that TNF-atreatment caused activation of
both caspase-3 and -12 in the HL-1 myocyte cell line.
There is also evidence that there is communication between
the ER and the mitochondrial death pathways. For instance,
ER-targeted Bcl-2 was reported to inhibit mitochondrial
membrane depolarization and cytochrome crelease in
apoptotic myelodysplastic syndrome erythroid precursor
cells [138]. BAP31 is an ER-associated protein that is
cleaved by caspase-8. The cleaved fragment directs pro-
apoptotic signals between the ER and mitochondria by
inducing release of Ca
2?
from the ER which gets taken up
by the mitochondria [139]. In addition, the BH3-only
542 Apoptosis (2009) 14:536–548
123
protein Bik has been reported to localize to the mito-
chondria where it initiates release of Ca
2?
via activation of
Bax/Bak in the ER membrane [140]. These studies suggest
that the ER communicates with the mitochondria by
releasing Ca
2?
.
Autophagy as a mediator of cell death
Autophagy is an important cellular process involved in
recycling of long-lived proteins and organelles. In the
heart, autophagy is important to maintain homeostasis and
disruption or a defect in this pathway leads to ventricular
dysfunction and heat failure [141]. It is also an important
survival response which is upregulated during starvation
when the cells need to recycle amino acid and fatty acids.
In the heart, autophagy is upregulated in response to
ischemia/reperfusion [58,142], and pressure overload
[143]. However, the functional significance of increased
autophagy in the heart is not clear and autophagy has been
reported to protect against cell death as well be the cause of
cell death [81,142,144].
Interestingly, recent studies suggest that there is cross
talk between autophagy and apoptosis. Atg5, an essential
autophagy protein, has been reported to activate apoptosis.
Yousefi et al. [145] found that overexpression of Atg5
increased the cell’s susceptibility to apoptosis following
stimulation with several death triggers, including antican-
cer drugs. They found that the death stimulation resulted in
calpain-mediated cleavage of Atg5, and truncated Atg5
induced cytochrome crelease and apoptosis. Overexpression
of Bcl-2 protected against Atg5-mediated mitochondrial
dysfunction. This suggests that Atg5 can serve as a molecular
switch between autophagy and apoptosis, but it is not clear
how truncated Atg5 triggers mitochondrial permeabiliza-
tion. Another study reported that Atg5 associated with the
Fas-associated death domain (FADD) protein to mediate
IFN-c-induced cell death. In addition, a mutant of Atg5
(Atg5K130R), which was unable to activate autophagy,
was able to induce cell death [146]. Similarly, calpain-
mediated truncated Atg5 was unable to promote autophagy,
but induced apoptosis [145]. These two studies suggest that
Atg5-mediated apoptosis does not require the formation of
autophagosomes. Further investigations of this molecular
link between autophagy and apoptosis are needed.
Moreover, anti-apoptotic Bcl-2 family members and pro-
apoptotic BH3-only proteins may participate in the inhibi-
tion and induction of autophagy, respectively. Beclin 1, an
essential autophagy protein, is regulated by the Bcl-2 pro-
teins. Under normal conditions, Bcl-2 and Bcl-X
L
suppress
autophagy by associating with Beclin 1 through a BH3
domain in Beclin 1 and the BH3 binding groove of Bcl-2/
Bcl-X
L
[147]. The BH3-only proteins can disrupt the
interaction between Beclin 1 and Bcl-2/Bcl-X
L
to induce
autophagy [148]. The BH3-only protein Bik was found to
cause enhanced cell death with autophagic features in
Bcl-2 deficient cells [149], and downregulation of Bcl-2
using siRNA induced autophagic cell death in MCF-7 cells
[150]. This suggests that with a lack of Bcl-2, there is no
break on Beclin-1 which can now be activated and induce
excess autophagy. The BH3-only protein Bnip3 has been
shown to be a potent inducer of autophagy in cardiac cells
[58]. Bnip3 has been shown to interact with Bcl-2 and Bcl-
X
L
[151], but it is not known whether Bnip3 disrupts the
interaction between Beclin 1 and Bcl-2/Bcl-X
L
. These
studies suggest that there is cross talk between autophagy
and apoptosis via Beclin 1 and Atg5. It is interesting that
Beclin 1 contains a BH3-only domain, but it is not known
whether it can act as a BH3-only protein and activate
apoptosis. However, Beclin 1 heterozygous mice (Beclin
1
?/-
), which have reduced autophagy, also have reduced
apoptosis and infarcts size after I/R injury [142], suggesting
that Beclin 1 might activate apoptosis.
Conclusion and perspective
It is clear that apoptosis plays a critical role in pathogenesis
of various cardiovascular diseases. Currently, three apop-
totic pathways (death receptor, mitochondrial, and ER-
stress) have been identified in the heart to contribute to
myocyte loss in various cardiovascular diseases. Since the
regenerative capacity of the myocardium is limited, there is
intense interest in the prevention of cardiomyocyte loss in
cardiovascular diseases to prevent development of heart
failure. Since apoptosis is a highly regulated process, it is a
good potential target for therapeutic intervention. Inhibi-
tion of cardiac myocyte apoptosis using novel research
technology including transgenic mice, gene deletion,
recombinant proteins, and pharmacological inhibitors
results in cardioprotection and prevention of heart failure.
However, many important questions regarding the effect of
each anti-apoptotic intervention remain to be answered. For
instance, loss of mitochondrial membrane integrity is
generally considered a point of no return in the cell death
process. Therefore, inhibiting downstream effectors such as
caspase-3 will delay, but not prevent cell death. Thus, it is
essential to preserve mitochondrial integrity for effective
therapy. Another complication is that multiple pro-apop-
totic proteins can be activated in response to injury. For
instance, multiple BH3-only proteins are activated during
I/R, each of which would have to be targeted for effective
therapy. In addition, it is not clear how much cross-talk
there is between necrosis, apoptosis, and autophagy in the
heart. Enhanced autophagy is often seen in cells where
apoptosis is inhibited, and it is unknown whether inhibition
Apoptosis (2009) 14:536–548 543
123
of apoptosis for cardioprotection against various heart
diseases will subsequently lead to non-apoptotic cell death
later. Although several potential therapeutic agents have
been tested in animal models of I/R injury with success,
nearly none of the specific anti-apoptotic agents have
reached the stage of clinical research. Thus, a better
understanding of the complex mechanisms associated with
cardiac myocyte apoptosis is necessary to identify potential
targets and to develop novel therapeutic strategies for
cardiovascular diseases.
Acknowledgments This manuscript was supported by a Scientist
Development Award from AHA, and NIH grant HL087023 to A
˚.B.G.
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