© 1999 American Heart Association, Inc.
Cellular Biology |
Regulation of Bcl-2 Family Proteins During Development and in Response to Oxidative Stress in Cardiac Myocytes
Association With Changes in Mitochondrial Membrane Potential
From the National Heart and Lung Institute, Cardiac Medicine Section (S.A.C., P.H.S.), and the Division of Biomedical Sciences, Molecular Pathology Section (A.C.), Imperial College School of Medicine, London, UK.
Correspondence to Angela Clerk, PhD, Division of Biomedical Sciences, Molecular Pathology Section, Imperial College School of Medicine, Sir Alexander Fleming Building, South Kensington, London SW7 2AZ, United Kingdom. E-mail s.a.cook{at}ic.ac.uk
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Abstract—Cardiac myocyte apoptosis is potentially important in many cardiac disorders. In other cells, Bcl-2 family proteins and mitochondrial dysfunction are probably key regulators of the apoptotic response. In the present study, we characterized the regulation of antiapoptotic (Bcl-2, Bcl-xL) and proapoptotic (Bad, Bax) Bcl-2 family proteins in the rat heart during development and in oxidative stress–induced apoptosis. Bcl-2 and Bcl-xL were expressed at high levels in the neonate, and their expression was sustained during development. In contrast, although Bad and Bax were present at high levels in neonatal hearts, they were barely detectable in adult hearts. We confirmed that H2O2 induced cardiac myocyte cell death, stimulating poly(ADP-ribose) polymerase proteolysis (from 2 hours), caspase-3 proteolysis (from 2 hours), and DNA fragmentation (from 8 hours). In unstimulated neonatal cardiac myocytes, Bcl-2 and Bcl-xL were associated with the mitochondria, but Bad and Bax were predominantly present in a crude cytosolic fraction. Exposure of myocytes to H2O2 stimulated rapid translocation of Bad (<5 minutes) to the mitochondria. This was followed by the subsequent degradation of Bad and Bcl-2 (from
30 minutes). The
levels of the mitochondrial membrane marker cytochrome oxidase remained
unchanged. H2O2 also induced translocation of
cytochrome c from the mitochondria to the cytosol within
15 to 30 minutes, which was indicative of mitochondrial dysfunction.
Myocytes exposed to H2O2 showed an early loss
of mitochondrial membrane potential (assessed by
fluorescence-activated cell sorter
analysis) from 15 to 30 minutes, which was partially restored
by 1 hour. However, a subsequent irreversible loss of mitochondrial
membrane potential occurred that correlated with cell death. These data
suggest that the regulation of Bcl-2 and mitochondrial function are
important factors in oxidative stress–induced cardiac myocyte
apoptosis.
Key Words: myocyte • mitochondrial membrane potential • apoptosis • oxidative stress • Bcl-2
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Introduction |
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Programmed cell death, or apoptosis, is an essential component of normal development and a response to pathological disease states. In the heart, cardiac myocyte apoptosis is a feature of many pathological disorders; it occurs, for example, in myocardial infarction and congestive heart failure (reviewed in Reference 11 ). Apoptosis allows surplus or damaged cells to be removed in the absence of an inflammatory response. The mechanisms involved are not fully understood. A key component is the activation of a family of proteases, the caspases, which participate in a cascade and ultimately cleave a set of proteins, causing disassembly of the cell (reviewed in Reference 22 ). Although the caspase cascade may be pivotal to the apoptotic response, caspase activation is regulated by a variety of other factors. These include established intracellular signaling pathways, such as the mitogen-activated protein kinases and protein kinase B (reviewed in Reference 11 ). The Bcl-2 family, whose functions are less well-defined, also regulate the apoptotic response (reviewed in References 3 and 43 4 ).
Bcl-2 family proteins may be either pro- or antiapoptotic.
Thus, Bcl-2 and Bcl-xL protect cells from apoptosis, whereas
Bax and Bad promote the response.3 4 At least 15 members
of this family have been identified; they each share homology within
4 regions (BH1 to 4). Although Bcl-2 and Bcl-xL contain all 4 regions
of homology, Bax lacks the BH4 domain, and others (eg, Bad) contain
only the BH3 "death" domain. The function of these proteins is not
clear, but Bcl-2, Bcl-xL, and Bax can form ion-conductive pores in
lipid bilayers, probably acting as dimers/oligomers.5 6
Because Bcl-2 and Bcl-xL are predominantly localized to the outer
mitochondrial membrane and Bax translocates to the mitochondria on
activation, they may function to regulate ion fluxes within the cell
(reviewed in References 3, 4, and 73 4 7 ). Alternatively, they may act
directly to regulate caspase activation. Structural studies indicate
that BH3 domains may fit into a hydrophobic pocket in Bcl-2, Bcl-xL, or
Bax, allowing dimerization and presumably modulating the function of
these proteins. The activities of Bcl-2 proteins are, therefore,
regulated by their subcellular localization (ie, cytosolic versus
mitochondrial) and their dimerization/oligomerization. Indeed,
dimerization of Bax triggers its translocation to the mitochondria and
induces apoptosis.8
The Bcl-2 family is also regulated by proteolytic
cleavage9 10 and by phosphorylation. Bad
has 
2 phosphorylation sites (Ser-112 and Ser-136)
and, in some cell types, phosphorylation of Ser-136 by
protein kinase B11 or through activation of
mitogen-activated protein kinase cascades12
results in binding to 14–3-3 proteins.13 This sequesters
Bad in the cytosol, preventing interaction with Bcl-2 and inhibiting
the proapoptotic function of Bad. Bad may also be
phosphorylated on Ser-112 at the mitochondria by
protein kinase A, causing it to dissociate from the mitochondria and
preventing its proapoptotic function.14
Conversely, apoptotic stimuli may induce
dephosphorylation of Bad, which would then translocate
to the mitochondria. Bcl-2 is phosphorylated on
multiple sites within an N-terminal unstructured
loop.15 16 17 Although phosphorylation of
Ser-70 is activating,17 phosphorylation of
other sites may be inactivating and lead to
apoptosis.16
The principal site of action of the Bcl-2 family proteins seems to be
the mitochondria, and it is increasingly apparent that most, if not
all, apoptotic responses involve mitochondrial dysfunction and
a loss of the mitochondrial membrane potential (
).7
Indeed, this step may be an irreversible commitment to cell death.
Recent studies indicate that apoptosis is associated with the
loss of cytochrome c (and potentially other
apoptotic factors18 ) from the intermembrane space
of the mitochondria and subsequent release into the cytosol. Cytochrome
c binds to apoptosis-activating factor 1 to form a
complex with caspase-9, which becomes activated.19
Caspase-9 triggers a proteolytic cascade, activating other caspases
(including caspase-3) and resulting in cell death.20 The
trigger for cytochrome c release is not known. Some
studies indicate that the mitochondrial permeability transition pore
(MPTP) may open and cause loss of . This may result in the
swelling of the mitochondria, rupturing the outer membrane and
releasing cytochrome c into the cytosol.21
However, in other studies, release of cytochrome c is
not associated with the loss of .7 Bcl-2 and/or
Bcl-xL may function in the mitochondrial membrane to prevent loss of
cytochrome c and thus inhibit
apoptosis,22 23 an effect that may be mediated by
direct interaction with apoptosis-activating factor
1.24 25
The Bcl-2 proteins are becoming increasingly recognized as important modulators of cardiac myocyte apoptosis. Bcl-2 mRNA is expressed in both developing and adult hearts,26 and the protein is upregulated after coronary occlusion in rat hearts27 or myocardial infarction in human hearts.28 As in other cells, overexpression of Bcl-2 protects cardiac myocytes from apoptosis.29 Proapoptotic Bax is upregulated in the rat heart after coronary occlusion,27 and its overexpression in the ventricles of spontaneously hypertensive rat hearts may contribute to apoptosis.30 Bcl-x and proapoptotic Bax are also expressed in cardiac myocytes and are upregulated during cytokine-induced cardiac myocyte apoptosis.31
In the heart, ischemia/reperfusion injury results in cardiac myocyte cell death by both necrotic and apoptotic mechanisms.1 One of the key features of this disorder is the generation of reactive oxygen species (ROS) during both the ischemic and reperfusion phases.32 Oxidative stress induces cardiac myocyte apoptosis in vitro23 33 34 35 and may be a major contributing factor in the apoptosis observed in ischemia/reperfusion injury and congestive heart failure. In the present study, we studied the regulation of members of the Bcl-2 family that are recognized as either antiapoptotic (Bcl-2, Bcl-xL) or proapoptotic (Bad, Bax). We characterized the developmental expression of these proteins in the rat heart and determined their subcellular localization in cardiac myocytes. We also examined the effects of oxidative stress on Bcl-2 proteins and on mitochondrial function. The role(s) of the Bcl-2 proteins in relation to mitochondrial dysfunction in cardiac myocyte apoptosis is discussed.
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Materials and Methods |
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Preparation of Heart and Myocyte Extracts
Hearts from Sprague-Dawley rats were extracted in buffer A (in mmol/L: ß-glycerophosphate 20 [pH 7.5], NaF 50, microcystin LR 0.002, EDTA 2, Na3VO4 0.2, benzamidine 10, leupeptin 0.2, trans-epoxy succinyl-L-leucylamido-(4-guanidino)butane 0.01, dithiothreitol 5, phenylmethylsulfonyl fluoride 0.3, 1% [vol/vol] Triton X-100), and samples were prepared as previously described.36 Myocytes were prepared by an adaptation37 of the method of Iwaki et al38 and plated on gelatin-coated dishes (1.4x103 cells/mm2). In all experiments, serum was withdrawn for 24 hours before treatment. For immunoblotting, myocytes were washed and scraped into buffer A. Samples were centrifuged, and the supernatants were boiled with sample buffer. Protein content was measured by the BioRad Bradford method.39
Terminal Deoxynucleotidyl dUTP Nick-End
Labeling Analysis of Cardiac Myocytes
Myocytes were plated in 8-well Laboratory-Tek tissue-culture
chamber slides precoated with laminin and gelatin (600
cells/mm2). After treatment, myocytes were fixed,
permeabilized, and labeled by
deoxynucleotidyl transferase (0.1 U/µL)
incorporation of biotinylated dUTP (2 nmol/L) in terminal transferase
buffer (Boehringer-Mannheim). Biotinylated dUTP was detected
with extravidin-FITC. Myocytes were counterstained with antibodies to
ß-myosin heavy chain (Vector Laboratories) and streptavidin-Texas Red
anti-mouse IgG, and they were examined by confocal microscopy (Leica
TCS-4D laser scanning confocal microscope). For each of 3 experiments,
3 fields were examined for each treatment performed in duplicate. Cells
were identified as apoptotic if they showed unequivocal
terminal deoxynucleotidyl dUTP nick-end labeling
(TUNEL) staining in the nucleus. Necrotic cells were not identified
because these cells become nonadherent and are removed when the
medium is decanted and the cell layer is washed in PBS.
Subcellular Fractionation
Cells were scraped into buffer B (in mmol/L: HEPES 10 [pH
7.5], mannitol 200, and sucrose 70), which contained protease and
phosphatase inhibitors. Samples were centrifuged
(500g) to pellet nuclei, unbroken cells, and plasma membrane
debris (nuclear fraction). The supernatants were recentrifuged
(10 000g) to separate the mitochondrial fraction from the
cytosolic fraction. The mitochondrial fraction was resuspended in
buffer B containing 1% (vol/vol) Triton X-100. The protein content of
each fraction was determined by the BioRad Bradford
assay.37 Samples were boiled with sample buffer.
Western Blot Analysis
Proteins were separated by SDS-PAGE and transferred to
nitrocellulose, as previously described.36 Nonspecific
binding was blocked, and the blots were incubated with primary
antibodies and then horseradish peroxidase–conjugated secondary
antibodies (Amersham, 1/5000 dilution). Bands were detected by enhanced
chemiluminescence, and blots were exposed to Hyperfilm MP
for 30 s to 2 minutes. Laser scanning densitometry was used for
semiquantitative analysis of the data.
Analysis of
Loss of was assessed by
fluorescence-activated cell sorter (FACS)
analysis of cells stained with Rhodamine 123 (R123) or
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolecarbocyanide
iodine (JC-1). Cells were incubated with 5 µmol/L R123
(Calbiochem) or 10 µg/mL JC-1 (Molecular Probes), harvested by
trypsinization, and analyzed by FACS (10 000 cells/sample).
The excitation wavelength was 488 nm. The emission fluorescence
for R123 was monitored at 582 nm, and JC-1 was monitored at 530 and 582
nm. The data were analyzed using Cell Quest (Becton Dickinson,
Immunocytometry Systems).
Statistical Analysis
Results are expressed as mean±SEM. Differences between means
were evaluated by unpaired 2-tailed Student’s t test.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
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Developmental Regulation of Bcl-2 Family Proteins
The expression of antiapoptotic (Bcl-2, Bcl-xL) and proapoptotic (Bad, Bax) members of the Bcl-2 family was assessed in rat heart ventricles by Western blot analysis (Figure 1
). In neonatal rat ventricles,
the Bcl-2 antibody detected 2 bands of 26 and 32 kDa (Figure 1A), which may represent different isoforms. Although
not apparent in Figure 1A, in some blots, both the 26- and
32-kDa forms appeared as multiple bands (see below), and
these may correspond to differentially phosphorylated
forms.16 Bcl-xL was detected as a 31-kDa band (Figure 1B), Bad was detected as a doublet of 25 kDa (Figure 1C), and Bax was detected as a doublet of 21 kDa (Figure 1D). The levels of Bcl-2 and Bcl-xL were sustained during
development (Figures 1A and 1B). In contrast, the expression of
Bad (Figures 1C and 2A) and Bax
(Figures 1D and 2B) declined substantially during
development.
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Subcellular Localization of Bcl-2 Family Proteins
Bcl-2 and Bcl-xL are predominantly localized to the mitochondria
in nonmyocytic cell types, whereas Bax and Bad are primarily cytosolic
(reviewed in References 3 and 73 7 ). We examined the subcellular
distribution of these proteins in primary cultures of neonatal myocytes
by differential centrifugation and Western blot
analysis. Bcl-2 and Bcl-xL were detected in the mitochondrial
fraction (Figure 3A and 3B), along with
the mitochondrial marker cytochrome oxidase (Figure 3E). Only a
small fraction of 32-kDa Bcl-2 immunoreactivity was detected in the
cytosolic fraction (Figure 3A). All proteins were detected in
the low-speed pellet (nuclear fraction) containing nuclei and unbroken
cells. Bad and Bax were detected in both the mitochondrial and
cytosolic fractions, with a greater proportion in the cytosol (Figure 3C and 3D). As expected, Bad and Bax were also detected in the
low-speed pellet.
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Stimulation of Cardiac Myocyte Apoptosis by Oxidative
Stress
Oxidative stress induces apoptosis in cardiac
myocytes.33 34 35 40 We confirmed that apoptosis
occurs in myocytes exposed to
H2O2 (0.5 mmol/L) by
examining the cleavage of poly(ADP-ribose) polymerase (PARP) and
caspase-3 and by TUNEL analysis. PARP is cleaved by caspase-3
to generate a 25-kDa fragment from the N-terminus and an 85-kDa
fragment from the C-terminus.41 We studied PARP using a
monoclonal antibody, C2-10, which detects an epitope at the N-terminus
of the 85-kDa cleavage product.42 In unstimulated
cells, the PARP holoenzyme was detected as a 117-kDa band on Western
blots (Figure 4A). We also detected bands
at 85 and 25 kDa (Figures 4A and 4B).
H2O2 stimulated a
significant decrease in both the 117-kDa holoenzyme and the 85-kDa band
between 2 and 4 hours (Figure 4A). A similar loss of both bands
was observed in myocytes exposed to proinflammatory
cytokines,31 although
H2O2/FeSO4
induced an increase in the 85-kDa cleavage product.35
The antibody used reportedly does not detect the 25-kDa PARP fragment
derived by caspase cleavage,42 but we observed an
increase in a 25-kDa band in response to
H2O2 (Figure 4B). We
also assessed cleavage (activation) of caspase-3. The caspase-3
holoenzyme was detected as a 32-kDa band in unstimulated myocytes, and
this band decreased in intensity after exposure to
H2O2 from 2 hours
(Figure 4C).
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We assessed DNA fragmentation by TUNEL analysis. Unstimulated
cells showed minimal DNA fragmentation (<1%) (Figure 5A), but myocytes exposed to
H2O2 for 16 hours were
predominantly TUNEL-positive (Figures 5B and 5C). The number of
TUNEL-positive cells increased from 8 hours (Figure 5C). We
also examined the dependence of DNA fragmentation on
H2O2 concentration at 16
hours (Figure 5D). Low concentrations (0.03 mmol/L) did
not stimulate any significant increase in TUNEL-positive cells, but
higher concentrations (0.1 and 0.5 mmol/L) induced a progressive
increase in DNA fragmentation. This was not inhibited by the
OH• radical scavenger dimethyl sulfoxide (1% [vol/vol]
or 140 mmol/L), suggesting that other ROS may be involved.
Alternatively, it is possible that
H2O2 may have nonspecific
toxic effects in addition to the generation of ROS. However, these data
are consistent with
H2O2-induced cardiac
myocyte apoptosis.
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Response of Bcl-2 and Bad to H2O2
Stimulation
The Bcl-2 family is a key regulator of the apoptotic
response (reviewed in References 3 and 43 4 ). We examined the response of
antiapoptotic Bcl-2 and proapoptotic Bad to
H2O2-induced cardiac
myocyte apoptosis. Both the 26- and 32-kDa immunoreactive Bcl-2
bands were lost from the mitochondrial fraction from 5 to 15 minutes,
with a substantial loss (50%) by 30 to 60 minutes (Figure 6A). A progressive increase in the 32-kDa
band in the cytosolic fraction occurred over this time course, but the
26-kDa isoform remained undetectable (Figure 6B). To confirm
that the loss of Bcl-2 from the mitochondrial fraction did not reflect
gross mitochondrial disruption, we determined the levels of cytochrome
oxidase in these samples. No change in cytochrome oxidase
immunoreactivity occurred after
H2O2 treatment for 120
minutes (Figure 6C).
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Bad was detected as a single band in the mitochondrial fraction (Figure 7A) but as a doublet in the cytosolic
fraction (Figure 7B).
H2O2 stimulated an increase
in Bad immunoreactivity in the mitochondrial fraction between 5 and 30
minutes, which declined thereafter to below basal levels (Figure 7A). In the cytosol, the lower band decreased in intensity from
5 to 15 minutes, and it was essentially lost by 60 minutes (Figure 7B). The intensity of the upper band was maintained for 60
minutes, but it was lost by 120 minutes. To identify the Bad species
appearing in the mitochondrial fraction, we directly compared the
relative molecular masses of the immunoreactive bands in the cytosolic
and mitochondrial fractions (Figure 7C). The band in the
mitochondrial fraction from unstimulated myocytes (lane 2) and myocytes
exposed to H2O2 (0.5
mmol/L, 5 minutes, lane 3) corresponded to the upper band in the
cytosolic fraction (lanes 1 and 4) and may represent a
phosphorylated form.
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Cytochrome c Redistribution and Loss of in
Myocytes Subjected to Oxidative Stress
Redistribution of cytochrome c from the
mitochondria to the cytosol is a critical event in the progression of
apoptosis in many systems (reviewed in Reference 1818 ). In
unstimulated myocytes, as expected, cytochrome c was
predominantly present in the mitochondrial fraction (Figure 8A and 8B).
H2O2 (0.5 mmol/L)
induced a loss of cytochrome c from the mitochondrial
fraction within 15 to 30 minutes (Figure 8A) and a corresponding
increase in the cytosolic fraction (Figure 8B).
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The redistribution of cytochrome c has been linked with
the loss of and the opening of the MPTP. However, this linkage
is contentious and may depend on cell type and stimulus.7
We assessed in
H2O2-stimulated myocytes
using R123 or JC-1. The fluorescence intensity of these dyes in
the mitochondria of a given cell is dependent on .
Maintenance of results in a high intensity of
fluorescence with R123 (population M1). As is lost, the
fluorescence intensity of R123 decreases (population
M2).21 In unstimulated myocytes, the M1 and M2 populations
were approximately equal (Figure 9A and 9F). Exposure of
myocytes to H2O2 (0.5
mmol/L) induced a shift in fluorescence from M1 to M2 at 15 and
30 minutes (Figure 9B and 9F). However, at 45 and 60 minutes,
evidence of repolarization existed, as shown by a redistribution from
the M2 to the M1 population, such that the 60-minute values of M1 were
significantly greater than (and the values of M2 were significantly
less than) the values at 30 minutes (Figure 9C and 9F). At later
times (>3 hours), the proportion of cells in the M1 population again
declined (Figure 9D and 9F). As a positive control, myocytes
were treated with the F1-ATPase
inhibitor oligomycin (10 µg/mL) and the mitochondrial
uncoupler carbonyl cyanide 3-chlorophenylhydrazine (50 µmol/L)
for 4 hours. As expected, virtually all cells were present in the
M2 population (Figure 9E and 9F).
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P<0.005 vs myocytes exposed to
H2O2 for 0.5 hours.
To confirm that an early loss of occurred, we used an
alternative fluorophore, JC-1, that concentrates in the mitochondria,
where it exists as monomers at a low membrane potential and as red
fluorescent J-aggregates at a high membrane potential. In
intact cells, JC-1 monomers accumulate as is lost. This is
associated with an increase in fluorescence intensity at 530 nm
(FL1) and a decrease in fluorescence at 585 nmol/L
(FL2).43 In isolated mitochondria, the FL2
fluorescence or the FL2/FL1 ratio may correlate more closely
with than the FL1 fluorescence.44
Consistent with the R123 data (Figure 9
),
H2O2 decreased the
fluorescence of FL2 from 0 minutes (Figure 10A) to 30 minutes (Figure 10B), and this was reflected in a decrease in the FL2/FL1 ratio
(Figure 10D). An increase in the fluorescence of FL1
also occurred (Figure 10A and 10B). At 60 minutes, an
indication of a restoration of existed (Figure 10C and 10D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is increasingly recognized that the Bcl-2 family and mitochondrial dysfunction are key components of the apoptotic process (reviewed in Reference 4545 ). There have been few studies of either in relation to cardiac myocyte apoptosis. From the present study, it is clear that both anti- and proapoptotic Bcl-2 family proteins are expressed in the intact heart (Figure 1
) and in isolated cardiac myocytes (Figure 3). However,
although the proteins that are generally recognized as
antiapoptotic (Bcl-2, Bcl-xL) are sustained during rat heart
development, the proapoptotic proteins (Bad, Bax) seem to be
downregulated. Recent evidence indicates that, in some situations,
Bcl-2 may promote withdrawal from the cell cycle,46 47 48
whereas Bax induces cell cycle progression.49 It is,
therefore, possible that the sustained expression of Bcl-2 and the
downregulation of Bax may be associated with the withdrawal of myocytes
from the cell cycle in the perinatal period. Reexpression of Bax in
terminally differentiated myocytes, as is seen in human heart
failure28 and in spontaneously hypertensive rat
hearts,30 may promote apoptosis.
Consistent with studies in other cell types, in unstimulated
myocytes, Bcl-2 and Bcl-xL were essentially localized to the
mitochondria, whereas Bad and Bax were predominantly present in the
crude cytosolic fraction (Figure 3).
At least 2 alternatively spliced isoforms of Bcl-2 have been
identified.50 Bcl-2
is composed of 236 amino acids,
whereas Bcl-2ß is lacking the C-terminal region and consists of 199
amino acids. The Bcl-2 antibody used in this study detected 2
immunoreactive species of 26 and 32 kDa (Figure 1A), both of
which may exist as multiple bands (Figure 6A and 6B). It is
generally accepted that Bcl-2 migrates at 26 kDa, and it has been
reported that phosphorylation produces a band of 30
kDa,51 which may correspond to our 32-kDa Bcl-2 band(s).
However, it is possible that this band may represent an
alternative isoform expressed in the rat heart or may be a
cross-reactive protein. Bcl-2 is phosphorylated on
multiple sites within the N-terminal unstructured
loop,16 17 which may account for the multiple bands within
each immunoreactive species. Although Bcl-xL is structurally similar to
Bcl-2 and possesses a similar N-terminal loop, we detected only a
single band of 31 kDa (Figure 1B) and found no evidence of
multiple bands. The Bad antibody detected 2 bands in the heart, which
is consistent with a phosphorylated form
(Figures 1C and 7); this is discussed in more detail
below. Bax exists as multiple isoforms,52 53 which may
account for the multiple bands detected in the heart (Figure 1D).
Others have shown that H2O2
induces cardiac myocyte apoptosis.34 35 We
confirmed that this was indeed the case by 3 different criteria: DNA
fragmentation, PARP proteolysis, and caspase-3 cleavage (Figures 4 and 5). In other studies,
H2O2 was used at
concentrations between 0.1 and 0.5 mmol/L, alone or in combination
with FeSO4.34 35 Our TUNEL data are
consistent with these studies, and we found that the majority
of myocytes were TUNEL-positive after 16 hours of incubation with
0.5 mmol/L H2O2
(Figures 5B, 5C, and 5D). The principal ROS produced by
H2O2 treatment is generally
accepted to be the OH• radical. However, dimethyl
sulfoxide, an OH• radical scavenger, had no effect on
H2O2-induced cardiac
myocyte cell death. It is possible that other ROS may be generated or
that H2O2 may have
nonspecific toxic effects in addition to the generation of ROS.
Alternatively, dimethyl sulfoxide (or, indeed, other free radical
scavengers) may themselves have toxic effects that contribute to cell
death. If this is the case, any inhibition of
H2O2-induced cardiac
myocyte cell death would be occluded by the effects of the
scavenger.
In addition to DNA fragmentation, apoptosis is characterized by
activation (proteolysis) of caspase-3, which cleaves a number of
cellular substrates, including PARP.1 We demonstrated that
H2O2 induced the loss of
the caspase-3 holoenzyme (Figure 4C) consistent with its
activation, although the antibody we used did not detect the cleavage
product. We also clearly observed a decrease in the 117-kDa PARP
holoenzyme, but we could not detect any increase in the 85-kDa cleavage
product (Figure 4A). Indeed, the 85-kDa band detectable in
unstimulated myocytes decreased in
H2O2-stimulated cells.
Using the same antibody (C2-10), Ing et al31 also showed a
parallel decrease in both the 117- and 85-kDa bands in cardiac myocytes
exposed to proinflammatory cytokines. However, von Harsdorf et
al35 detected the 85-kDa cleavage product in myocytes
exposed to
H2O2/FeSO4.
This may reflect differences between sustained or transient cellular
stresses. In the present experiments (as in those of
others31 34 ), the medium was not changed for the duration
of the experiment, so myocytes were subjected to a prolonged cellular
stress, whereas von Harsdorf et al35 transiently exposed
myocytes to
H2O2/FeSO4
for 1 hour before incubating them in
H2O2-free medium for a
further 19 hours. The prolonged stress may activate additional
pathways, resulting in further degradation of the 85-kDa cleavage
product. In support of this, we detected an increase in a 25-kDa
band (Figure 4B). This band has not been previously reported,
but we suggest it may represent further degradation of the
85-kDa cleavage product.
The mechanisms that lead to caspase activation are not clear. In other
cells, apoptosis is usually associated with the translocation
of cytochrome c into the cytosol, often accompanied by a
loss of . Whether loss of precedes or is a consequence of
cytochrome c translocation remains controversial.
Although some studies infer that the MPTP and loss of are
essential for cytochrome c release,54 55
others indicate that alternative mechanisms are
involved.56 57 From this study, it is clear that oxidative
stress stimulates cytochrome c translocation in cardiac
myocytes (Figure 8). We found that the time course of this
translocation (within 30 minutes) corresponded to a loss of
(Figures 9 and 10), but cyclosporin A (which inhibits
MPTP opening) did not inhibit cytochrome c translocation
(results not shown). Unexpectedly, we observed a partial restoration of
between 45 and 60 minutes (Figures 9 and 10),
suggesting that cardiac myocytes may have some capacity for recovery.
Similar recoveries of both and energy levels have been
demonstrated in other cell types.58 59 However, it is also
possible that the early loss in may be indicative of a
subpopulation of myocytes undergoing more rapid cell death. These cells
would fragment and be lost from the population subjected to FACS
analysis at later time points. Because the remaining population
of cells may be less damaged, the FACS data may suggest a partial
recovery, when a change in the gated population is the true cause of
the observed differences. The distinction between these 2 possibilities
is extremely important, because in the first scenario, loss of
precedes or parallels the loss of cytochrome c from the
mitochondria, whereas the in the second, loss of cytochrome
c clearly precedes the reduction in . Further
studies of isolated cardiac myocyte mitochondria are necessary to
clarify this issue. In myocytes, a subsequent progressive loss of
occurred over 6 hours (Figure 9), the time course of
which correlates with PARP proteolysis (Figure 4A and 4B). We
suggest that the loss of cytochrome c from the
mitochondria disrupts the electron transport chain, generating ROS that
propagate cellular injury and may induce this phase of mitochondrial
dysfunction. In support of this, it has been shown that mitochondrial
electron transport is a significant source of oxidative stress in
cardiac myocytes and other cells.60 61
The function of Bcl-2 in the mitochondria is not understood, although
it has ion-conducting properties in lipid bilayers6 and
appears to regulate proton flux.62 Bcl-2 also prevents the
release of cytochrome c56 61 and subsequent
activation of caspase-9.63 We observed that
H2O2 induced the loss of
Bcl-2 from the mitochondria (Figure 6), with a similar time
course as the initial loss of (Figures 9 and 10)
and cytochrome c release (Figure 8). It is
tempting to speculate that the loss of Bcl-2 may trigger the loss of
and result in release of cytochrome c. The
mechanisms by which Bcl-2 is lost are unknown. We were unable to
inhibit its loss by the caspase inhibitor ZVAD-fmk,
suggesting that its degradation is not mediated by caspases (results
not shown).
Phosphorylation of Bad at Ser-136 targets it to 14–3-3
proteins, which sequester Bad away from the
mitochondria.58 Dephosphorylation of this
site results in translocation to and association with Bcl-2, promoting
apoptosis (reviewed in Reference 6464 ). In cardiac myocytes,
H2O2 induced the
translocation of Bad to the mitochondria within 5 minutes (Figure 7A). In contrast to the accepted dogma,58 we found
that the form of Bad present in the mitochondria comigrated with
the upper band detected in the cytosol; it may represent a
phosphorylated form (Figure 7C). A recent study
suggested that Bad is phosphorylated on Ser-112 by
protein kinase A at the mitochondria.14 Our data would fit
with a model of Bad phosphorylation occurring at the
mitochondria and with subsequent release into the cytosol. Subsequent
to Bad translocation, it is lost from both the cytosolic and
mitochondrial fractions (Figures 7A and 7B). The time course
parallels the loss of Bcl-2 from the mitochondria (Figure 6A),
suggesting that they may be associated. This is currently under
investigation. Our data, showing that
H2O2 induces the loss of
both Bcl-2 and Bad (Figures 6 and 7), contrasts with the
recent study by von Harsdorf et al,35 which showed no loss
of either protein during oxidative stress–induced cardiac myocyte
apoptosis.35 However, as mentioned above, we
subjected our cells to a more prolonged cellular stress.
It is now generally recognized that cardiac myocyte apoptosis
is an important contributing factor to many disease states in the
heart, although only limited studies exist on the mechanisms that may
be involved. It is clear, however, that overexpression of Bcl-2 can
protect cardiac myocytes29 and that
ischemia/reperfusion injury results in the opening of the MPTP,
which is associated with mitochondrial dysfunction.65 On
the basis of the present study, we propose that Bcl-2 maintains the
homeostasis of the mitochondria. Translocation of Bad in response to
H2O2 induces loss of Bcl-2
from the mitochondria, causing transient membrane depolarization and
translocation of cytochrome c. Although damaged myocytes
may recover to an extent, as illustrated by partial restoration of
, cytochrome c cannot be recovered, resulting in
irreversible mitochondrial dysfunction. Cell death then ensues. Further
studies will clarify whether such mechanisms occur in vivo.
|
Acknowledgments |
|---|
This work was funded by the British Heart Foundation. We thank Steve Rothery for assistance with the confocal microscopy.
|
Footnotes |
|---|
Reprint requests to Stuart A. Cook, MBBS, National Heart and Lung Institute, Cardiac Medicine Section, Dovehouse Street, London SW3 6LY, UK.
Received July 13, 1999; accepted September 13, 1999.
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References |
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S. A. Cook, T. Matsui, L. Li, and A. Rosenzweig Transcriptional Effects of Chronic Akt Activation in the Heart J. Biol. Chem., June 14, 2002; 277(25): 22528 - 22533. [Abstract] [Full Text] [PDF]
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K. Kunisada, E. Tone, S. Negoro, Y. Nakaoka, Y. Oshima, T. Osugi, M. Funamoto, M. Izumi, Y. Fujio, H. Hirota, et al. Bcl-xl reduces doxorubicin-induced myocardial damage but fails to control cardiac gene downregulation Cardiovasc Res, March 1, 2002; 53(4): 936 - 943. [Abstract] [Full Text] [PDF]
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X. Zhang, G. Azhar, K. Nagano, and J. Y. Wei Differential vulnerability to oxidative stress in rat cardiac myocytes versus fibroblasts J. Am. Coll. Cardiol., December 1, 2001; 38(7): 2055 - 2062. [Abstract] [Full Text] [PDF]
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J. Shiraishi, T. Tatsumi, N. Keira, K. Akashi, A. Mano, S. Yamanaka, S. Matoba, J. Asayama, T. Yaoi, S. Fushiki, et al. Important role of energy-dependent mitochondrial pathways in cultured rat cardiac myocyte apoptosis Am J Physiol Heart Circ Physiol, October 1, 2001; 281(4): H1637 - H1647. [Abstract] [Full Text] [PDF]
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F. Qin, N. K. Rounds, W. Mao, K. Kawai, and C.-s. Liang Antioxidant vitamins prevent cardiomyocyte apoptosis produced by norepinephrine infusion in ferrets Cardiovasc Res, September 1, 2001; 51(4): 736 - 748. [Abstract] [Full Text] [PDF]
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D. Ekhterae, O. Platoshyn, S. Krick, Y. Yu, S. S. McDaniel, and J. X.-J. Yuan Bcl-2 decreases voltage-gated K+ channel activity and enhances survival in vascular smooth muscle cells Am J Physiol Cell Physiol, July 1, 2001; 281(1): C157 - C165. [Abstract] [Full Text] [PDF]
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P. H. Sugden Mechanotransduction in Cardiomyocyte Hypertrophy Circulation, March 13, 2001; 103(10): 1375 - 1377. [Full Text] [PDF]
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M. Xu, Y. Wang, K. Hirai, A. Ayub, and M. Ashraf Calcium preconditioning inhibits mitochondrial permeability transition and apoptosis Am J Physiol Heart Circ Physiol, February 1, 2001; 280(2): H899 - H908. [Abstract] [Full Text] [PDF]
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J. W. Adams, A. L. Pagel, C. K. Means, D. Oksenberg, R. C. Armstrong, and J. H. Brown Cardiomyocyte Apoptosis Induced by G{alpha}q Signaling Is Mediated by Permeability Transition Pore Formation and Activation of the Mitochondrial Death Pathway Circ. Res., December 8, 2000; 87(12): 1180 - 1187. [Abstract] [Full Text] [PDF]
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J. He, Y. Xiao, C. A. Casiano, and L. Zhang Role of Mitochondrial Cytochrome c in Cocaine-Induced Apoptosis in Coronary Artery Endothelial Cells J. Pharmacol. Exp. Ther., December 1, 2000; 295(3): 896 - 903. [Abstract] [Full Text]
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F. H. Pham, P. H. Sugden, and A. Clerk Regulation of Protein Kinase B and 4E-BP1 by Oxidative Stress in Cardiac Myocytes Circ. Res., June 23, 2000; 86(12): 1252 - 1258. [Abstract] [Full Text] [PDF]
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B. O’Rourke Apoptosis : Rekindling the Mitochondrial Fire Circ. Res., November 12, 1999; 85(10): 880 - 883. [Full Text] [PDF]
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S. Zhuang, J. T. Demirs, and I. E. Kochevar p38 Mitogen-activated Protein Kinase Mediates Bid Cleavage, Mitochondrial Dysfunction, and Caspase-3 Activation during Apoptosis Induced by Singlet Oxygen but Not by Hydrogen Peroxide J. Biol. Chem., August 18, 2000; 275(34): 25939 - 25948. [Abstract] [Full Text] [PDF]
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 A. Dirks and C. Leeuwenburgh Apoptosis in skeletal muscle with aging Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2002; 282(2): R519 - R527. [Abstract] [Full Text] [PDF]
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M. Akao, A. Ohler, B. O'Rourke, and E. Marban Mitochondrial ATP-Sensitive Potassium Channels Inhibit Apoptosis Induced by Oxidative Stress in Cardiac Cells Circ. Res., June 22, 2001; 88(12): 1267 - 1275. [Abstract] [Full Text] [PDF]
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