Am J Physiol Heart Circ Physiol
281: H1637–H1647, 2001.
Important role of energy-dependent mitochondrial
pathways in cultured rat cardiac myocyte apoptosis
JUN SHIRAISHI,1 TETSUYA TATSUMI,1 NATSUYA KEIRA,1 KAZUKO AKASHI,1
AKIKO MANO,1 SATOSHI YAMANAKA,1 SATOAKI MATOBA,1 JUN ASAYAMA,3
TAKESHI YAOI,2 SHINJI FUSHIKI,2 HENRY FLISS,4 AND MASAO NAKAGAWA1
1
Second Department of Medicine and 2Department of Dynamic Pathology, Kyoto Prefectural
University of Medicine, Kyoto 602-8566; 3Department of Clinical Pharmacology, Kyoto
Pharmaceutical University, Kyoto 607-8414, Japan; and 4Department of Cellular and Molecular
Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada
Received 19 January 2001; accepted in final form 18 June 2001
APOPTOSIS IS A GENETICALLY determined form of cell
death, which can be triggered by a number of physiological and pathological conditions. Apoptosis is char-
acterized by condensation, margination, and degradation of chromatin, as well as cytoskeletal disruption,
cell shrinkage, and membrane blebbing. Ultimately,
the cell is fragmented into membrane-enclosed apoptotic bodies (4, 50). In contrast, necrosis is a passive
process characterized morphologically by mitochondrial swelling and the loss of plasma membrane integrity without concomitant severe damage to the nuclei
(3). Apoptosis and necrosis are therefore considered to
be conceptually and morphologically distinct forms of
cell death. However, recent reports (1, 21, 22, 40) have
indicated that these two processes can occur simultaneously in tissues or cell cultures exposed to the same
stimulus.
It is well documented (6, 18) that myocardial ischemia triggers cardiac cell death, which may possess the
properties of both apoptosis and necrosis. Studies from
our laboratory and studies by others have demonstrated
that myocardial apoptosis is observed in the early phases
of ischemic injury, whereas necrosis appears in the later
stages. Moreover, the data also suggest that apoptotic
myocytes are generally absent from profoundly ischemic regions of the myocardium but are more prevalent
in moderately perfused regions such as the hypoperfused border zone between the central infarct area and
the noninjured myocardial tissue and that apoptotic
death of ischemic myocytes may be hastened by reperfusion (10, 11, 37, 39, 47). These data therefore suggest
that intracellular energy production may be required
to fuel the apoptotic machinery in ischemically injured
myocytes. It was recently reported (9, 23) that intracellular ATP levels are a determinant of cell death
modes in actively proliferating cells (i.e., Jurkat and
HeLa). However, it is still uncertain whether the intracellular energy levels also regulate the manifestation of cell death in postmitotic cells such as cardiac
myocytes and whether the mitochondrial permeability
transition (PT) is an important step in the induction of
myocyte death similarly to those used in mitotic cells.
Address for reprint requests and other correspondence: T. Tatsumi, Second Dept. of Medicine, Kyoto Prefectural Univ. of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan
(E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
cytochrome c; mitochondria; necrosis; staurosporine
http://www.ajpheart.org
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society
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Shiraishi, Jun, Tetsuya Tatsumi, Natsuya Keira, Kazuko Akashi, Akiko Mano, Satoshi Yamanaka, Satoaki
Matoba, Jun Asayama, Takeshi Yaoi, Shinji Fushiki,
Henry Fliss, and Masao Nakagawa. Important role of energy-dependent mitochondrial pathways in cultured rat cardiac
myocyte apoptosis. Am J Physiol Heart Circ Physiol 281:
H1637–H1647, 2001.—Recent studies have suggested that apoptosis and necrosis share common features in their signaling
pathway and that apoptosis requires intracellular ATP for its
mitochondrial/apoptotic protease-activating factor-1 suicide
cascade. The present study was, therefore, designed to examine
the role of intracellular energy levels in determining the form of
cell death in cardiac myocytes. Neonatal rat cardiac myocytes
were first incubated for 1 h in glucose-free medium containing
oligomycin to achieve metabolic inhibition. The cells were then
incubated for another 4 h in similar medium containing staurosporine and graded concentrations of glucose to manipulate
intracellular ATP levels. Under ATP-depleting conditions, the
cell death caused by staurosporine was primarily necrotic, as
determined by creatine kinase release and nuclear staining
with ethidium homodimer-1. However, under ATP-replenishing
conditions, staurosporine increased the percentage of apoptotic
cells, as determined by nuclear morphology and DNA fragmentation. Caspase-3 activation by staurosporine was also ATP
dependent. However, loss of mitochondrial transmembrane potential (⌬⌿m), Bax translocation, and cytochrome c release were
observed in both apoptotic and necrotic cells. Moreover, cyclosporin A, an inhibitor of mitochondrial permeability transition,
attenuated staurosporine-induced apoptosis and necrosis
through the inhibition of ⌬⌿m reduction, cytochrome c release,
and caspase-3 activation. Our data therefore suggest that staurosporine induces cell demise through a mitochondrial death
signaling pathway and that the presence of intracellular ATP
favors a shift from necrosis to apoptosis through caspase activation.
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INTRACELLULAR ATP AND APOPTOSIS
MATERIALS AND METHODS
Chemicals
Staurosporine was a generous gift from Kyowa Medex.
Oligomycin was purchased from Nacalai Tesque. Acetyl-AspGlu-Val-Asp-aldehyde (DEVD-CHO) and cyclosporin A were
from Biomol and Sigma, respectively. Stock solutions of staurosporine and DEVD-CHO were prepared in dimethyl sulfoxide (DMSO). Oligomycin and cyclosporin A were dissolved in
ethanol. The final concentration of ethanol and DMSO was
0.1% (vol/vol). Stock solutions were stored at ⫺80°C.
Neonatal Rat Cardiac Myocytes
Primary cultures of neonatal rat cardiac myocytes were
prepared as previously described with some modifications
(29). Briefly, 1- to 2-day-old Wistar rats were anesthetized
with ether, and the hearts were removed under aseptic conditions and placed in Ca2⫹- and Mg2⫹-free phosphate-buffered saline (PBS). The atria and aorta were discarded, and
the ventricles were minced into 1- to 3-mm3 fragments and
were enzymatically digested four times for 10–15 min each
with 7.5 ml of PBS containing 0.2% type I collagenase
(Sigma). Cell suspensions from each digestion were pooled,
centrifuged at 300 g for 5 min, and resuspended in HEPES
buffer containing (in mM) 116 NaCl, 5.4 KCl, 0.8 MgSO4, 1.0
NaH2PO4, 20 HEPES, and 5.5 glucose, with a pH of 7.35. The
cells were then layered onto a Percoll density gradient (density 1.059/1.082) and were centrifuged at 1,000 g for 30 min
(42). The myocyte layer at the Percoll interface was carefully
collected and washed twice in HEPES buffer. The cells were
then resuspended in Dulbecco’s modified Eagle’s medium
(DMEM; Nissui Pharmaceutical) supplemented with 10%
fetal bovine serum (FBS) (Bioserum) and 1% antibiotics
(5,000 ␮g/ml gentamicin, 5,000 ␮g/ml ampicillin, and 100
␮g/ml amphotericin B). The myocytes were seeded into
60-mm culture dishes (1 ⫻ 105 cells/cm2) and were incubated
AJP-Heart Circ Physiol • VOL
at 37°C in a humidified atmosphere containing 5% CO2-95%
room air. Bromodeoxyuridine (BrdU; 100 ␮M) was added
during the first 48 h to inhibit proliferation of nonmyocytes.
The myocytes were then incubated in DMEM containing
0.5% FBS without BrdU, and all experiments were done
36–48 h after this incubation. By using this approach, we
routinely obtained contractile cultures with ⬎95% myocytes,
as assessed by immunofluorescence staining with a monoclonal antibody against ␤-myosin heavy chain (29). Desmin
staining was also used to distinguish cardiac myocytes from
nonmyocytes as described previously (2). Cardiac myocytes
on type I collagen-coated coverslips were fixed with 2% paraformaldehyde at 0°C, permeabilized with ice-cold methanolacetone (1:1, vol/vol), blocked with 10% heat-inactivated goat
serum, and sequentially incubated with anti-desmin polyclonal antibody [1:50 dilution in 1% bovine serum albumin
(BSA)-buffered PBS, Monosan], fluorescein isothiocyanatelabeled goat anti-rabbit IgG F(ab⬘) (1:100 dilution in 1%
BSA-buffered PBS, ICN) and 0.5 ␮g/ml Hoechst 33258. The
cells were visualized using fluorescence microscopy, and the
images were generated using dual-exposure photography.
Experimental Protocols
Cell cultures were washed twice with PBS before the start
of metabolic inhibition (MI), which was achieved by incubating the myocytes with 0.05 ␮M oligomycin in glucose-free
DMEM (GIBCO-BRL), pH 7.4, at 37°C for 1 h. The myocytes
were then washed twice with PBS and were incubated for
another 4 h in DMEM containing 10 (MI10 group), 30 (MI30
group), 50 (MI50 group), or 100 (MI100 group) mg/dl of
glucose, without oligomycin, to manipulate the intracellular
ATP content. Staurosporine (1 ␮M) was added at the start of
this 4-h incubation period to induce cell death. Myocytes that
were treated with staurosporine and 100 mg/dl glucose for
4 h but without the prior MI were designated as the MI(⫺)
group. Control myocytes were incubated in DMEM containing 100 mg/dl glucose but were not subjected to either MI or
staurosporine treatment (control group). To examine
whether staurosporine induces either apoptosis or necrosis
through the mitochondrial PT, myocytes were treated with
cyclosporin A (0.2 ␮M) 1 h before exposure to staurosporine
and then were treated with staurosporine in the presence of
cyclosporin A.
Measurement of ATP Content
The ATP content of myocytes was measured before, immediately after, or 2 and 4 h after MI without staurosporine.
Cardiac myocytes (0.96 ⫻ 105 cells/cm2) were treated with
0.25 ml of 0.6 N ice-cold perchloric acid and centrifuged at
1,000 g for 5 min at 4°C. The supernatant was neutralized
with KOH to pH 5.0–7.0 and, after 10 min, was centrifuged
at 8,000 g for 5 min at 4°C to remove the KClO4. The
supernatant was used for the assays. ATP was measured by
high-performance liquid chromatography (LC-9A liquid chromatograph, Shimadzu) with a column of STR ODS-M (Shimadzu) (45). The protein content of the samples was determined in the acid precipitated by the method of Lowry, using
BSA as standard.
Histochemical Determination of Cell Viability
and Apoptosis
Living and dead cells were distinguished by using the
Viability/Cytotoxicity Kit (Molecular Probes) (12). Myocytes
were grown on type I collagen-coated glass coverslips. The
culture medium was replaced with 2 ␮M calcein-acetoxy-
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The aim of the present study was, therefore, to investigate the role of intracellular energy levels in determining whether injured cardiac myocytes die by
apoptosis or necrosis through the mitochondrial death
signaling pathway. For the injury agent, we selected
staurosporine, a protein kinase inhibitor and a wellestablished inducer of apoptosis that causes the release
of mitochondrial cytochrome c into the cytosol (49). To
examine the role of energy status in myocyte cell death,
we first blocked both mitochondrial ATPase with oligomycin and glycolysis with glucose-free medium and
then manipulated the level of intracellular ATP by
incubating the staurosporine-injured cells in a medium
containing different concentrations of glucose. We determined the type of cell death by histochemical and
biochemical methods. We also assessed the time course
of mitochondrial transmembrane potential (⌬⌿m), Bax
translocation from the cytosol to the mitochondria, and
cytochrome c release from the mitochondria into the
cytosol. In addition, we investigated whether cyclosporin A, an inhibitor of mitochondrial PT (54), suppresses staurosporine-induced loss of ⌬⌿m, Bax translocation, cytochrome c release, and cell death. Finally,
we examined whether the activation of caspase-3, an
effector caspase (28, 36), is also dependent on energy
levels in the myocytes.
INTRACELLULAR ATP AND APOPTOSIS
methyl ester and 4 ␮M ethidium homodimer-1, and the cells
were incubated for 45 min at room temperature. Cells with
permeabilized membranes (necrotic) take up ethidium homodimer-1 and their nuclei fluoresce red, whereas viable
cells with intact membranes show green fluorescence. The
number of viable and necrotic cells in 10 random microscopic
fields was counted in each coverslip, and the percentage of
viable cells was calculated. Apoptotic cells were identified by
the distinctive condensed or fragmented nuclear morphology
in cells stained with Hoechst 33258. An average of 800–1,000
nuclei from random fields were analyzed for each experiment, and apoptotic cell counts were expressed as a percentage of the total number of nuclei counted.
Creatine Kinase Release
Agarose Gel Electrophoresis
Double-stranded DNA breaks were assessed by agarose gel
electrophoresis of low-molecular-weight genomic DNA from
myocytes (14). Briefly, the culture medium was removed.
Myocytes were then treated with a lysis buffer (10 mM
EDTA, 10 mM Tris 䡠 HCl, pH 7.4, 0.5% Triton X-100), collected by scraping, and centrifuged at 18,000 g for 20 min.
The supernatant was treated with RNase A (400 ␮g/ml) for
1 h at 37°C and was then treated with proteinase K (400
␮g/ml) for 1 h at 37°C. The DNA was precipitated with 0.5
mM NaCl-isopropanol solution (1:1 vol/vol) at ⫺20°C. The
precipitated DNA was centrifuged at 18,000 g for 15 min, and
the isopropanol was thoroughly removed. The nucleic acid
pellet was resuspended in 10 mM Tris 䡠 HCl buffer containing
1 mM EDTA (pH 8.0). The samples were normalized for cell
number and subjected to electrophoresis on 2% agarose gel.
Gels were stained with SYBR green (Molecular Probes), and
DNA was detected by visualization under ultraviolet light.
Mitochondrial Transmembrane Potential
Loss of ⌬⌿m was assessed with the use of the dye JC-1
(Molecular Probes) (1). Cells grown on coverslips were incubated in PBS containing 10 ␮M JC-1 at 37°C for 5 min.
Fluorescence emission at 527 and 590 nm was determined
after excitation at 480 nm.
Immunoblotting
For detection of cytochrome c and Bax, myocytes were
scraped and pelleted by centrifugation at 800 g for 5 min. The
cells were suspended in 150 ␮l of cold lysis buffer containing
(in mM) 250 sucrose, 20 HEPES, 10 KCl, 1 MgCl2, 1 EDTA,
1 EGTA, 1 dithiothreitol, and 1 phenylmethylsulfonyl fluoride, pH 7.5, and were incubated for 5 min on ice. The cells
were then homogenized, and the suspension was centrifuged
at 750 g for 10 min at 4°C to sediment the nuclear fraction.
The supernatant was centrifuged at 12,000 g for 10 min at
4°C to sediment the mitochondrial fraction and then centrifuged for 60 min at 100,000 g for 4°C. The resultant supernatant was used as the cytosolic fraction (51). To detect the
cleavage of procaspase-3, the scraped and pelleted myocytes
were resuspended in 50 ␮l of cold lysis buffer (2⫻ PBS, 1%
NP40, 0.5% deoxycholic acid, and 0.1% sodium dodecyl sulfate; pH 7.5) and incubated for 10 min on ice. The suspension
was centrifuged at 14,000 g for 10 min at 4°C, and the
AJP-Heart Circ Physiol • VOL
supernatant was collected for analysis. Immunoblotting was
performed using standard protocols. Samples containing
equal amounts of protein were subjected to electrophoresis on
12% sodium dodecyl sulfate-polyacrylamide gel and blotted
onto polyvinylidene difluoride membrane (AE-6665, ATTO;
Tokyo, Japan). After being blocked with 5% skim milk in PBS
containing 0.1% Tween 20 at room temperature for 1 h, the
membranes were probed with antibodies specific to cytochrome c (7H8.2C12, PharMingen; San Diego, CA), Bax (sc526, Santa Cruz Biotechnology, Santa Cruz, CA), or
caspase-3 (sc-7148, Santa Cruz Biotechnology) at 4°C overnight, followed by horseradish peroxidase-conjugated antimouse IgG or horseradish peroxidase-conjugated anti-rabbit
IgG (Amersham; Little Chalfont, UK) at room temperature
for 1 h. Detection of chemoluminescence was performed with
ECL Western blot detection kits (Amersham) according to
the supplier’s recommendations.
Caspase-3 Activity
Caspase-3 enzymatic activity was determined with the
CPP32 assay kit (MBL), which detects the production of the
chromophore p-nitroanilide after its cleavage from the peptide substrate DEVD-p-nitroanilide as previously described
(7). In brief, 2.7 ⫻ 106 cells were solubilized and aliquots of
lysate containing equal amounts of protein were reacted with
200 ␮M DEVD-p-nitroanilide at 37°C for 2 h. The activity
was read in a microtiter plate reader at 400 nm. DEVD-CHO
was used as a specific caspase-3 inhibitor (48).
Statistical Analysis
Data are expressed as means ⫾ SE of ⬎6 samples derived
from ⬎6 separate experiments. Differences were analyzed by
one-way analysis of variance combined with Bonferroni test.
A P value of ⬍0.05 was considered to be statistically significant.
RESULTS
Effect of Glucose Concentration on ATP Content
In contrast to the MI(⫺) group, in which ATP levels
remained constant at ⬃22.4 ⫾ 0.7 nmol/mg protein for
the duration of the 5-h experiment, the ATP content in
the MI-treated groups declined to 24.6 ⫾ 1.6% of baseline (Fig. 1). Subsequent incubation of the MI-treated
cells for 4 h with varying concentrations of glucose
resulted in a time- and concentration-dependent restoration of ATP, such that 10, 30, 50, and 100 mg/dl of
glucose increased the ATP to 25.6 ⫾ 2.5, 50.4 ⫾ 3.3,
66.6 ⫾ 2.3, and 89.0 ⫾ 4.8% of baseline, respectively
(Fig. 1).
Cell Viability in Staurosporine-Treated Myocytes
The effect of staurosporine treatment on cell viability
is illustrated in Fig. 2A. The MI10 group contained a
high fraction of nonviable cells (red fluorescent nuclei),
compared with the control group or MI(⫺) group, in
which the fraction of viable cells (green fluorescent)
was consistently ⬎95%. However, with increasing glucose availability, the fraction of viable cells increased
and reached levels not significantly different from control. In the absence of staurosporine, incubation of the
MI-treated cells for 4 h with 10, 30, 50, or 100 mg/dl
glucose (MI and recovery) alone did not significantly
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Creatine kinase (CK) activity in culture media was measured spectrophotometrically as an index of necrotic cell
death after the 4-h incubation with staurosporine, according
to Rosalki’s procedure. [CK activity was expressed as
IU 䡠 l⫺1 䡠 mg protein⫺1 (29).]
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INTRACELLULAR ATP AND APOPTOSIS
decrease the viability of myocytes, such that 10, 30, 50,
and 100 mg/dl glucose resulted in 97.3 ⫾ 0.1, 96.9 ⫾
0.3, 97.8 ⫾ 0.4, and 97.6 ⫾ 0.5% of viability, respectively. The MI10 group also showed a significant release of CK into the culture media compared with
control (Table 1). No significant increase in CK was
observed with any of the other groups. Similarly, MI
and recovery alone did not significantly increased CK
release (data not shown).
Apoptosis in Staurosporine-treated Myocytes
Fig. 2. A: histochemical determination of cell
viability. Cardiac myocyte groups were subjected to MI for 1 h, followed by a 4-h incubation in medium containing staurosporine and
10 (MI10), 30 (MI30), 50 (MI50), or 100 mg/dl
(MI100) glucose. CONT, myocytes subjected
to neither MI nor staurosporine and incubated in medium containing 100 mg/dl glucose. Cells were then labeled with calceinacetoxymethyl ester and ethidium homodimer-1,
visualized by fluorescence microscopy as described in MATERIALS AND METHODS, and then
scored as either viable (green fluorescent) or
nonviable (red nuclear fluorescent). Left: representative micrographs (magnification ⫻200).
Right: percentage of viable and nonviable cells
present in 10 random microscopic fields in each
group, per condition, per experiment, was calculated using fluorescence microscopy, as described in MATERIALS AND METHODS (n ⫽ 6). *P ⬍
0.01 vs. CONT. B: histochemical characterization of apoptotic myocytes. Cardiac myocytes
were subjected to metabolic inhibition, followed
by staurosporine treatment, as described in A.
Myocytes were stained with an anti-desmin
polyclonal antibody and Hoechst 33258 and
were visualized by fluorescence microscopy.
Left: representative micrographs (magnification ⫻400). Right: percentage of apoptosis in
cardiac myocytes. Apoptotic myocytes were
identified by their fragmented nuclei, and the
percentage of apoptotic cells was calculated as
described in MATERIALS AND METHODS (n ⫽ 6).
*P ⬍ 0.01 vs. CONT; †P ⬍ 0.01 vs. MI(⫺); ‡P ⬍
0.05 vs. MI10; §P ⬍ 0.01 vs. MI50.
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Fig. 1. ATP content in cardiac myocytes. Cardiac myocyte groups
were subjected to metabolic inhibition (MI) for 1 h, followed by 4-h
incubation (Recovery) in medium containing (in mg/dl) 10 (MI10), 30
(MI30), 50 (MI50), or 100 (MI100) glucose. MI(⫺), myocytes not
subjected to MI but incubated in medium containing 100 mg/dl
glucose. ATP content in the myocytes was determined as described in
MATERIALS AND METHODS (n ⫽ 6). *P ⬍ 0.01 vs. MI(⫺); **P ⬍ 0.05 vs.
MI(⫺); †P ⬍ 0.01 vs. MI10; ‡P ⬍ 0.01 vs. MI30; §P ⬍ 0.01 vs. MI50.
Histochemical staining of the myocytes with Hoechst
33258 and an antibody to desmin showed typical fragmented nuclei in staurosporine-treated groups, as
illustrated in Fig. 2B. However, the frequency of apoptotic cells increased with increasing glucose concentration (Fig. 2B). The highest percentage of apoptotic
myocytes (23.5 ⫾ 2.0%) was observed in the MI(⫺)
group, when contrasted with control (1.2 ⫾ 0.2%). In
the MI10 group, the percentage of apoptotic myocytes
was only 2.2 ⫾ 0.3%. However, the percentage of apoptotic nuclei increased significantly to 6.5 ⫾ 0.4, 9.1 ⫾
1.0, and 15.7 ⫾ 1.0% in the MI30, MI50, and MI100
groups, respectively. MI and recovery, without staurosporine treatment, did not result in myocyte apoptosis,
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INTRACELLULAR ATP AND APOPTOSIS
Table 1. CK release from cardiac myocytes into culture media
Group
CK release, IU 䡠 l
⫺1
䡠 mg protein
⫺1
Control
MI(⫺)
MI10
MI30
MI50
MI100
0.23 ⫾ 0.01
0.23 ⫾ 0.01
0.28 ⫾ 0.01*
0.24 ⫾ 0.01
0.25 ⫾ 0.01
0.24 ⫾ 0.01
Values are means ⫾ SE; n ⫽ 6. Cardiac myocytes were subjected to metabolic inhibition (MI), followed by staurosporine treatment (see
Fig. 2A). Creatine kinase (CK) release from the myocytes was measured as described in MATERIALS AND METHODS. * P ⬍ 0.01 vs. control.
Mitochondrial Transmembrane Potential
JC-1, a potential sensitive mitochondrial probe, exists as a green fluorescent monomer at low membrane
potential. However, at higher potentials, JC-1 forms
red fluorescent “J-aggregates.” The fluorescent emission of this dye can therefore be used to monitor ⌬⌿m in
apoptotic cardiac myocytes (8). In the present study,
control myocytes showed red-orange mitochondrial
staining, indicative of normal high membrane potentials (Fig. 4). Staurosporine treatment caused loss of
⌬⌿m in the first hour of treatment (Fig. 4). Moreover,
myocytes treated with staurosporine in either the ATP-
replenishing MI(⫺) or ATP-depleting MI10 group
showed green fluorescence indicating the loss of ⌬⌿m
at 1 h after the initiation of staurosporine treatment
(Fig. 5).
Translocation of Bax and Cytochrome c
The time-dependent effects of staurosporine on the
intracellular localization of Bax and cytochrome c in
the MI(⫺) group are illustrated in Fig. 4. Bax was
constitutively expressed in the cytosolic fraction of the
myocytes. However, staurosporine treatment caused a
rapid loss of Bax from the cytosolic fraction in the first
hour of treatment (to 12.4 ⫾ 0.9% of 0 h) and a concomitant increase in Bax in the mitochondrial fraction
during the same period (to 1264.2 ⫾ 257.9% of 0 h). The
mitochondrial Bax immunoreactivity was depressed
after 4 h of staurosporine treatment. In contrast, before staurosporine treatment, cytochrome c was detected exclusively in the mitochondrial fraction. Staurosporine treatment caused a time-dependent decrease
in cytochrome c immunoreactivity in the mitochondrial
fraction (to 19.6 ⫾ 1.8% of 0 h after 4 h of treatment),
with a concomitant increase in the cytosolic fraction,
reaching maximum levels after 4 h of staurosporine
treatment (to 636.8 ⫾ 64.5% of 0 h). To examine the
effect of intracellular ATP content on the translocation
of Bax and cytochrome c, a similar analysis was done
with the control, MI(⫺), and MI10 groups at 1 h (for
Bax) and 4 h (for cytochrome c) after the initiation of
staurosporine treatment (Fig. 5). The data show that
staurosporine induced a translocation of Bax from the
cytosol to the mitochondrial fraction and a translocation of cytochrome c from the mitochondrial fraction
into the cytosolic fraction under conditions of both high
[MI(⫺) group] or low ATP content (MI10 group).
Activation of Caspase-3
Fig. 3. Electrophoretic analysis of DNA fragmentation. Cardiac myocytes were subjected to MI, followed by staurosporine treatment as
described in Fig. 2. The low-molecular-weight genomic DNA was
extracted from the myocytes in each group and was subjected to
agarose gel electrophoresis as described in MATERIALS AND METHODS.
“Size marker” lane shows a 123-bp ladder. Results are representative of 2 independent experiments.
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Caspase-3 activity increased as a function of glucose
concentration after 4 h of treatment with staurosporine. Whereas caspase-3 activity in the MI10 group
increased by a factor of 1.9 over control, it increased
4.5-fold in the MI(⫺) group (Fig. 6A). The addition of
DEVD-CHO, a specific caspase-3 inhibitor, to the medium of the MI(⫺) group 1 h before the initiation of
staurosporine treatment suppressed the activities of
caspase-3 to control level (Fig. 6A). The formation of
caspase-3 from its precursor was also analyzed by
Western blots. As shown in Fig. 6B, procaspase-3 (32
kDa) was constitutively expressed in the myocytes. No
cleavage of procaspase-3 was apparent in the control
group, and only modest cleavage of procaspase-3 was
detected in the MI10 group. However, significant cleav-
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such that 10, 30, 50, and 100 mg/dl of glucose resulted
in 1.7 ⫾ 0.4, 1.6 ⫾ 0.1, 1.6 ⫾ 0.3, and 1.7 ⫾ 0.2% of
apoptosis, respectively. The histological evidence for
apoptosis in staurosporine-treated groups was confirmed by the DNA analysis data. As shown in Fig. 3,
control myocytes showed no DNA fragmentation. In
contrast, DNA isolated from cardiac myocytes treated
with 1 ␮M staurosporine for 4 h exhibited extensive
DNA fragmentation, thus producing the characteristic
DNA ladders. The intensity of the ladders in the MI
groups increased with increasing glucose concentration. MI and recovery alone did not cause DNA fragmentation (data not shown).
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INTRACELLULAR ATP AND APOPTOSIS
age of procaspase, and the concomitant appearance of
the 17-kDa caspase-3 subunit, was observed in the
MI(⫺) group.
Effect of Cyclosporin A on Cell Death
Cyclosporin A inhibited the staurosporine-induced
loss of ⌬⌿m in the MI(⫺) group (data not shown).
Moreover, it suppressed cytochrome c release (Fig. 7),
caspase-3 activity [3.00 ⫾ 0.07-fold vs. 4.52 ⫾ 0.14-fold
for the MI(⫺) group, P ⬍ 0.0001] and the percentage of
apoptosis [9.3 ⫾ 0.5 vs. 23.5 ⫾ 2.0% for the MI(⫺)
group, P ⬍ 0.0001]. Furthermore, cyclosporin A significantly increased cell viability (68.0 ⫾ 1.4 vs. 30.0 ⫾
2.1% for the MI10 group, P ⬍ 0.0001), estimated by
calcein and ethidium homodimer-1 staining. However,
cyclosporin A did not suppress Bax translocation (Fig.
7).
DISCUSSION
The present study demonstrates for the first time
that staurosporine can cause both apoptotic and neAJP-Heart Circ Physiol • VOL
crotic cell death in neonatal rat cardiac myocytes. Both
types of cell death are associated with a loss of ⌬⌿m, a
translocation of Bax from the cytosol into the mitochondria, and a release of cytochrome c from the mitochondria into the cytosol. Significantly, our data show
that intracellular energy levels regulate the form of cell
death, with low cellular ATP effecting necrosis, and
high ATP causing the activation of caspase-3 and promoting apoptosis. Our data therefore suggest that
myocyte apoptosis and necrosis share common mitochondrial death signaling pathways, primarily those
mediated by the mitochondrial PT, and that ATPdependent steps in the apoptotic signal transduction
pathway exist upstream of caspase-3 activation, as
well as downstream of cytochrome c release.
In the present study, the intracellular ATP level in
the myocytes was adjusted by first inhibiting mitochondrial energy production and subsequently by varying the glucose content in the medium during the
staurosporine injury phase. With glucose concentrations depleted to ⬍30 mg/dl, the fraction of nonviable
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Fig. 4. Time course of mitochondrial transmembrane potential, translocation of Bax, and cytochrome c (Cyt C)
release. A: cardiac myocytes in the MI(⫺) group were harvested at the given time intervals. Myocytes were stained
with JC-1 dye, as described in MATERIALS AND METHODS, and fluorescence was monitored at 527 and 590 nm.
Mitochondrial and cytosolic fractions were prepared, and aliquots containing 20 ␮g of protein were subjected to
Western blot analysis and were probed with antibodies for Bax and Cyt C, as described in MATERIALS AND METHODS.
Results are representative of 3 independent experiments. STS, staurosporine. B: densitometric analysis of Bax
translocation and Cyt C release. Levels of Bax and Cyt C are shown as a percentage of change in average from 3
independent experiments compared with unstimulated myocytes (0 h). Top left: *P ⬍ 0.01 vs. 0 h; †P ⬍ 0.05 vs. 0 h;
‡P ⬍ 0.05 vs. 1 h; §P ⬍ 0.01 vs. 1 h. Top right: *P ⬍ 0.01 vs. 0 h; †P ⬍ 0.05 vs. 1 h. Bottom left: *P ⬍ 0.01 vs. 0 h;
†P ⬍ 0.01 vs. 1 h; ‡P ⬍ 0.01 vs. 2 h. Bottom right: *P ⬍ 0.01 vs. 0 h; †P ⬍ 0.05 vs. 2 h.
INTRACELLULAR ATP AND APOPTOSIS
H1643
cells increased significantly after staurosporine treatment, accompanied by a significant increase in CK
release, and enhanced staining by nuclear dyes. In our
study, the increase in CK was quite modest compared
with the loss of cell viability, estimated by calceinacetoxymethyl ester and ethidium homodimer-1 staining. Modest CK increase was probably due to the lower
sensitivity compared with staining for cell viability.
These data suggest that under conditions of low ATP,
the injured cells lost membrane integrity and succumbed to necrotic cell death. In contrast, with glucose
concentrations ⬎30 mg/dl, there was an increase in
cellular ATP, which resulted in a concomitant increase
in the frequency of apoptotic cell death as illustrated by
the increased nuclear and DNA fragmentation.
Apoptosis differs from necrosis in that apoptosis is
an active, genetically regulated, and energy-requiring
process, whereas necrosis is generally viewed as an
AJP-Heart Circ Physiol • VOL
unregulated, passive phenomenon normally caused by
catastrophic and overwhelming injury. Several recent
studies (9, 23) have confirmed that increased ATP
levels favor an apoptotic form of cell death, whereas
low energy levels promote necrosis in several forms of
injury, including staurosporine. In an effort to identify
the ATP-requiring steps within the apoptotic death
signaling pathway in injured myocytes, we examined
the activity of caspase-3, a cysteine protease, because
of its established critical role in apoptosis in general
and staurosporine-induced apoptosis in particular (28,
36, 53). In the present study, the activity of caspase-3
was significantly increased in myocytes treated with
staurosporine, as confirmed by both enzymatic activity
assays and cleavage of the 32-kDa precursor to the
17-kDa caspase-3 isoform. More importantly, the level
of caspase-3 activity appeared to correlate with the
percentage of apoptotic cells, as well as with the myo-
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Fig. 5. Loss of mitochondrial transmembrane potential, translocation of Bax, and Cyt C release. A: myocytes in
CONT, MI(⫺), and MI10 groups, as defined in Fig. 2, were stained with JC-1 at 1 h after the initiation of
staurosporine treatment as described in MATERIALS AND METHODS, and fluorescence was monitored at 527 and 590
nm. Cardiac myocytes from the CONT, MI(⫺), and MI10 groups were harvested at 1 h and 4 h after the initiation
of staurosporine treatment for detection of Bax and Cyt C, respectively. Mitochondrial and cytosolic fractions were
then prepared and aliquots containing 20 ␮g of protein were subjected to Western blot analysis and were probed
with antibodies for Bax and Cyt C as described in MATERIALS AND METHODS. Results are representative of 3
independent experiments. B: densitometric analysis of Bax translocation and Cyt C release. Levels of Bax and Cyt
C are shown as the percentage of change in average from 3 independent experiments compared with CONT. Top
left: *P ⬍ 0.01 vs. CONT. Top right: *P ⬍ 0.01 vs. CONT. Bottom left: *P ⬍ 0.01 vs. CONT. Bottom right: *P ⬍ 0.01
vs. CONT.
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INTRACELLULAR ATP AND APOPTOSIS
cyte ATP content. The data, therefore, strongly suggest
that caspase-3 activation is involved in staurosporineinduced myocyte apoptosis and that ATP-dependent
steps exist upstream of this activation.
Besides the well-established CD95 or tumor necrosis
factor receptor/caspase-8-mediated apoptotic pathway,
recent reports (56) have identified a novel cascade that
is regulated by the apoptotic protease activating factor-1 (Apaf-1), thereby suggesting an important role for
mitochondria in the induction of apoptosis (46, 51).
With cardiac myocytes, an important mitochondrial
contribution has already been reported (5, 8) under
conditions of serum or glucose deprivation as well as
oxidative stress. Recent observations (1, 15, 19) have
also shown that an early critical event of both apoptotic
and necrotic cell death appears to be mitochondrial PT,
which is associated with several potentially lethal consequences, including the reduction of ⌬⌿m (38), the
uncoupling of the respiratory chain with increased
production of reactive oxygen species (ROS) (27), and
the liberation of preformed apoptogenic proteins such
AJP-Heart Circ Physiol • VOL
Fig. 7. Effect of cyclosporin A (CsA) on Bax translocation and Cyt C
release. Myocytes were incubated with 1 ␮M staurosporine for 1 or
4 h in the presence or absence of 0.2 ␮M CsA in the MI(⫺) group. At
1 or 4 h after the initiation of staurosporine treatment, mitochondrial and cytosolic fractions were prepared, and aliquots containing
20 ␮g of protein were subjected to Western blot analysis and were
probed with an antibody for Bax or Cyt C, respectively, as described
in MATERIALS AND METHODS. Results are representative of 3 independent experiments.
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Fig. 6. A: caspase-3 activity in cardiac myocytes. Cardiac myocytes
were subjected to MI, followed by staurosporine treatment as described in Fig. 2. Caspase-3 activity was determined as described in
MATERIALS AND METHODS (n ⫽ 6). *P ⬍ 0.01 vs. MI(⫺); †P ⬍ 0.05 vs.
MI10; ‡P ⬍ 0.01 vs. MI50. B: Western blot analysis of procaspase-3
cleavage. Cell extracts were prepared from the CONT, MI(⫺), and
MI10 groups, and aliquots containing 100 ␮g of protein were subjected to Western blot analysis and were probed with an antibody to
caspase-3 as described in MATERIALS AND METHODS. Arrow, location of
caspase-3. Results are representative of 3 independent experiments.
DEVD-CHO, acetyl-Asp-Glu-Val-Asp-aldehyde.
as cytochrome c and apoptosis-inducing factor (43, 55).
In addition, recent reports (41) have suggested that one
of the triggers of mitochondrial PT is translocation of
Bax, a proapoptotic protein, from the cytosol into mitochondria, and Bax may form selective channels for
cytochrome c release. In the present study, we show
that staurosporine caused the loss of ⌬⌿m as estimated
by JC-1 staining, a concomitant translocation of Bax
into the mitochondria, and the release of cytochrome c
into the cytosol, under ATP-supplying and ATP-depleting conditions. Moreover, cyclosporin A, an inhibitor of
mitochondrial PT, suppressed the staurosporine-induced apoptosis or necrosis through the inhibition of
the ⌬⌿m reduction, cytochrome c release, and caspase-3
activation, whereas it did not prevent Bax translocation.
This suggests that staurosporine caused Bax translocation into the mitochondria, raised the mitochondrial PT,
reduced ⌬⌿m, and liberated apoptogenic proteins from
the mitochondria independently of intracellular ATP levels. Our data also suggest that mitochondrial PT is an
early common step in the death signaling pathways of
apoptosis and necrosis in the myocytes, in agreement
with the previous observations (1, 15, 19), and that the
ATP-dependent steps occur downstream of the release of
cytochrome c from the mitochondria in staurosporineinduced apoptosis. Therefore, under conditions in which
intracellular energy levels are preserved, mitochondrial
PT induced by staurosporine can trigger apoptosis,
whereas under conditions in which protease activation is
precluded by loss of ATP, PT causes necrosis.
The precise mechanism by which ATP regulates the
apoptotic pathway remains unclear. However, recent
in vitro analysis has indicated that cytochrome c can
bind to Apaf-1 and this event unmasks the caspase
recruitment domain (CARD) motif in Apaf-1 permit-
INTRACELLULAR ATP AND APOPTOSIS
Study Limitations
Neonatal versus adult cardiac myocytes. Hypoxia-ischemia, with or without reoxygenation, is a potent
stimulator of apoptotic death in both neonatal and
adult cardiac myocytes in vitro and in vivo. In most of
the reports (25, 33, 52) on the induction of apoptosis,
neonatal myocytes are more resistant to either ischemia or hypoxia than adult ventricular myocytes. For
staurosporine-induced apoptosis, there are no data indicating comparison between neonatal and adult cardiac myocytes. As described in the previous reports (25,
32), both hypoxia and staurosporine stimulation cause
apoptosis via a mitochondria-dependent release of cytochrome c. Taken together, it is therefore speculated
AJP-Heart Circ Physiol • VOL
that neonatal myocytes are more resistant to staurosporine-treatment than adult ventricular myocytes.
Effect of staurosporine. The bacterial alkaloid staurosporine is reported to induce apoptosis in a variety of
cells. Staurosporine was initially described as an inhibitor of protein kinase C (44) and has been shown to
inhibit many different protein kinases (13). Although it
is well known that activation of protein kinases, such
as the mitogen-activated protein kinase family, affects
the induction of apoptosis, we have no data demonstrating the effect of protein kinases modulated by
staurosporine on apoptosis in the present study. The
mechanisms of staurosporine-induced apoptosis are
largely unknown. However, previous reports (20) indicate that staurosporine can cause mitochondrial ROS
production and elevation of intracellular free calcium
levels very early in the apoptotic process that might be
relevant to hypoxic or ischemia-reperfusion injury.
Effect of oligomycin. Apoptosis induced by overexpression of Bax has been reported to be prevented by
oligomycin-induced inhibition of F0F1-ATPase (30). As
described in previous reports (9, 23), inhibition of F0F1ATPase by oligomycin in glucose-containing media
does not necessarily prevent the induction of apoptosis.
In conclusion, the present study demonstrates that
staurosporine induces the translocation of cytosolic
Bax into the mitochondria and the loss of ⌬⌿m with a
concomitant release of mitochondrial cytochrome c into
the cytosol, and that it accelerates myocyte apoptosis
through the activation of caspase-3 under ATP-rich
conditions, but that it induces necrosis under ATPdepleting conditions. Thus, although mitochondrial PT
is an early common critical event in both types of
myocyte demise, intracellular ATP levels are an important determinant in the regulation of myocyte cell
death.
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