Yonago Acta medica 2000;43:11–18

Induction of Apoptosis of Rat Neonatal Cardiomyocytes by Chemical
Ischemia and Reoxygenation: The Role of Phosphatidylserine
Kimiyo Ono, Shingo Ishiguro, Hiroaki Kuroda and Shigetsugu Ohgi
Second Department of Surgery, Faculty of Medicine, Tottori University, Yonago 683-0826,
Japan
Ischemia/reperfusion injury plays a crucial role in the induction of the cell death of
myocytes. The precise mechanism of the cell death, however, has not been elucidated
enough. This study examined the cell death of rat neonatal myocytes induced by chemical
ischemia and reoxygenation with an in vitro model, in terms of apoptosis, and the roleof phosphatidylserine, which is recognized with annexin V. Chemical ischemia andreoxygenation were conducted on the cultured myocytes obtained from 1- or 2-day-old
Wistar rats. The cells were divided into 4 groups exposed to chemical ischemia for 9 h(Group A), 18 h (Group B) and 24 h (Group C) and one group not exposed to chemical
ischemia (Control Group). DNA ladder formation on agarose gel electrophoresis wasnoted in Groups B and C followed by reoxygenation, but not in Group A, as well as all
4 groups without reoxygenation. There were cells positive to terminal deoxynucleotidyltransferase-mediated dUDP-biotin nick end labeling in all 3 groups except for the Control
Group; after reoxygenation, the number of cells became larger in Groups B and C thanin Group A. Flow cytometry revealed that annexin V-positive cells were 1.15 ± 0.82%
in the Control Group, 4.07 ± 3.8% in Group A without reoxygenation and 15.5 ± 6.3%
in Group A after 30-min reoxygenation, respectively; the value was significantly higher
in the latter than the former two (P < 0.01). Although 18-h and 24-h ischemia increased
the annexin V-positive cells, reoxygenation did not alter the number of cells in Groups
B and C. These results indicate that i) chemical ischemia followed by reoxygenation
variably induces apoptosis of rat myocytes, ii) long-term ischemia causes phosphatidylserine translocation on the cell surface membrane, regardless of reoxygenationand iii) mild ischemia necessitates reoxygenation to translocate phosphatidylserine,
which might play a crucial role in the initiation of apoptosis of the myocytes.
Key words: annexin V; apoptosis; cardiomyocyte; phosphatidylserine; reoxygenation injury

Recent reports have indicated that apoptosis of
myocytes plays an important role in cardiac
failure as well as ischemia/reperfusion injury inthe myocardium (Olivetti et al, 1994; Kajstura
et al., 1996; Dong et al., 1997; Anversa andKajstura, 1998; Takemura, 1998). Although
myocardial infarction was considered to be
characterized by necrotic cell death due to thebreakdown of cellular energy metabolism, there
is some evidence that myocyte loss during theacute stages of myocardial ischemia and reperfusion involves both apoptotic and non-apoptotic cell death (Gottlieb et al., 1994, 1996;
Saraste et al., 1997). The role of apoptosis inreperfusion injury has been addressed in rat andrabbit models (Gottlieb et al., 1994; Fliss andGattinger, 1996) in which reperfusion wasshown to accelerate the occurrence of apoptotic
cell death. In cardiomyocytes, however, themechanism of apoptosis has not been elucidated
enough (Gottlieb et al., 1996; Misao et al.,
1996; Haunstetter and Izumo, 1998; Yaoita etal., 1998; Yue et al., 1998). Therefore, it isimportant to examine each signal transduction

Abbreviations: FITC, fluorescein isothiocyanate; PS, phosphatidylserine; TUNEL, terminal deoxynucleotidyl transferase-mediated dUDP-biotin nick end labeling11

K. Ono et al.

pathway of cell death with reproducible
simulated models.
Apoptosis is characterized by chromatincondensation, reduction in cell volume, andcleavage of DNA by endonuclease into oligonucleosomal fragments. The early phase of
apoptosis, on the other hand, is accompanied by
the translocation of phosphatidylserine (PS) at
the surface of the cell (Koopman et al., 1994;
Martin et al., 1995; Van den Eijnde, 1997). The
expression of PS on the cell surface plays animportant role in the recognition and removal of
apoptotic cells by macrophages (Verhoven etal., 1995, 1999). A new method for the detection of early phase-apoptotic cells has beenused in a variety of organs and tumors (Koopman
et al., 1994; Martin et al., 1995; Castedo et al.,
1996; van Engeland et al., 1996). Researchers
have used a PS-binding protein as a specificprobe to detect the redistribution of this phospholipid. Annexins have been found to have ahigh affinity for aminophospholipids in thepresence of Ca2+ ions (Bratton et al., 1997; Van
den Eijnde et al., 1997). A member of thisfamily, annexin V, preferentially binds PS.
In the present study, we attempted to ascertain whether short-term chemical ischemia followed by reoxygenation of cultured rat neonatal
cardiomyocytes induces myocyte apoptosis ornot.

at 37˚C. The digestion medium was discarded.
The cells were washed twice with Hanks solution and resuspended in medium 199 (M199,
Bio-Whittaker, Walkersville, MD), supplemented with 4% fetal bovine serum (JRH
biosciences, Lenexa, KS), 100-U/mL penicillinand 100-µg/mL streptomycin (Bio-Whittaker),
10-µg/mL transferrin from bovine plasma(Wako Chemical) and 10-µg/mL bovinepancreas insulin (Wako Chemical). The cellsuspension was preplated on a 60-mm diameterplastic dish for 30 min at 37˚C in a 5% CO2
incubator for isolating fibroblasts. The nonattached cells were sowed onto collagen-coated
60-mm, 35-mm and 24-well culture dishes at aconcentration of 2.5 × 10 4 per 1 mm 2 andincubated in a 5% CO2 incubator at 37˚C for 48
h prior to use.

Chemical ischemia
The culture medium was replaced with a
glucose-free ischemic solution containing 136mmol/L NaCl, 5.4-mmol/L KCl, 1-mmol/L
CaCl 2 , 0.53-mmol/L MgCl 2 , 5.5-mmol/L
HEPES and 5-mmol/L NaCN to inhibit oxidative phosphorylation adjusted to pH 6.7. We
confirmed that the myocytes stopped beating at
an early stage. They were then incubated in thesame incubator for 9 h (Group A), 18 h (GroupB) and 24 h (Group C). The control Group was
not exposed to chemical ischemia.

Materials and Methods

Reoxygenation period

All animal procedures were conducted in accordance with the Guidelines for Animal Experimentation at the Faculty of Medicine,
Tottori University.

The ischemic solution was removed and myocytes were reoxygenated with M199. They
were incubated in the same incubator for 30 min
or 6 h.

Cell cultures and treatments
DNA extraction and electrophoresis

Hearts were removed from 1- or 2-day-old
Wistar rats and the ventricles were separated
from the atria and vessels. Neonatal ventricular
myocytes were cultured by the modification ofa method previously described (Simpson and
Savion, 1982; Ichiba et al., 1998). Briefly,
myocytes were dissociated with 0.1% collagenase type I (Wako Chemical, Osaka, Japan)

At the end of each incubation period, cardiomyocytes were harvested and washed twicewith phosphate-buffered saline. DNA wasextracted with a DNA Extractor WB Kit (Wako
Chemical, Osaka, Japan). Cells were kept in a
37˚C water bath for 60 min with 1-mg/mL
protease. DNA was extracted with sodium
12PS and apoptosis of rat cardiomyocytes

iodide and isopropyl alcohol (Wako Chemical)
and then precipitated with ethanol (Wako
Chemical). The precipitate was resuspended in
10-mmol buffer of Tris-HCl + 1-mmol EDTA.
DNA electrophoresis was carried out with 2%
agarose gel for 1.5 h at 50 V. As a size marker,
a 200-base pair DNA StepLadder (lot #87203,
Promega Corporation, Madison, WI) was used.
The DNA was then visualized with ethidium
bromide in UV light.

were analyzed with the standard Lysis II andCellfit software (Becton Dickinson). Damaged
myocytes exhibit PS on their surface to which
FITC-labeled annexin V attaches. Normal cells
are annexinV-negative. This method can beused as a specific marker for early cell damage
where the cell membrane is still intact.

Statistical analysis
All data are expressed as mean ± SD. The
significance of difference was evaluated byStudent’s t-test. Statistical analysis was performed using the software program StatView
version 5.0 on a Macintosh computer. Differences were considered significant if P was lessthan 0.05.

TUNEL assay
Cardiomyocytes were fixed in 90% ethanol for
4 h at room temperature and washed twice withphosphate-buffered saline. Terminal deoxynucleotidyl transferase-mediated dUDP-biotin
nick end labeling (TUNEL) staining was performed with a commercially available kit(ApopTag Peroxidase Kit, Intergen Company,
Purchase, NY) according to the manufacturer’s
instructions.

Results
The prepared rat neonatal cardiomyocytes
contracted spontaneously and synchronously at
an average rate of 179 ± 23 beats/min at 37˚C.
They were cultured under the chemical ischemia followed by reoxygenation, which provoked the cardiomyocytes to pulsate. For example,

Measurement of PS exposure by annexin
V-FITC
Translocation of PS in rat neonatal myocytes
was measured with annexin V-fluorescein isothiocyanate (FITC) using an Annexin V-FITC
Kit (Immunotech, MC, France). In brief, 5-µL/
well annexin V-FITC solution was added to thecells cultured in a 24-well plate with a 500-µL
medium and the plate was incubated for 15 minat room temperature. The cells were then harvested and washed twice with M199 containing
2.0-mmol Ca2+ ions and resuspended in M199
(106 cells/mL). Propidium iodide solution wasadded 2.55 µL/mL to the samples and the cells
were incubated for 15 min on ice. We
employed a confocal laser scanning system(INSIGHT PLUS, Meridian Instruments,
Okemos, MI) equipped with an Olympus IMT2 inverted microscope. The fluorescence
images were recorded using a CCD camera(Meridian Instruments) and analyzed using
INSIGHT-IQ software. For flow cytometricanalysis, a FACSort (Becton Dickinson, San
Jose, CA) equipped with a single argon ion
laser was used. Ten thousand cells per sample

M

1

2

3

4

5

(bp)

1000
800
600
400

200

Fig. 1. Internucleosomal DNA cleavage induced bysimulated ischemia and/or reoxygenation on 2%
agarose gel electrophoresis. A 200-base pair (bp)DNA molecular size marker (Lane M), ischemiafree control (Lane 1), ischemia for 9 h (Lane 2),
ischemia for 9 h + reoxygenation for 6 h (Lane 3),
ischemia for 18 h (Lane 4) and ischemia for 18 h +reoxygenation for 6 h (Lane 5). Lane 5 shows theladder pattern.

13

K. Ono et al.

Fig. 2. Light micrograph of the incubated cardiomyocytes with terminal deoxynucleotidyl transferasemediated dUDP-biotin nick end labeling (TUNEL) staining. The ischemia for 9 h + reoxygenation for 30 min
increases TUNEL-positive cells (arrowheads). Original magnification, × 400.

the cardiomyocytes exposed to 9-h chemical
ischemia pulsated at an average rate of 176 ± 19
and 226 ± 8.5 beats/min after 30-min and 60min reoxygenation, respectively.

(Fig. 2) and C, followed by 6-h reoxygenation.
The number of TUNEL signal-positive cells
was smaller in the groups which were exposed
to 0- and 9-h chemical ischemia (Group A), followed by 6-h reoxygenation.

DNA fragmentation
PS exposition after chemical ischemia
and reoxygenation

First, DNA ladder formation was examined on
the 2% agarose gel electrophoresis. No DNA
ladder was noted in the cardiomyocytes of the 4groups obtained immediately after chemical
ischemia. The cardiomyocytes showed DNA
ladders, only when the cells were harvested
after 18-h and 24-h chemical ischemia (Groups
B and C) followed by 6-h reoxygenation (Fig. 1
lane 5). On the other hand, no DNA ladder wasnoted in the Control Group without chemical ischemia or in Group A followed by 6-h reoxygenation (Fig.1).
Next, the cardiomyocytes were analyzed by
the TUNEL method to confirm apoptotic cell
death morphologically. Only a few TUNEL
signal-positive cells were noted in all 4 groups,
prepared immediately after ischemia without
reoxygenation. A good number of TUNEL
signal-positive cells were observed in Groups B

The cardiomyocytes were stained with annexin
V-FITC after chemical ischemia with/without
reoxygenation. Fluorescence-activated cell
sorter analysis showed no signal for FITC or
propidium iodide in the myocytes of the Control Group without chemical ischemia and reoxygenation (Fig. 3A). Figure 3B shows autofluorescence of the cardiomyocytes which were
exposed to 9-h ischemia and 30-min reoxygenation. On the other hand, annexin V-FITCpositive cardiomyocytes increased in the group
exposed to 9-h ischemia and 30-min reoxygenation, and outnumbered the positive cells in thegroup exposed to 9-h ischemia without reoxygenation (Figs. 3C and D).
Figure 4 shows the percentage of annexin
V-positive cells after ischemia with/without re14

FL2 – H/FL2 – Height→Secondarynecrotic

Viable Apoptotic

Annexin V-FITC

102

101

100
100

101

102

103

104

FL1 – H/FL1 –Height →104

FL2 – H/FL2 – Height→FL2 – H/FL2 – Height→

103

A

104

103

102

101

100
100

B

104

104

FL2 – H/FL2 – Height→Propidium iodide

PS and apoptosis of rat cardiomyocytes

103

102

101

100
101

102

103

104

102

101

100
10 0

101

C

FL1 – H/FL1 –Height →

103

102

103

FL1 – H/FL1 –Height →

10 4

100

D

101

102

103

104

FL1 – H/FL1 –Height →

Fig. 3. Schematic presentation of the flowcytometric results obtained with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide. A: results of control. B: ischemia for 9 h, autofluorescence. C:
ischemia for 9 h. D: ischemia for 9 h + reoxygenation for 30 min.

oxygenation. The average percentage of thepositive cells was 1.15 ± 0.82 in the Control
Group (n = 20), 4.07 ± 3.8 in Group A (n = 8)
without reoxygenation, and 15.5 ± 6.3 in GroupA (n = 8) with 30-min reoxygenation; the value
was significantly higher in the latter than in theformer two (P < 0.01). Thus, reoxygenation en-

hances translocation of annexin V (PS) at thecell surface. Although a number of the annexin
V-positive cells significantly increased after
18-h and 24-h ischemia (P < 0.05), reoxygenation did not increase the cells in Groups B and C(Fig. 4).

(%)
20

*

*

*

15

10

, with annexin V, with propidium iodide
and annexin V

5

0

Con

–

Group A

+

Con

–

+

Con

Group B–+

Group C

Fig. 4. Effects of chemical ischemia and reoxygenation on the viability of neonatal rat cardiomyocytes.
Group A, 9-h ischemia; Group B, 18-h ischemia; Group C, 24-h ischemia. +, with 30-min reoxygenation;–, without reoxygenation; Con, control. * P < 0.05.

15

K. Ono et al.

1994; Martin et al., 1995; van Engeland et al.,1996). We used FITC-labeled annexin V forthe detection of PS translocation as an initiationof cardiomyocyte apoptosis (Verhoven et al.,
1995; Fadok et al., 1998; Verhoven et al., 1999).
This process has been thought to precede DNA
fragmentation and loss of membrane integrity.
The mechanisms regulating the distribution oftransmembrane phospholipids and the implications of transbilayer lipid scrambling and cell
death are still under consideration (Castedo etal., 1996; Van den Eijnde et al., 1997; Bevers etal., 1998). The DNA stain propidium iodide
was included in the assay mixture to discriminate between the different types of cell death in
the present study. Annexin V-positive cells canbe divided into 2 populations based on the
integrity of the plasma membrane: cells with adamaged plasma membrane and cells with anintact membrane. The former are labeled withpropidium iodide and the latter with annexin V.
In response to the addition of cyanide and to
the glucose-free conditions during the ischemic
phase, the intracellular ATP levels decreased
and intracellular Ca2+ levels increased in cardiomyocytes. Moreover, reoxygenation markedly increases intracellular Ca2+ concentrationand causes the hypercontracture of cardiomyocytes within several minutes (Quaife et al.,
1991; Siegmund et al., 1994). This might be themain cause of the myocyte injury in the present
model because there are few influences fromthe extracellular environment such as cytokines. Some reports showed that an ATPdependent transporter called translocase isresponsible for the rapid inward movement ofphospholipids in normal cells and that the increased levels of intracellular Ca2+ of damaged
cells accelerates the redistribution of all phospholipids via Ca2+ dependent “scramblase”(Bratton et al., 1997; Bevers et al., 1998).
Ischemia reduced myocyte ATP levels. Whenreoxygenation starts, the intracellular ATP
level is still low and therefore, an overload of
intracellular Ca2+ might accelerate the translocation of PS via activation of “scramblase”.
In our study, 9-h ischemia with 30-min
reoxygenation induced the translocation of PS.
But 18-h ischemia without reoxygenation

Discussion
The present study clearly demonstrates thatchemical ischemia followed by reoxygenationinduces cell death in cultured rat neonatal
ventricular myocytes. This model has beenwell-established and was conducted to elucidate the mechanism of myocardial cell death byischemia as well as reoxygenation.
Long et al. (1997) used a cultured neonatal
rat cardiomyocyte model and showed that exposure of myocytes to hypoxia for 48 h resulted
in an intranucleosomal cleavage of genomic
DNA characteristic of apoptosis in 60% of thecells. This result is similar to that of Tanaka etal. (1994) who demonstrated that exposure ofcultured neonatal cardiomyocytes to more than
12 h of hypoxia induces apoptosis. On the other
hand, Karwatowska-Prokopczuk et al. (1998)
found that chemical ischemia for 3 h induced
myocyte apoptosis in 65% of rat neonatal cardiomyocytes. These results indicate that ischemia alone can induce apoptosis in rat cardiomyocytes. In the present study, it was foundthat chemical ischemia for 18 and 24 h followedby reoxygenation induces apoptosis, which was
confirmed by DNA ladder formation on agarose
gel electrophoresis, a hallmark of apoptosis.
No DNA ladder was noted in any other group,in which chemical ischemia was loaded for 9 h.
On the other hand, the nuclear TUNEL signal
has been demonstrated in various levels in thecultured rat myocytes of all 3 groups. Thus, 9-hischemia followed by reoxygenation did notshow any DNA ladder, in spite of the presenceof TUNEL signal-positive cells. This discrepancy might be explained by the small number of
the TUNEL-positive cells in Group A. Although the standard method for measuring apoptosis is the microscopic detection of morphological changes of the cells, this method is notavailable for quantitative analysis.
Conceptionally, apoptosis is divided into 3
phases: initiation, propagation and execution.
Recent studies have shown that changes in theplasma membrane have been recognized as an
attractive target for the analysis of apoptosis ofvarious organs or tumors (Koopman et al.,16PS and apoptosis of rat cardiomyocytes

potentiated apoptosis or necrosis (secondary
necrosis) and the influence of reoxygenation
alone was less extensive. Chen et al. (1998)
showed that 18-h chemical ischemia triggered
apoptosis of isolated neonatal rat cardiomyocytes and they distinguished between a reversible initiation phase and a subsequent irreversible execution phase. Our results suggestthat reoxygenation in the early stages of ischemia might result in the additional cell death ofpotentially viable myocytes. This would explain why apoptotic cardiomyocytes were observed in the transitional areas of the infarcted
myocardium of an in vivo experimental model(Saraste et al., 1997). In other words, ischemia
for more than 9 h induces myocardial injury andfurther reoxygenation promotes the damage.
The choice of strategies to prevent reperfusion
injury might differ with the degree of cell viability.
This study was based on the hypothesis that
PS exposure is an early signal of cardiomyocyte
apoptosis. However, few reports have investigated whether the transmembrane mobility ofphospholipids activates the signal transduction
pathways of cell death especially in cardiomyocytes. We did not obtain a DNA ladder at
9-h ischemia with reoxygenation. A variety ofstimuli induce apoptosis, acting through several
signaling pathways that eventually converge toa sequence of execution steps (Shimizu et al.,1996). This is why results may differ between a
DNA ladder and PS exposition. It is necessary
to examine each event leading up to myocyte
apoptosis with reproducible models.
In summary, chemical ischemia followed byreoxygenation induces apoptosis of neonatal rat
cardiomyocytes. The early phase of apoptosis
can be initiated by PS, which can then be successfully recognized with annexin V.

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(Received December 14, Accepted December 17, 1999)

18