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Methods Find Exp Clin Pharmacol 2007, 29(10): 681-687
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2007 Prous Science
CCC: 0379-0355/2007
DOI: 10.1358/mf.2007.29.10.1147767
Application of Flow Cytometry in the Study of Apoptosis in Neonatal
Rat Cardiomyocytes
H. Kniewald1, I. Mal
ci
c1, K. Rado
sevi
c2, V. Gaurina Sr
cek2, I. Slivac2, D. Polan
cec3,
M. Matija
si
c3, J. Kniewald2 and Z. Kniewald2
1
Department of Pediatrics, Clinical Hospital Center Zagreb; 2Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia; 3GlaxoSmithKline Research Center Zagreb, Biology, Zagreb, Croatia
SUMMARY
Depending on the concentration, catecholamines activate various intracellular signaling pathways and can induce apoptosis in cardiac myocytes. Although 5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolocarbocyanine iodide (JC-1) has been previously used to study mitochondria in
intact cardiomyocytes, there have been no reports on the detection of apoptosis in neonatal cardiomyocytes in combination with flow cytometry and
confocal microscopy. In our study, neonatal rat cardiomyocytes were exposed to norepinephrine (NE) and isoproterenol (ISO) in concentrations of 1
and 10 mM for 48 h. NE concentrations of 1 and 10 mM decreased the number of viable cardiomyocytes by 18% (*p < 0.05) and 24% (**p ¼ 0.01),
respectively. ISO in a concentration of 1 mM increased the number of viable cardiomyocytes by 13% while 10 mM decreased the number of viable
cardiomyocytes by 43% (***p < 0.001). Apoptotic cells were detected by flow cytometry and confocal microscopy. NE in concentrations of 1 and
10 mM increased the percentage of apoptotic cells by 12.2% and 34.3%, respectively, while ISO alone in a concentration of 10 mM increased the percentage of apoptotic cells by 11.3%. The results demonstrated that these two methods are reliable and suitable for the detection and study of apoptosis in cultures of neonatal cardiomyocytes. ' 2007 Prous Science. All rights reserved.
Key words: Apoptosis - Confocal microscopy - Flow cytometry - Isoproterenol - Neonatal cardiomyocytes Norepinephrine
INTRODUCTION
Apoptosis, that is, programmed cell death, plays an
important role in causing heart failure (1-3), but the
mechanism is still unclear. Many investigations have
demonstrated that apoptosis occurs in the myocardium
in a variety of pathological conditions but there is still
much debate concerning the nature of its role (4). There
is evidence, both in human and animal models, suggesting that apoptosis may be an important mode of cell
death during heart failure. The number of apoptotic
myocytes is elevated in myocardia obtained not only
from patients with end-stage heart failure and myocardial infarction but also from experimental models of
myocardial hypertrophy and failure, including aorticbanded rats (5), spontaneously hypertensive rats (6) and
rats with myocardial infarction (7). Experiments with
cultured cardiac myocytes have demonstrated that apoptosis can be stimulated in vitro by several endogenous
peptides that are increased in the hypertrophied or failing myocardium, including tumor necrosis factor-alpha
(TNF-a), angiotensin II and atrial natriuretic peptide, as
well as norepinephrine (NE) and isoproterenol (ISO)
(8). NE is the primary transmitter of the sympathetic
nervous system and stimulates apoptosis of cardiomyocytes through the a- and b-adrenergic pathways. The
effect of NE is seen through its binding to G-proteincoupled adrenergic receptors and the subsequent activation of the cAMP-protein kinase A pathway (9-11).
ISO is a b-adrenergic receptor agonist that mimics the
effect of NE by increasing the number of apoptotic
cells (10).
MATERIALS AND METHODS
Cardiomyocytes isolation
Neonatal cardiomyocytes were isolated using the
neonatal cardiomyocyte isolation system (NCIS) purchased from Worthington Biochemical Corporation
(Lakewood, NJ, USA). Primary ventricular cardiac
myocytes were prepared from 2- to 5-day-old Fisher rat
pups. The hearts from 6 to 10 rats were excised, the ventricles pooled and the ventricular cells isolated according to NCIS protocol. The beating hearts were surgically
excised and placed in a tube with ice-cold CMF HBSS
buffer. The hearts were rinsed in the solution and transferred to a petri dish, having been kept on ice. The tissue
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H. Kniewald et al./Methods Find Exp Clin Pharmacol 2007, 29(10): 681-687
was minced with a blade to <1 mm3 pieces, and CMF
HBSS buffer was added. Dissolved trypsin was transferred to the petri dish with the minced tissue, mixed
thoroughly by swirling and placed in the refrigerator
overnight (16–20 h) at 2–8 8C. The tissue and buffer
were then transferred to a centrifuge tube, and dissolved
trypsin inhibitor was added. The contents were mixed
and the oxygenation of the tissue was preformed for
1 min with O2 passed over the surface of the liquid. The
mixture was then warmed to 30–37 8C, and dissolved
purified collagenase was added. The tube with its content was placed on a magnetic stirrer and incubated at
37 8C for 30–45 min. The following steps were performed in a sterile hood at room temperature. Trituration
with a pipette was repeated approx. 10 times to release
the cells. After allowing the tissue residue to settle for a
few minutes, the supernatant was filtered through a cell
strainer into a new tube. An additional amount of Leibovitz L-15 media was added to the tissue residue to repeat
the trituration step and filtered through the cell strainer
into the same tube as mentioned earlier. Culture media
was added, and the cells were oxygenated for 1 min and
then left undisturbed for 20 min at room temperature.
The cells were swirled gently. If there was no clumping
and the appearance was uniform, sedimentation was performed at 50–100g for 5 min, and the cell pellet was
suspended in the culture medium. To reduce the background signal from the subcellular debris during flow
cytometry analysis, the red blood cells were lysed in
ammonium chloride buffer (8.29 g l1 NH4Cl, 1 g l1
KHCO3 and 0.037 g l1 Na2EDTA; pH ¼ 7.2) for 3 min
at room temperature. After final centrifugation, the cell
pellet was suspended in suitable culture medium with
added serum and pipetted gently to disperse the cells
before counting them in a haemocytometer. The cell
concentration was adjusted, and the cells were transferred to tissue culture ware and placed in an incubator
at 37 8C.
Cultured cells, conditions and treatment
After isolation, the cells were plated in 24-well culture plates (Costar, Corning Incorporated, NY, USA) at
a density of 1.25 105 cells/cm2 in DMEM/F:12 (1:1),
supplemented with 10% fetal bovine serum (FBS), both
from Gibco (Paisley, Scotland). A mixture of antibiotic/
antimycotic (0.1%; Sigma, St. Louis, MO, USA) was
also added. The cells were cultured in a humidified
atmosphere with 5% CO2 at 37 8C. After 48 h, the cells
mostly demonstrated regenerated contracting activity
and were treated with selected stimuli, norepinephrine
hydrochloride named Aterenol (1 and 10 mM; Aventis
Pharma, Germany) and isoproterenol hydrochloride
(1 and 10 mM; Abbott Laboratories, IL, USA). After 48 h,
the cells were harvested, counted in a hemocytometer
using the Trypan-blue exclusion method and collected
for further analysis.
Cell counting
The cell number was determined by counting in a
Fuchs-Rosenthal hemocytometer, using the Trypan blue
method. The nonadherent cells were removed by washing with DMEM/F:12, trypsinized, resuspended in the
culture medium and counted. The cells that remain
unstained are considered to be viable.
Immunocytochemistry assay
Mouse monoclonal anti-a-sarcomeric actin (Acris
Antibodies GmbH, Hiddenhausen, Germany) was used
as the primary antibody to identify the cardiomyocytes
in the culture. Staining was followed by the use of Rphycoerythrin (R-PE)-conjugated rat anti-mouse monoclonal IgM (BD Biosciences Pharmingen, San Diego,
USA) as the secondary antibody according to the manufacturer’s instructions with a minor modification.
DRAQ-5 dye (Biostatus, Leicestershire, UK) was used
to counterstain cell nuclei according to the manufacturer’s protocol. Samples were analyzed using an
LSM5210 META confocal microscope (Carl Zeiss,
Jena, Germany). The images were photographed and
further processed using AutoDeblur software (AutoQuant Imaging).
Flow cytometry
Cardiomyocytes were treated with NE and ISO for
48 h and prepared for analysis. 5,50 ,6,60 -Tetrachloro1,10 ,3,30 -tetraethylbenzimidazolocarbocyanine
iodide
(JC-1) dye (Invitrogen-Molecular Probes, Eugene, OR,
USA) was used for the detection of depolarized mitochondria in apoptotic cells. Staining was performed
according to the manufacturer’s instructions. One milliliter of each cell sample (1 106 cells/ml) was transferred to a 15-ml centrifuge tube and centrifuged at
room temperature. To each pellet, 0.5 ml of JC-1 working solution was added and incubated for 15 min at
37 8C in a CO2 incubator. Each sample was washed
twice, first with 2 ml and then with 1 ml of 1 assay
buffer at room temperature. The cells were then gently
mixed and incubated for 15 min at room temperature in
the dark. The samples were resuspended in 0.5 ml of 1
assay buffer following centrifugation at 400g for 5 min
and kept on ice until flow cytometric analysis. The samples were analyzed on a FACScan flow cytometer (BD,
San Diego, CA, USA), using CellQuest Pro software
(BD). The data were analyzed using Summit software
(Dako, Copenhagen, Denmark) and compiled using
Microsoft Excel software.
Confocal microscopy
Part of the sample prepared with JC-1 dye for flow
cytometry was also used for confocal microscopy.
Images were taken with an LSM510 META (Carl Zeiss,
Jena, Germany) and processed using AutoDeblur software (AutoQuant Imaging).
H. Kniewald et al./Methods Find Exp Clin Pharmacol 2007, 29(10): 681-687
683
FIG. 1. Scanning electron microscopy of a primary culture of neonatal cardiomyocytes at the time of inoculation (A) and the developed monolayer (B).
Statistical analysis
All data are expressed as mean 6 SEM. Comparisons between the control and NE-, ISO-treated cells
were performed using a Student’s unpaired t-test. Statistical analysis was performed using the ANOVA procedure. Differences between two groups were considered
to be statistically significant if *p < 0.05, **p < 0.01 or
***p < 0.001.
RESULTS
Identification of cultured cardiomyocytes
A culture of neonatal cardiomyocytes (Fig. 1) was
established according to the NCIS protocol. The attach-
FIG. 2. Confocal microscopy of neonatal cardiomyocytes stained
with anti-a-sarcomeric actin mouse antibody (green) and DRAQ-5 dye
for nucleus staining (red); magnification 400.
ment of the cardiomyocytes occurred within 24 h of isolation and inoculation, and the cells began to spread,
showing specific morphological properties. After 48 h
of inoculation, the cardiomyocyte cells were elongated
with cross connections between the cells. At the same
time, beating activity appeared. The purity of the cardiomyocytes was verified by immunocytochemistry assay
using an anti-a-sarcomeric actin. The stained cells were
visualized by confocal microscopy (Fig. 2). Most of the
cells were flattened. Strands of well-organized crossstriated myofibrils ran in various directions (green color)
and the myocytes possessed one or two nuclei (red color).
Cell growth inhibition effect of NE and ISO on
cardiomyocyte cells
To quantify the effect of NE and ISO on cardiomyocyte viability, the cell number was estimated after 48-h
exposure according to the Trypan-blue exclusion
method (Fig. 3). The data presented are the means of
FIG. 3. Effect of NE and ISO on cardiomyocyte viability. The data
shown are the means of three to five experiments, each performed
in duplicate. *p < 0.05 versus control; **p < 0.01 versus control;
***p < 0.001 versus control.
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H. Kniewald et al./Methods Find Exp Clin Pharmacol 2007, 29(10): 681-687
FIG. 4. Effect of NE on apoptosis in neonatal cardiomyocytes, as assessed by flow cytometry. Control cells (A); 1 mM NE (B); 10 mM NE (C),
1 mM ISO (D); 10 mM ISO (E).
four experiments, each performed in duplicate. NE in
concentrations of 1 and 10 mM significantly decreased
the number of viable cardiomyocytes by 18% (*p <
0.05) and 24% (**p ¼ 0.01). ISO in a concentration of
10 mM decreased the number of viable cardiomyocytes
by 43% (***p < 0.001), while the exposure of cardiomyocytes to 1 mM ISO resulted in an increased number
of cells (13%).
Analysis of the mitochondrial membrane
potential (DCm)
The control and treated cells were stained with JC-1
and then analyzed by flow cytometry to obtain an apoptosis scatter plot. A two-parameter fluorescence display
of control cells (Fig. 4A) revealed that the apoptotic
cells mostly emitted green fluorescence characteristic of
the JC-1 monomer, whereas viable cells (region R2)
H. Kniewald et al./Methods Find Exp Clin Pharmacol 2007, 29(10): 681-687
685
FIG. 5. Confocal microscopy of cardiomyocytes labeled with JC-1. Cardiomyocytes were grown on cover slips and stained with JC-1 in the absence
control (A) and presence of 10 mM of NE (B).
emitted relatively high levels of both green and orangered fluorescence.
The percentage of apoptotic cells ranged from 12.2%
for 1 mM of NE to 34.3% for 10 mM of NE, respectively
(Fig. 4B and C). The cultured cells treated with 1 mM of
ISO did not affect the percentage of apoptotic cells,
while 10 mM increased the percentage of apoptotic cells
by 11.3% (Fig. 4D and E).
Confocal microscopy was used to confirm the results
obtained by flow cytometry. Images of the control and
treated cardiomyocytes exposed to 10 mM of NE and
stained with JC-1 are shown in Figure 5. The control
cells show heterogeneous staining of the cytoplasm with
both green and red fluorescence coexisting in the same
cell (Fig. 5A), while the apoptotic cells exhibit characteristic green fluorescence (Fig. 5B).
DISCUSSION
The primary culture of neonatal cardiomyocytes is a
valuable tool for various studies in the field of cardiac
investigation. This cell model has great potential for
studying the cellular and molecular aspects of cardiac
alterations due to injury, with emphasis on the fact that
the apoptosis of cardiomyocytes plays an important role
in causing heart failure (12). The original method for
establishing a neonatal rat cardiomyocyte culture (13)
has been modified by many scientists (14, 15), and several
commercial kits for the isolation of neonatal rat cardiomyocytes are available today. Using the NCIS protocol,
as described in the Materials and methods section, 2 106 cells/ml myocytes were obtained from a single heart,
according to the manufacturer’s instructions and literature
data (16). Beating activity, the main feature of a cardiomyocyte culture, was observed under a light microscope
48 h after inoculation when a monolayer was formed.
It is important to verify that the purity of the cardiomyocytes is high enough to eliminate the presence of
other cell types in the heart, mainly cardiac fibroblasts.
Figure 2 shows the confocal microscopy of neonatal cardiomyocytes stained with anti-a-sarcomeric actin mouse
antibody, which was specifically expressed in the myocytes (green color) but not the cardiac fibroblasts. The
immunocytochemistry assay confirmed that 95% of
the adherent cells was a population of cardiomyocytes.
The cell nuclei (red color) were counterstained with
DRAQ-5 dye. This DNA-interactive agent with a fluorescence signature is often used as a discriminating parameter
for nucleated cells, in combination with fluorochromelabeled antibodies for subpopulation recognition (17).
The exposure of cardiomyocytes to 1 and 10 mM of
NE, respectively, during 48 h showed a significant
decrease in cell number (Fig. 3). These data were related
to the results obtained when adult rat ventricular myocytes were exposed to 10 mM of NE (10). At the same
time, ISO in a concentration of 1 mM increased cardiomyocyte growth, while 10 mM ISO decreased the number of viable cardiomyocytes. Literature data showed
that the treatment with 5 mM ISO increased cardiomyocyte growth after 48 h by about 40% (18). It is
known that catecholamines activate various intracellularsignaling pathways, depending on their concentration
or on the cell stimulation by other peptides or growth
factors (19).
When apoptosis is induced in a population of cells,
they undergo morphologically apoptotic changes in an
asynchronous manner. Therefore, flow cytometry is the
method of choice because it allows the cell-by-cell analysis of an entire cell population. Flow cytometry can be
used for the analysis of the mitochondrial membrane
potential (DCm) in whole cells (20). Because the maintenance of DCm is fundamental to the normal function
and survival of cells that have a high-energy requirement, such as beating cardiomyocytes, measurement of
DCm is essential for understanding the mechanisms
involved in cardiomyocyte function. Loss of DCm as an
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early apoptotic event was studied using JC-1, a mitochondrial depolarization sensitive dye (21). Although
JC-1 has been previously used to study mitochondria in
intact cardiomyocytes (22-24), there have been no
reports on the use of this dye in combination with flow
cytometry to evaluate changes of DCm in neonatal cardiomyocytes. The carbocyanine dye JC-1 is permeable
to the plasma membrane and is generally thought to
accumulate specifically in active mitochondria in proportion to the magnitude of their DCm (24, 25). The
small percentage of the control population of cells with
depolarized DCm may reflect the basal level of apoptosis or the presence of other cellular processes that are
associated with depolarized DCm, because changes in
the DCm have also been described during necrosis (26)
and cell-cycle arrest (27).
To observe apoptosis induced by NE and ISO in neonatal cardiomyocytes, cultured cells were treated with
these stimuli in concentrations of 1 and 10 mM and
examined by flow cytometry 48 h after treatment. The
results obtained in a scatter plot (Fig. 4) demonstrate
that the neonatal cardiomyocytes lost their mitochondrial
membrane potential due to NE and ISO treatment. The
number of apoptotic cells was significantly increased in
both the NE concentrations (Fig. 4B and C) and in the
ISO concentration of 10 mM (Fig. 4E) when compared
with the control cells. ISO in a concentration of 1 mM
(Fig. 4D) did not show any effect on the treated cell population. Although ISO may have an activating or protective effect on programmed cell death (19), the possible
dose-dependent effect should be investigated further.
The results obtained by flow cytometry were confirmed by confocal microscopy. In living cells, JC-1
penetrates the plasma membrane of cells as a monomer.
Because the normal mitochondria of healthy cells are
polarized, JC-1 is rapidly taken up, increasing the concentration of the dye and leading to the formation of Jaggregates within the mitochondria. JC-1 aggregates
accumulated in the control, nontreated cells showed
higher levels of red fluorescence (Fig. 5A). JC-1 does
not accumulate in apoptotic cells with depolarized mitochondria and remains in the cytoplasm as a monomer,
which exhibits fluorescence in the green end of the spectrum, does not have a red spectral shift and therefore has
lowered red fluorescence. In cells treated with NE, apoptosis was induced, and these cells exhibited the green
fluorescence characteristic of the JC-1 monomer, as
shown in Figure 5B.
The results demonstrated that neonatal cardiomyocyte culture is a valuable model for studying apoptosis
in vitro, as well as for the investigation of potential therapeutic compounds and other biomedical researches
related to cardiopathogenesis. A combination of these
two methods, flow cytometry as a quantitative method
and confocal microscopy as a qualitative method,
proved to be useful for the analysis of apoptosis in vitro.
Furthermore, we can conclude that NE and ISO have a
significant antiproliferation effect, probably related to
the induction of apoptosis and the disruption of mitochondrial membrane potential. The molecular basis of
these findings is under our active investigation and represents a major challenge for our future research.
ACKNOWLEDGMENTS
This work was supported by the Ministry of Science,
Education and Sports of the Republic of Croatia (Grants
No. 0058001 and No. 0058010).
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Address for correspondence: Zlatko Kniewald, Head of the Laboratory for Cell Culture Technology, Application and Biotransformation,
Faculty of Food Technology and Biotechnology, University of
Zagreb, Pierottijeva 6, HR-10000 Zagreb, Croatia. E-mail: zlatko.
[email protected]