Download Experimental study on the function of cardiomyocytes

Clinical Hemorheology and Microcirculation 29 (2003) 369–374
IOS Press
369
Experimental study on the function of
cardiomyocytes
Cheng Wang, Hongwei Li, Ailing Li, Jing Zhang and Ruijuan Xiu ∗
Institute of Microcirculation, Chinese Academy of Medical Sciences & Peking Union Medical College,
Beijing 100005, China
Abstract. This paper aimed at investigating the properties of cultured cardiomyocytes using microcirculatory and molecular
technology to culture cardiomyocytes from different parts of the neonate Wistar rat’s heart and record their spontaneous pulsation under time-lapse video microscopy, then analyze their activity and inspect their survival rate and apoptotic rate under natural
and nitric oxide conditions. The pulsation frequency in cardiomtocytes of different parts in heart are: 78.5 ± 11.0 beats/min in
the atrium, 88.4 ± 6.3 beats/min in the left ventricle, 90.3 ± 7.9 beats/min in the right ventricle and 115.3 ±11.4 beats/min in
the cardiac apex, respectively, with an average frequency of 81.3 beats/min. Different concentrations of nitric oxide showed no
effect on the frequency of cardiomyocyte pulsation. The survival rates of the above cardiomyocytes are 96.0%, 95.0%, 95.0%,
and 95.3% respectively and 95.0% for the whole heart. The apoptotic rates are 1.3%, 1.1%, 4.8%, and 1.8% respectively and
5.1% for the whole heart. Different concentrations of nitric oxide had no effect on these results. Our study showed that cultured
myocardial cells from different parts of the heart displayed various pulsation frequencies, and the frequency of the cardiac apex
is the highest while the atrium is lowest. We also found that there is no statistically significant difference in the survival rates
and apoptotic rates of different parts of the heart, and that nitric oxide has no effect on the beating frequency, survival rates or
apoptotic rates of the cardiomyocytes in vitro.
Keywords: Cardiomyocytes, beating rate, nitric oxide
1. Introduction
Cardiovascular disease is one of the major diseases throughout the world, and myocardial ischemia
is the early phenomena of heart disease in clinical trials [1]. Due to the complicated effects of hemodynamics, hormones and neural reactions in vivo, the myocardial ischemic model cannot reflect the effects
of some factors or elements during this process accurately, which influences the reliability and accuracy
of the research data. Recently, cell culture technology has been widely used in studies of the function of
human organs. A large number of tests have shown that cardiomyocytes primarily cultured in vitro could
reflect the function of the heart, while cells at neonatal stage are thought to be an effective model for the
study of myocardial ischemia [2].
In this study, we used molecular techniques to study the relationship between cardiomyocytes with
different beating frequencies, cell apoptosis and cell death under ischemic conditions and also to explore
the function of nitric oxide.
The current tendency is to combine research techniques from different medical fields to study the
function of cardiomyocytes from different angles [3–5], to expound on the function of cardiomyocytes
from a molecular level, reveal the pathophysiological processes that take place in cells during ischemia,
thus opening up new ways to prevent heart diseases.
*
Corresponding author: E-mail: [email protected].
1386-0291/03/$8.00  2003 – IOS Press. All rights reserved
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2. Materials and methods
2.1. Animal and apparatus
We used pure-bred Wistar rats aged 1–2 days (provided by the Institute of Medical Experimental
Animals, Chinese Academy of Medical Sciences, Peking Union Medical College, China). We used
DMEM/F12 medium (Gibco, USA), Fatal calf serum (Gibco, USA), 5 -BrdU (Sigma, USA), and L-AA
(Beijing Chemical Reagents Co., China). As experimental apparatus, we used a Time Lapse Recorder8050 (National, Japan), a 5000-series television camera (National, Japan), an inverted phase-contrast
microscope (Nikon, Japan), a water-jacketed incubator (Forma Scientific Model, USA), monitor (JVC,
Japan), and centrifuge (Heraeus, Germany).
2.2. Procedures
2.2.1. Cell cultivation [6,7]
We divided the cardiomyocytes into two groups: Group I consisted of cells from the whole heart,
Group II of cells from the atrium, left ventricle, right ventricle and cardiac apex, respectively. After we
had cut the whole heart into pieces, we rinsed the pieces with iced PBS several times to clean red blood
cells, digested them with 0.08% trypsin for 6 minutes at 37◦ C and abandoned the supernatant. Then we
added another 0.08% trypsin for 6 minutes and translocated the supernatant into a new tube with the
same volume of iced DMEM/F12 medium containing 10% FCS to neutralize the trypsin, and digested
the remained tissue with the trypsin 8–10 times to reach full digestion. We centrifuged the supernatants
for 10 minutes at 1500 rpm and collected the digested cells, observed the living state, then seeded the
cells into 25 cm2 cultivation flasks, and incubated at 37◦ C 5% CO2 air. We collected the medium after
90 minutes, centrifuged for another 10 minutes at 1500 rpm, and the cells left are cardiomyocytes. We
added 0.1 mM 5-BrduR into the medium and cultured the cells at 37◦ C, with 5% CO2 air. All the above
steps were carried out under sterile conditions. Finally, we identified the cardiomyocytes with HE dye
and α-actin dye.
2.2.2. Time-lapse video microscopy and image analysis
We added different concentrations of L-AA to the medium, and placed the flask on the stage of the
inverted phase-contrast microscope housed inside a temperature-controlled chamber. We recorded the
action state of the selected cells using a time-lapse recorder 8050 for a continuous 4 hours, then analyzed
the data.
2.2.3. Cell necrosis rate and cell apoptotic rate [8,9]
We blended the 4% Trypan Blue and 1.8% sodium chloride equally to produce 2% Trypan Blue solution, then mixed together a drop of cell suspension and a drop of Trypan Blue, deposited this for
2 minutes and counted 150 cells with the cell arithmometer. For cell apoptotic rats, we referred to the
manual from the Beijing Baosai Biological Reagent Co.
3. Results
3.1. The culture and identity of cardiomyocytes
The cardiomyocytes from the whole heart and each part of the neonatal Wistar rats began to grow
along the flask after 6 hours’ culture, and their shape changed from circles to polygons. Observed at
C. Wang et al. / The function of cardiomyocytes
371
normal display speed, we found that some cells exist individually, beat with their immobile frequency
and protrude around, while some cells couple together and take on a rhythmic synchronous beating
(Figs 1–3).
Figure 2 shows that the cardiomyocytes hold out pseudopod, have 1∼2 nuclei agreeing with the hypothesis that cardiomyocytes are polynucleus cells. Figure 3 shows that the α-actin dye is positive, which
proves that the cells cultured in this experiment are cardiomyocytes.
Fig. 1. Cardiomyocytes observed under an inverted phase-contrast microscope. (A: 10 × 10; B: 10 × 10.)
Fig. 2. Cardiomyocytes with HE dye. (A: 10 × 10; B: 10 × 10.)
Fig. 3. Cardiomyocytes with α-action dye. (A: 10 × 10; B: 10 × 10.)
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3.2. The beating frequency of different cardiomyocytes and the effect of L-AA
Table 1 indicates that there are differences in the beating frequency of cardiomyocytes from different
parts of the heart: there are significant statistical differences between cardiac apex and atrium, cardiac
apex and left ventricle, cardiac apex and right ventricle (P < 0.01). There are also statistic differences
between atrium and left ventricle and atrium and right ventricle (P < 0.05), while there is no statistical
difference between left and right ventricles. Under normal conditions, different concentrations of L-AA
have no effect on the beating frequency of each part of the heart (Table 1).
3.3. The necrosis rate of different cardiomyocytes and the effect of L-AA
Table 2 shows that there are no significant differences in necrosis rate among cardiomyocytes from
different parts of the heart under normal conditions, while different concentrations of L-AA have no
effect on the necrosis rate of each part of the heart (P > 0.05). Living cells could not be dyed, while
dead cells could be dyed blue by Trypan Blue (Table 2).
Table 1
The beating frequency of different cardiomyocytes and the effect of L-AA
Normal condition
Atrium
78.5 ± 11.0
Left ventricle
88.4 ± 6.3∗
Right ventricle
90.3 ± 10.9∗
Cardiac apex
115.34 ± 1.36∗∗
Whole heart
81.3 ± 7.3
∗
P < 0.05, ∗∗ P < 0.01.
Arginine (1 mmol/l)
81.5 ± 9.2
87.6 ± 10.1∗
93.3 ± 9.2∗
120.1 ± 9.3∗∗
85.0 ± 6.1
Arginine (5 mmol/l)
79.2 ± 15.5
94.3 ± 14.2∗
95.6 ± 10.6∗
122.8 ± 5.33∗∗
84.3 ± 9.8
Table 2
The necrosis rate of different parts of the heart under different conditions
Atrium
Left ventricle
Right ventricle
Cardiac apex
Whole heart
Normal condition
4.0%
5.0%
5.0%
4.6%
4.0%
Arginine (1 mmol/l)
4.2%
4.0%
5.2%
4.8%
3.2%
Arginine (5 mmol/l)
2.6%
4.8%
4.8%
3.4%
3.8%
Table 3
The apoptotic rate of different parts of the heart under different conditions
Atrium
Left ventricle
Right ventricle
Cardiac apex
Whole heart
P > 0.05.
Normal condition
1.3%
1.1%
4.8%
1.8%
5.1%
Arginine (1 mmol/l)
1.3%
5.8%
6.7%
3.1%
6.3%
Arginine (5 mmol/)
1.9%
4.5%
3.7%
6.7%
5.3%
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3.4. The apoptotic rate of different cardiomyocytes and the effect of L-AA
Table 3 shows that there are no significant differences in apoptotic rate among cardiomyocytes from
different parts of the heart under normal conditions, while different concentrations of L-AA have no
effect on the apoptotic rate of each part of the heart (P > 0.05) (Table 3).
4. Discussion
Cardiomyocytes consist of working cells and autorhythmic cells. Although working cells have excitability and contractility, they have no autonomy, and cannot beat spontaneously without extraneous
stimulation. Autorhythmic cells have autonomy, thus could beat spontaneously without extraneous stimulation. Because we have not distinguished working cells from autorhythmic cells, the beating cells here
are a mix of these two kinds of cells.
As we know, the autorhythmic signal starts from the sinoatrial node, and transfers to the Purkinje system via the atrioventricular node and atrioventricular bundle. We found in our experiment that the beating
frequency of cardiomyocytes in the atrium is slowest in the original part of the signal and fastest in the
final parts of signal transfer, so that there is a significant statistical difference between different parts of
the heart. We proposed that the differences in beating frequency of cardiomyocytes in our experiment
may be related to the metabolic level of cells per se, which might influence the physiological function of
cardiomyocytes. The cardiac apex lies in the bottom of the ventricule which has a high metabolism, so
the beating frequency of this area is highest in our experiment; the atrium lies at the top of the ventriculus
with a low metabolism, so the beating frequency of this area is lowest.
Nitric oxide is a new kind of signal transfer gas which exerts a profound influence on the cardiovascular system, immunity system and nerve system [10]. In a normal heart, nitric oxide is synthesized by
endothelial NO synthase through a series of reactions. NO can then activate Gc, increase the level of
cGMP and relax the smooth muscle. Moreover, it has been found that nitric oxide participates in every
phase of ischemic preconditioning as a signal transfer molecule under ischemic/reperfusion conditions,
playing an important role in this process [11–13].
As we know, myocardial ischemic injury is the early presentation of heart disease in clinical trials.
It can induce not only cell necrosis but also cell apoptosis. As one means of death, apoptosis occurs
mainly in the left ventricle and may lead to ventricle dysfunction or heart failure [14], suggesting that the
apoptosis of cardiomyocytes might be one means of the loss of cardiomyocytes in heart failure [14,15].
Although the active factor for apoptosis is not yet clear, we believe that this process must involve many
factors that work in coordination [16]. We found in our experiment that, under normal conditions, there
is no significant difference in necrosis rate and apoptotic rate among cardiomyocytes from different parts
of the heart, while different concentrations of L-AA have no effect on those results; but under ischemic
conditions, nitric oxide can enhance the tolerance of the myocardium to long-term ischemic stress, that
is, ischemic preconditioning. It can reduce the severity of ischemia and lethal arrhythmia, recover heart
function, delay myocardiac infarction and reduce infarcted heart size [17–19]. We speculate that these
results may help to clarify the effect of nitric oxide as an endogeneous signal molecule that can regulate
the cardiovascular function of cardiomyocytes under ischemic conditions.
Acknowledgement
This research was supported by the Foundation of National Innovative Projects of China, Chinese
Academy of Sciences.
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