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Kidney and Hypertension Research Center, Department of Physiology, Medical Center of Fudan University, Shanghai 200032, People's Republic of China
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We used cultured
neonatal rat cardiac myocytes to test the hypothesis that
all-trans retinoic acid (atRA) may act to
modulate ANG II actions in inducing myocyte hypertrophy. Our
observations were as follows. 1) atRA
(10
7 to ~105 M ) inhibited ANG II-induced
hyperplasia of fibroblasts in a dose-dependent manner. 2)
Treatment of atRA attenuated the ANG II-induced increase in
total cell protein content. 3) Treated with ANG II
(107 M) for 5 days, the cultured neonatal rat cardiac
myocytes demonstrated an apparent accumulation of sarcomeric fiber
proteins and Golgi's complex, as well as reorganization of the
sarcomeric unit within individual myocytes. atRA
(106 M) treatment reduced the accumulation of contractile
proteins and Golgi's complex without affecting the ANG II-induced
reorganization of the sarcomeric unit. 4) atRA
attenuated the ANG II-induced increase in intracellular
Ca2+. Our results show that atRA inhibits some
effects of ANG II on neonatal rat cardiac myocytes and suggest that
atRA may be a therapeutic candidate for the prevention and
therapy of cardiac hypertrophy and remodeling.
cardiac hypertrophy; dedifferentiation; intracellular calcium; hyperplasia, induction of myocyte differentiation
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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CARDIAC HYPERTROPHY IS A
FUNDAMENTAL adaptive process in response to hemodynamic overload
and is thought to be one of the most important phases proceeding heart
failure. In response to increased demands for cardiac work or a variety
of pathological stimuli, the remodeling process of myocytes is evoked.
This response is characterized by an increase in myocyte size,
accumulation of contractile proteins, activation of embryonic gene
marker expression, and lack of a concomitant effect on muscle cell
proliferation. The hypertrophied myocyte contains more fetal type
proteins such as 
-myosin heavy chain (MHC), which induce the heart
to show a decreased contractile property with an increased oxygen
consumption (10, 21). Therefore, we consider the process
of cardiac remodeling as a course of myocyte dedifferentiation and
further hypothesized that cardiac hypertrophy may be reversed by
induction of myocyte differentiation. To test this hypothesis, we
selected all-trans retinoic acid
(atRA) to see whether atRA has any effect on the process of myocyte remodeling in an in vitro model.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Cardiac cell culture. Cardiac cells were dissociated from 1-day-old Wistar rat pups with 0.1% trypsin. To selectively enrich for myocytes, dissociated cells were preplated in a cabinet at 37°C with 5% CO2 for 1 h. The nonmyocytes attached readily to the bottom of the culture dish (21, 27). The resultant suspension of myocytes was plated into culture dishes at a density of 5 × 105 cells/ml. One hundred micromolar bromodeoxyuridine was added during the first 24-36 h to prevent proliferation of nonmyocytes (26). Using this method, we routinely obtained cultures with ~95% myocytes, as assessed by microscopic observation of cell beating.
Cells adherent to the culture dishes during the preplating procedure were resuspended with culture medium and then allowed to reattach for 30 min at 37°C. After two repetitions of this procedure, the cells were distributed into culture dishes and incubated in the culture medium described above, except for using 10% calf serum without bromodeoxyuridine. In the third passage, the cells were mainly cardiac fibroblasts (21). Cells in the third or fourth passage were used for experiments.Cell proliferation assay. Subconfluent nonmyocytes were seeded in 96-well plates at a density of 4,000 cells per well and cultured for 12 h in the presence of 10% calf serum. Subsequently, cells were washed twice with serum-free medium and growth arrested in the serum-free medium for 24 h. Then the quiescent cells were treated with ANG II and/or atRA for 24 h. ANG II was supplemented at 12 h to compensate for a decrease due to degradation by endogenous angiotensinase. Control cultures contained DMSO vehicle. To terminate the experiment, the plates were rinsed twice with 0.8 ml of PBS, fixed with 4% formaldehyde for 1 h, washed twice with PBS, stained with 0.5% crystal violet for 2 h, distained with distilled water, and solubilized in 36% acetic acid. Absorbency at 650 nm was measured on a multiwell scanning spectrophotometer. Absorbency at 650 nm was directly proportional to the number of cells in the wells (8). Results were presented as means ± SE of multiple cultures.
Quantification of total cell protein content. Myocytes collected from primary cell cultures were suspended in culture medium, and cell numbers were counted for four times. Same amounts of cell suspension were seeded in each well of the six-well plates. After incubation in serum-free medium for 24 h, cardiac myocytes were stimulated with ANG II (100 nM) for 5 days in the presence of indicated concentrations of atRA. ANG II was supplemented every 12 h to compensate for the decrease due to degradation, and atRA was replenished when the culture medium was replaced with fresh serum-free medium every other day. Dishes were washed three times with chilled PBS to remove trace medium, and the cells were dissolved in 2% SDS (1 loading buffer). Protein concentrations were measured by a modification of the method of Lowry et al. (18).
Analysis of MHC profile.
Cultured myocytes were homogenized (1:10, wt/vol) in 75 mM Tris buffer,
pH 6.8. The homogenate was placed in a 100°C heating block for 10 min
and immediately placed on ice for 5 min. The sample was then vortexed
and centrifuged at 12,000 rpm for 10 min. An aliquot of the supernatant
was used to determine protein concentration by a modification of the
method of Lowry et al. (18), and the rest was used for
SDS-PAGE or stored at 
80°C for later use (13).
-mercaptoethanol, and
0.001% bromphenol blue. Samples were boiled for 3 min, immediately placed on ice for 1 min, and centrifuged at 12,000 rpm for 5 min before
loading the gel.
The stacking gels consisted of 5% glycerol, 4%
acrylamide-N,N'-methylene-bis-acrylamide
(bis) (50:1), 70 mM Tris (pH 6.8), 4 mM EDTA, and 0.4% SDS. Separating
gels were composed of 5% glycerol, 8% acrylamide-bis (50:1), 200 mM
Tris (pH 8.8), 100 mM glycine, and 0.4% SDS. The upper electrode
buffer consisted of 0.1 M Tris (base), 150 mM glycine, 0.1% SDS, and
10 mM -mercaptoethanol, and the lower electrode buffer of 50 mM Tris
(base), 75 mM glycine, and 0.05% SDS (30). The gel was
run in a V-16 unit at 4°C. After electrophoresis, the gels were
stained with Coomasie blue R-250 for at least 4 h and destained
until the bands were apparent. Gels were photographed and subsequently
placed in 30% glycerol for storage. The photographs were scanned and
analyzed by using Leica image analysis system to determine the relative
quantities of 
- and -MHC.
Ultramicroscopic structure observation. Cultured neonatal rat cardiac myocytes were detached from the cultured flasks and collected by centrifugation. The cells were fixed, dehydrated, infiltrated, and embedded in epoxy resin. Fifty- to sixty-nanometer ultra thin sections were cut on LKB-1 ultramicrotome and stained before they were observed and photographed by a JEM-1200 EX transmission electron microscopy.
Measurement of intracellular Ca2+ levels. Cardiac myocytes were cultured on glass coverslips in serum-free medium. Before experiments, those coverslips were placed in a Petri dish containing 1 ml of serum-free medium. Fluo 3-AM loading of the cells was performed by adding 0.33 ml of labeling stock solution to the culture medium, which consisted of 10 parts of 1 mM fluo 3-AM in dry DMSO, 15 parts of 10% Pluronic F-127 solution, and 975 parts of culture medium containing 2% BSA. After the parts were mixed, the dishes were incubated in a cabinet containing 5% CO2 at 37°C for 30 min. After two repetitions, a coverslip containing fluo 3-loaded cells was mounted on the stage of a fluorescence microscope. The excitation wavelength was 500 nm, and the fluorescence signals emitted by the cells were observed and photographed by an auto-photography system. The signals remained stable for about 1.5 h at 28°C. The photographs were scaned and analyzed by a Leica image analysis system, and the results were presented as integrated fluorescence density. The background was determined with the nonlabeled cells (25).
Statistical analysis. Data are given as means ± SE. When data were amenable to statistical analysis, a one-way ANOVA was performed with Student-Newman-Keuls test for significance. Significance was accepted at P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Hyperplasia of nonmyocytes.
ANG II induced a significant increase in cell number over 24 h in
a dose-dependent manner (Fig.
1A). The values of absorbency in the wells of control and the wells treated with 1010,
109, 108, and 107 M ANG II
were 0.1778 ± 0.0096, 0.2032 ± 0.0237, 0.2592 ± 0.0189, 0.3147 ± 0.0158, and 0.2923 ± 0.0153, respectively.
As shown in Fig. 1B, atRA dose dependently
inhibited ANG II-induced hyperplasia of nonmyocytes. ANG II induced a
64% increase in cell number over 24 h, and this increase was
abolished by 106 and 105 M atRA.
The values of absorbency in the control group and in the groups of
108 M ANG II, 108 M ANG II + 107 M atRA, 108 M ANG II + 106 M atRA, and 108 M ANG
II + 105 M atRA were 0.1778 ± 0.0096, 0.3147 ± 0.0158, 0.2588 ± 0.0139, 0.2168 ±
0.0173, and 0.1867 ± 0.0098, respectively.
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7, 106, and
105 M atRA were 0.1778 ± 0.0096, 0.1808 ± 0.0133, 0.1772 ± 0.0153, and 0.166 ± 0.0199, respectively.
Myocyte protein content.
Total protein content increased by 30% in the myocytes incubated with
106 M ANG II for 5 days. Treatment with 106
M atRA abolished this increase, and 106 M
atRA alone caused no significant change in protein content (Fig. 2). The values of total protein
content of the group of control and the groups of 107 M
ANG II, 107 M ANG II + 106 M
atRA, and 106 M atRA were
1.520 ± 0.104, 1.965 ± 0.226, 1.592 ± 0.172, and 1.639 ± 0.161 µg/ml, respectively. However, 105 M
atRA caused a significant reduction in protein content in
not only the ANG II-treated group but also the control cultures.
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Cardiac MHC profile.
The display of MHC profiles of cultured neonatal rat cardiac myocytes
showed that treatment with 107 M ANG II for 5 days caused
a shift of the profile to the expression of -MHC. The ratio of
-MHC to -MHC in the group of control and the groups of
107 M ANG II, 107 M ANG II + 105 M atRA, 107 M ANG II + 106 M atRA, 105 M
atRA, and 106 M atRA was 0.710 ± 0.039, 0.887 ± 0.069, 0.868 ± 0.063, 0.815 ±
0.072, 0.923 ± 0.103, and 0.896 ± 0.091, respectively (Fig. 3). There was no detectable effect of
atRA on ANG II-induced shift in MHC profile within our
dosage range.
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Ultramicroscopic structure.
In the control cells, there were some sparsely distributed Golgi
complexes and sarcomeric fibers, but no typical Z band was observed
(Fig. 4A). Treated with ANG II
(107 M) for 5 days, the cultured neonatal rat cardiac
myocytes demonstrated apparently an accumulation of Golgi complexes and
nonparallel sarcomeric fibers as well as reorganization of the
sarcomeric unit within individual myocytes (Fig. 4B).
atRA (106 M) treatment reduced the
accumulation of sarcomeric fibers without affecting the ANG II-induced
reorganization of the sarcomeric unit (Fig. 4C). There is no
apparent difference in the quantity and distribution of sarcomeric
fibers and Golgi's complexes between the cells treated with
atRA alone and the control group (Fig. 4D).
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Intracellular Ca2+ levels in cultured
cardiac myocytes.
Fluo 3-loaded cardiac myocytes were analyzed for fluorescence emission
and photographed. Parallel experiments were conducted in which fluo
3-untreated cells were analyzed with the same protocol. Untreated
control myocytes formed monolayer sheets of synchronously beating
cells, with an average beating rate of 20 beats/min at 28°C. Fluo
3-loaded cells were incubated with 106 M ANG II for 4 min
at 28°C. This resulted in a rapid increase in intracellular
fluorescence. This elevated level of fluorescence maintained constant
for about 1.5 h at 28°C. The increase in fluorescence was in the
range of 1.5- to 2.0-fold. atRA treatment (106
M) attenuated the increase of intracellular fluorescence induced by ANG
II (106 M) (Figs. 5 and
6). atRA alone had no
detectable effect on the intracellular Ca2+ levels of the
cultured neonatal rat cardiac myocytes.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Hyperplasia of nonmyocytes. Cardiac fibroblast is the major cell type in the extracellular matrix of myocardium with the potent capacity to proliferate and secrete collagen. Hyperplasia of cardiac fibroblasts is an important pathological phenomenon in cardiac hypertrophy and remodeling. During cardiac hypertrophy, cardiac fibroblasts are documented to proliferate and produce much collagen, which is accumulated in the extracellular matrix and, in turn, causes myocardial fibrosis and the dysfunction of the myocardium (29). Our results showed that atRA can inhibit ANG II-induced proliferation of the cardiac fibroblast. Taking the evidence that ANG II can directly activate collagen synthesis in isolated adult rat cardiac fibroblasts, we hypothesize that atRA has a beneficial effect on the remodeling process of cardiac extracellular matrix.
Myocyte protein content. The increase in protein content in the cardiac myocytes is an important character of cardiac hypertrophy and remodeling (23). Sodashima and Izumo (24) reported that, in the cultured neonatal rat cardiac myocytes, treatment with ANG II for 5 days caused a dramatic increase in the protein content.
atRA plays an important role in cell growth and differentiation during development. It is documented that atRA plays a very important role in embryonic stem cell-derived cardiac differentiation and the development of ventricular cardiomyocytes (19, 33). atRA has two nuclear receptors: rapidly adapting receptor (RAR) and retinoid X receptor (RXR). Both of these receptors can be further categorized into three subtypes:, , and 
. RAR and RXR can form homodimers and
heterodimers, which can bind DNA in the nucleus with high affinity. RXR
can also form heterodimers with the thyroid and vitamin D3
receptors, and play a wider role in various biological processes. In
cardiovascular studies, research on RXR knockout mice provided direct
evidence that RXR is essential in the development of heart in the
homozygous RXR knockout mice. Deletion of RAR (i.e., RAR and
RAR) was shown to amplify some features of the RXR /
phenotype, suggesting that retinoid receptor-signaled activity may
converge at key points in embryogenesis to promote normal development
of the cardiovascular system (11, 15, 28).
In our study, atRA inhibited the increase in total protein
content during ANG II-induced cardiac hypertrophy. There are also reports showing that atRA can inhibit other phenotypes in
cardiac hypertrophy. Zhou et al. (36) reported that, in
cultured neonatal rat cardiac myocytes, atRA suppressed
phenyphrine and endothelin-1 (ET-1)-induced increase in cell size and
induction of atrial natriuretic factor (ANF). Wu et al.
(34) documented that atRA antagonized ET-1-induced ANF secretion and increase in cell size in cultured neonatal rat cardiac myocytes.
When it comes to the receptor type (RAR or RXR) by which
atRA inhibits cardiac hypertrophy, there are contradictory
receptors. Zhou et al. (36) showed that it is through RARs
that atRA inhibit phenylephrine-induced ANF secretion.
However, the results of Wu et al. are contradictory to those of Zhou et
al., and Wu et al. concluded that it is not RARs but RXRs that mediate
the inhibitory effects of atRA on ET-1-induced cardiac
hypertrophy (34).
Cardiac MHC profile.
Between 12 and 24 h after hypertrophic stimulation, the expression
of secondary response genes in cardiac myocytes is activated, which
include -MHC, ANF, S · · actin, myosin light
chains, and so forth. These genes are all "fetal-type genes," which
are expressed at high levels in the embryos, and their expression decreases or even ceases after birth. Their expression is responsible for the changes of morphology and function of the heart during cardiac
remodeling (10, 14).
-MHC increased dramatically, similar to our findings. During the induction of cardiac hypertrophy, the ratio of -MHC to -MHC mRNA increased because of the enhanced expression of -MHC, and the ratio has been a marker at the molecular level in cardiac hypertrophy and remodeling (14).
In our experiments, atRA treatment has no detectable effect
on ANG II-induced decrease in the ratio of -MHC-to--MHC. However, Rohrer et al. (22) reported that treating cardiac myocytes
with similar concentrations of atRA for 16 h increased
the expression of the -MHC gene significantly. The differences
between Rohrer et al.'s study and ours are that, besides the levels
(protein and mRNA) and time points studied, in Rohrer et al.'s
experiments, thyroid-free serum was used in cell culture. In such a
culture medium, the -MHC gene is hardly expressed and -MHC is
predominant. So if there is an increase in -MHC gene expression, it
will be easier to detect.
Ultramicroscopic structure.
Cultured in the serum- free medium, cardiac myocytes show a dispersed
myofiber and nontypical sarcomeric unit. In response to hormones or
growth factors such as ANG II, there will be an accumulation of
contractile proteins (including myosin light chain2, S · · actin, MHCs, and so forth) and myofibrillar
organization (9, 31). These events corresponded to the
pressure overload or the hormone- and growth factor-induced cardiac
hypertrophy and remodeling.
Intracellular Ca2+ levels. It has been shown that Ca2+ may be an initiator of cardiac hypertrophy and participate in the signal pathway activated by ANG II and ET-1 (21).
Increase in the extracellular Ca2+ concentration, activator of Ca2+ channel, and electric stimuli all can cause an elevation in the intracellular Ca2+ concentration and further induce cardiac hypertrophy and remodeling. Moreover, decreased capacity of reception of Ca2+ by the sarcoplasmic reticulum and the elevation of the basal intracellular Ca2+ are both correlated with the induction of cardiac hypertrophy and heart failure (4, 6, 17). During the transition from cardiac hypertrophy to heart failure, there is a dramatic increase in the intracellular Ca2+ in the diastolic phase and dysfunction in the diastolic phase of the heart (1, 3). ANG II can induce the increase in Ca2+ concentration either by activating the L-type calcium channel or releasing calcium stored in the sarcoplasmic reticulum (1, 2, 16). In human epidermal fibroblasts, release of stored Ca2+ alone is enough to activate the extracellular signal-regulated kinase, which is an important or even essential signal molecule in cardiac hypertrophy (5, 7, 35). The recent study on the role of Ca2+ and nuclear factors of activated lymphocytes in the hypertrophic responses showed that Ca2+-dependent pathways are very important signaling pathways in the induction of cardiac hypertrophy (20). Currently, there are very few reports on the effect of atRA on intracellular Ca2+. Varani et al.'s study (32) on the dermal fibroblasts showed that treatment of the cells with atRA alone did not alter the concentration of intracellular Ca2+ resulting from changes in extracellualr Ca2+ concentration. They further documented that this inhibitory effect was due to interference of Ca2+ movement across the plasma membrane, and it was specific for Ca2+ and has no effect for Ba2+. In the present study, atRA inhibited the ANG II-induced increase in the intracellular Ca2+ concentration in the cardiac myocytes. This inhibition is a direct evidence that atRA interfers with the transduction pathways stimulated by ANG II, and it might account for the inhibitory effects of atRA on cardiac hypertrophy and remodeling. The present study showed for the first time that atRA could attenuate ANG II-induced changes of cultured cardiac cells, suggesting a possibility of a new approach for cardiac hypertrophy. Further investigations should be performed to elucidate the mechanism of actions of atRA on cardiac cells as well as to demonstrate the effect of atRA on cardiac hypertrophy in vivo.| |
FOOTNOTES |
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Address for reprint requests and other correspondence: Y.-C. Zhu, Dept. of Physiology, Medical Center, Fudan Univ., 138 Yi Xue Yuan Rd., 200032 Shanghai, People's Republic of China (E-mail: yczhu{at}shmu.edu.cn).
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.
10.1152/japplphysiol.01192.2001
Received 3 December 2001; accepted in final form 31 December 2001.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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