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Introduction
Cardiac hypertrophy is recognized as an adaptive
response characterized by the growth of individual
cardio-myocytes in size rather than the increase in cell number.
Initially beneficial, sustaining cardiac hypertrophy
eventually leads to decompensation and results in dilated
cardio-myopathy, arrhythmia, fibrotic disease, heart failure, and
even sudden death[1]. Furthermore, some studies have indicated
that hypertrophy may not be required for a successful
adaptation to increased workload[2]. Clinical studies have found
that several classes of drugs, including
angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers,
calcium channel blockers[3_5], and nitric oxide
(NO)[6] could have some beneficial effects in the prevention or treatment
of cardiac hypertrophy. However, more studies should be
done to provide more therapeutic choices for cardiac
hypertrophy.
It has been reported that ginsenosides extracted from
the root of the herb Panax ginseng CA. Mey have many
pharmacological effects, including increasing the activity of
superoxide dismutase[7] and protecting the brain and heart
from ischemic and reperfusion
injuries[8,9]. Notably, ginseno-side
Rb1 (Rb1), a major component in ginsenosides, has been
shown to elevate the release of NO in rat ventricular
myo-cytes[10] and decrease intracellular free
Ca2+ in cardiac myo-cytes and other
tissues[11_13], which indicates that
Rb1 may be a potential drug for anticardiac hypertrophy.
Prostaglandin F2α (PGF2α) has been shown to induce
cardiac myocyte hypertrophy in vitro and cardiac growth
in vivo and is a good candidate to mediate the growth of
cardiac cells[14]. Meanwhile,
Ca2+ signaling has been reported to play a critical role in the development of cardiac
hypertrophy induced by various hypertrophic
stimuli[15]. The increased intracellular
Ca2+ binds to calmodulin and regulates several downstream effectors, such as calcineurin (CaN),
which is a key mediator of cardiac
hypertrophy[16,17]. CaN dephosphorylates the nuclear factor of activated T cells
(NFAT), and then later translocates to the nucleus where it
acts with other transcription factors (eg
GATA4) for the activation of downstream target genes to induce cardiac
hypertrophy[18,19]. A series of studies has shown that the
neuro-protective activities and anti-aging function of
Rb1 were related to decreasing intracellular
Ca2+[11_13]. However, whether
Rb1 has antihypertrophic effects on cardiac hypertrophy and
inhibits the Ca2+_CaN signal pathway has not known as yet.
Materials and methods
Primary culture of myocytes Ventricular myocytes from
1_3-d-old rats (Animal Center of Institute of Surgery
Research of the Third Military Medical University, Chongqing,
China) were prepared and cultured for 48 h in Dulbecco's
modified Eagle's medium (DMEM) containing 20% fetal
bovine serum and 0.1 mmol/L bromodeoxyuridine (Sigma, St
Louis, Missouri, USA) as described
previously[20]. The cells were adjusted to
1.5×106_3×106 cells/mL for measuring
intracellular free calcium concentration
([Ca2+]i), or to
0.5×106_1×105 cells/mL for measuring cell diameter and protein
content. The medium was replaced by serum-free DMEM
for a further 48 h before the treatment of drugs. 100 nmol/L
PGF2α (Cayman Chemical, Ann Arbor, Michigan, USA) was
used to stimulate the cardiomyocytes,
Rb1 with 99% purity and final concentrations of 50, 100, and 200 µg/mL (Division
of Chinese Material Medical and Natural Products, National
Institute for the Control of Pharmaceutical and Biological
Products, Ministry of Public Health, Beijing, China) and
L-arginine with final concentration of 1 mmol/L (Alexis, Lausen,
Switzerland) were used to investigate their antihypertrophic
effects. NG-nitro-L-arginine-methyl ester
(L-NAME; Alexis, Lausen, Switzerland) 1 mmol/L was used to investigate the
relationship between the antihypertrophic effects of
Rb1 and NO.
Measurement of cardiomyocytic diameters The
cardio-myocytes were fixed in 4% polyformaldehyde solution and
stained with HE. The diameter of single cells was measured
by the BI2000 Imaging Analytic System (Chengdu Taimeng
Sci-Tec, Chengdu, China). Five random fields (10-15 cells
per field) from each slide were analyzed. The experiments
were repeated 3 times.
Measurement of cardiomyocytic protein contents
The cardiomyocytes were digested by trypsinase and counted.
The cells were then washed 3 times with Hanks' balanced
salt solution (HBSS) without Ca2+ and
Mg2+ (D-HBSS; in mmol/L: NaCl 137.0, KCl 5.0,
Na2HPO4 0.6,
KH2PO4 0.4,
NaHCO3 3.0, glucose 5.6, pH 7.2) by centrifuging at
400×g for 2 min. The cardiomyocytes were homogenized with RIPA lysis buffer
(Upstate, Lake Placid, New York, USA) and centrifuged at
12 000×g for 20 min at 4 °C. The protein concentration in the
supernatant was determined by the Bradford assay using
bovine γ-globulin as the standard, then the protein
concentration per cell was calculated.
Measurement of
[Ca2+]i The
[Ca2+]i was measured by the method described
before[21]. Briefly, the cells
(1×106) were incubated in the medium with Fura 2/AM (5 µmol/L; Sigma,
St Louis, USA) for 50 min at 37 °C, then washed 3 times with
HBSS (D-HBSS plus 1.30 mmol/L CaCl2 and 0.5 mmol/L
MgCl2) containing 0.2% bovine serum albumin by
centrifuging at 500×g for 2 min. The fluorescence value from 1 mL cell
suspension was measured by a Shimadzu RF-5000
spectrofluorometer (Kyoto, Japan) with dual excitation wavelengths
at 340 and 380 nm and emission wavelengths at 510 nm. The
[Ca2+]i was calculated by the following equation:
[Ca2+]i=Kd×(
F_Fmin)/(Fmax
_F). Here, Kd was the dissociation constant
of Fura 2/AM for Ca2+ (about 224 nmol/L at 37 °C),
F was the basal fluorescence value of the cells,
Fmax was the fluorescence value under the presence of excess calcium in the cells
due to the lysis of the cellular membrane caused by 0.98 g/L
Triton-X 100 (Sigma, St Louis, Missori, USA),
Fmin was the fluorescence value under the presence of minimal calcium
using 5 mmol/L ethyleneglycotetraacetic acid (EGTA) to
chelate the Ca2+ in the cells after the lysis of the cellular
membrane by Triton-X 100.
RT-PCR analysis of mRNA Total RNA was extracted
from the cardiomyocytes by use of an RNeasy mini kit
(Qiagen, Valencia, California, USA). RT-PCR was performed
with an RT-PCR kit (Promega, San Jose, California, USA)
according to the manufacturer's instructions. The
nucleotide sequence of the primers were as
follows[22,23]: (i) atrial natriuretic peptide (ANP): sense 5'- GCC CTG AGC GAG CAG
ACC GA -3', antisense 5'-CGG AAG CTG TTG CAG CCT
A-3'; (ii) CaN: sense 5'-ACT GGC ATG CTC CCC AGC GGA-3',
antisense 5'-GTG CCG TTA GTC TCT GAG GCG-3'; and (iii)
β-actin: sense 5'-GAC TAC CTC ATG AAG ATC CTG ACC-3', antisense 5'-TGA TCT TCA TGG TGC TAG GAG CC-3'.
The predicted products in size were 202, 244, and 423 bp,
respectively. These primers were synthesized by Beijing
Dingguo Biotech (Beijing, China). The following conditions
of the RT-PCR reactions were met: (i) 1 cycle at 48 °C for 45
min, 94 °C for 2 min; (ii) 35 cycles at 94 °C for 30 s, 60 °C for 30
s, and 72 °C for 1 min; and (iii) 72 °C for 7 min. The products
were separated by electrophoresis on 1% agarose gel
containing ethidium bromide, and photographed. The integral
optical density values for each band of ANP, CaN, and
β-actin on the gel were assayed by the BI2000 Imaging
Analysis System (Chengdu Taimeng Sci-Tec, China).
β-actin was used as an internal control for the semiquantitative assay.
Western blotting The protein (30 µg) from
cardiomyo-cytes separated by 10% SDS-PAGE was transferred onto
polyvinylidene difluoride nylon membranes.
The blots were probed with mouse
anti-CaNA-α (1:200 dilution) or
anti-NFAT3 (1:100 dilution) or
anti-GATA4 antibodies (1:100 dilution; (Santa Cruz Biotechnology, Santa Cruz, California,
USA), and then with horseradish peroxidase-conjugated goat
anti-mouse immunoglobulin G (1:2500 dilution) antibodies.
Immunodetection was carried out using the BI2000 Imaging
Analysis System.
Statistical analysis All of the data were expressed as
mean±SD and analyzed by either ANOVA or Student's
t-test with SPSS 11.5 software (SPSS Inc, Chicago, Illinois,
USA. Differences were considered statistically
significant at P<0.05.
Results
Effects of Rb1 on PGF2α-induced cardiomyocyte
hypertrophy Light microscopic findings of the cardiomyocytes
showed that the cardiomyocytes treated with
PGF2α (100 nmol/L) became swollen and enlarged with undistinguishable
borders among the cells (Figure 1B).
Rb1 (200 µg/mL) markedly alleviated the morphological changes induced by
PGF2α (Figure 1C). The addition of
L-NAME (1 mmol/L) could not antagonize the effect of
Rb1 on the hypertrophic myocyte (data not shown).
Table 1 showed that the diameters and protein contents
of the cardiomyocytes treated with PGF2α
significantly increased, compared with that of the control
(P<0.01). The treatment of
Rb1 with a variety of concentrations (50, 100,
and 200 µg/mL) significantly relieved the changes induced
by PGF2α in a concentration-dependent manner
(P<0.05). L-arginine (1 mmol/L) also lowered these changes induced
by PGF2a (P<0.01). L-NAME (1 mmol/L) abolished the
effects of L-arginine, but failed to abolish the effects of
Rb1 (200 µg/mL) on the cardiomyocyte diameter and protein
content.
There was a low fundamental expression of ANP mRNA
in the cardiac myocytes (0.005±0.002,
n=3). PGF2α treatment obviously increased the ANP mRNA expression, which
was significantly antagonized by
Rb1 (200 µg/mL) treatment (Figure 2A).
Effects of Rb1 on
PGF2α-induced
[Ca2+]i in cardio-myocytes
The resting [Ca2+]i was 149.7±26.2
nmol/L (n=6), and it increased by 83% after the cardiomyocytes were treated
with PGF2α (100 nmol/L) for 48 h
(P<0.01). Treatments with either
Rb1 at the concentrations of 50, 100, and 200 µg/mL or
L-arginine (1 mmol/L) strongly blocked the
[Ca2+]i increase caused by
PGF2α. Once again, L-NAME (1 mmol/L)
treatment abolished the effect of L-arginine
(P<0.01), but failed to antagonize the effect of
Rb1 (P>0.05; Figure 3).
Effects of Rb1 on transcription of CaN and expressions
of CaN, NFAT3, and GATA4 proteins from cardiomyocytes
treated by PGF2α The relative CaN mRNA expression was
0.225±0.023 in the control and increased by 52% in the
PGF2α-treated cardiomyocytes (Figure 2B;
n=3, P<0.01). Similar treatments with
PGF2α also significantly increased the
expressions of the CaN, NFAT3, and
GATA4 proteins of cardiomyo-cytes. The treatment of
Rb1 (200 µg/mL) markedly decreased
the mRNA expression of CaN and the protein expressions of
CaN, NFAT3, and GATA4 from cardiomyocytes treated by
PGF2α. L-arginine (1 mmol/L) also inhibited the protein
expressions of CaN and its downstream factors
(P<0.05;
Figure 4, n=3).
Discussion
It has been reported that the morphological changes of
cardiomyocyte hypertrophy can be induced in
vitro by stimulating cultured neonatal cardiomyocytes with various
growth factors and cytokines, such as angiotensin II,
endothelin-1, and PGF2α, which was similar to those induced
by pressure or volume load[14,24_26]. The characteristic
phenotype of hypertrophy following growth factor stimulation
includes an increase of cell volume and protein synthesis
with an accumulation of contractile proteins, organization of
the contractile proteins into sarcomeric
units, as well as the re-expression of fetal cardiac genes, including
ANP[27]. In this study, the findings from measuring the diameter,
protein content, and ANP mRNA expression of the cardiac
myocytes suggested that PGF2α could induce cardiomyocyte
hypertrophy resembling that described by Lai et
al[14], and that Rb1 could significantly decrease the elevated
cardiomyo-cyte volume, protein content, and ANP mRNA expression
caused by PGF2α.
Our results showed that PGF2α induced cardiomyocyte
hypertrophy with the elevating
[Ca2+]i level. Meanwhile, the
antihypertrophic effects of Rb1 were accompanied
simultaneously with the alleviating
[Ca2+]i level. L-NAME, an NO
synthase inhibitor, did not antagonize both effects of
Rb1 on hypertrophy and
[Ca2+]i of cardiomyocytes; on the contrary,
it abolished the antihypertrophic and decreasing
[Ca2+]i
effects of L-arginine. These results suggested that the
directly decreasing [Ca2+]i effect rather than the NO release
may be responsible for the antihypertrophic effects of
Rb1.
In the past several years, a number of experiments have
implicated that the CaN signal transduction pathway may
play an important role in the cardiomyocyte hypertrophy
process[28]. We have previously reported that cardiac
hypertrophy by PGF2α may be mediated by the CaN signal
pathway in rats[29]. In the present paper, the fact that the
transcription and expression of CaN, as well as the
expression of the CaN downstream factors increased with elevating
[Ca2+]i under the stimulation of
PGF2α to the cardiomyocytes, which were blunted by
Rb1, suggested that the interference of the CaN signaling pathway might be involved in the
antihyper-trophic mechanisms of Rb1.
L-arginine, an NO donor, was observed to inhibit
cardio-myocyte hypertrophy[30], which could be abolished by
L-NAME. Surprisingly, L-NAME had no influence on
either the antihypertrophic effect or on the inhibiting
[Ca2+]i rise from
Rb1. The results suggested that the antihypertrophic effect
of Rb1 might be different from L-arginine, but the
relationship between the Rb1 effects and NO still needs much
investigation.
In conclusion, our study demonstrates that
Rb1 can alleviate cardiac hypertrophy
in vitro, which may be mediated in part by an inhibitive effect on elevated
[Ca2+]i due to the inactivation of the CaN transduction pathway.
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