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Herba Epimedii is an important traditional Chinese herbal medicine that is widely used as a tonic, aphrodisiac, and
antirheumatic in China. The content of icariin, its effective ingredient, has been used as a quality control standard. In our
previous work, the specific properties of embryonic stem (ES) cells and standard culture
methods[1] were used to confirm the inducible effects of icariin and its two metabolites on the differentiation of ES cells into
cardiomyocytes in vitro[2]. Icariin at
a concentration of 1×10-7 mol/L facilitates the directional differentiation of ES cells into cardiomyocytes. Cardiomyocytes are
characterized by the expression of
sarcomericproteins, a-actinin and cardiac troponin T. Icariin has been found to have
effects on regulation of the cell cycle, induction of apoptosis, as well as modulation of
p53 during the early differentiation of
ES cells and cardiac development, by using flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR) and
Western blot analysis[3]. But the other possible mechanisms by which icariin inducibly affects the directional differentiation
of ES cells into cardiomyocytes are still not completely understood and it is worthwhile to investigate them further.
Cardiac-specific genes, proteins, receptors and signaling molecules are expressed in a developmentally controlled
manner during cardiac differentiation of ES cells
in vitro, which closely recapitulate the developmental pattern of early cardiogenesis
in vivo[4]. With a view to detecting the targets of icariin action during the promotion of ES cells to differentiate, the expression
of cardiac developmental-
dependent genes was measured using RT-PCR, and the chronotropic
responsesof cardiomyocytes to b-adrenoceptor
stimulation were determined.
The second messengers, cAMP and cGMP, play an important part in regulating cellular proliferation and differen-tiation.
It is crucial that the correct cAMP/cGMP ratio is maintained. Moreover, if a compound heightens the cAMP level of cells, it
may facilitate differentiation and inhibit
proliferation[5,6]. Therefore, the levels of cAMP and cGMP in ES cells treated with
icariin were measured to clarify the possible actions of icariin on early differentiation events before a shift to the cardiomyocyte
phenotype.
NO, a potential differentiation signal molecule, is important for cardiac development. It has been reported that incubation
of embryoid body (EB) with nitric oxide synthase (NOS) inhibitors resulted in a pronounced differentiation arrest of
cardiomyocytes, whereas this effect could be reversed by coapplication of the NO donor. In the development of cardiogenesis,
the classic mode of action of NO is through intracellular signaling cascades via the activation of
cGMP[7]. When administered orally, icariin can increase the mRNA and protein expression of eNOS and iNOS as a result of regulating the endogenous NO
level to enhance erectile function[8]. In the present study, we tested the hypothesis that icariin promoted the differentiation
of ES cells into cardiomyocytes in part through regulating the endogenous NO signaling pathway. The concentration of
endogenous NO in ES cells during differentiation was evaluated by using the Griess reaction. In addition, aminoguanidine
(AG), an effective and selective inhibitor of iNOS, was used to explore the influence of icariin on endogenous NO levels in the
differentiation of ES cells into cardiomyocytes, and to confirm the targets of icariin action.
Materials and methods Cells, drugs, and reagents The permanent ES cell line D3 (ES-D3) was obtained from the American Type Culture
Collection (CRL-1934). Icariin was obtained from the Drug Biology Product Examination Bureau, Beijing, China (batch
No 0737-200011, purity 99%); retinoic acid (RA),
b-mercapto-ethanol, aminoguanidine (AG) and monoclonal
anti-sarcomeric-actinin were purchased from Sigma (St Louis, MO, USA); Dulbecco¡¯s modified Eagle¡¯s minimal essential medium (DMEM)
was purchased from Gibco Invitrogen Corporation (Grand Island, NY, USA). Fetal calf serum (FCS) and the non-essential
amino acids (NEAA) were purchased from Hyclone (Logan, UT, USA). Mitomycin C was purchased from Kyowa Hakko
Kogyo (Tokyo, Japan); and recombinant mouse leukemia inhibitory factor (LIF) was purchased from Chemicon International
(Temecula, CA, USA). The fluorescein isothiocyanate (FITC)-conjugated
F(ab)2 fragment of affinity-purified goat
anti-mouse IgG was obtained from Rockland (Gilbertsville, PA, USA). The reagents for the RT-PCR were purchased from the
Shanghai Sangon Biological Engineering Technological and Service Company (Shanghai, China).
125I-cAMP and 125I-cGMP were obtained from the Shanghai University of Traditional Chinese Medicine.
ES cell culture and icariin/aminoguanidine
treatment ES-D3 cells were cultured in an undifferentiated state on primary
cultures of mouse embryonic fibroblasts (MEF) cells in DMEM, supplemented with 10% FCS, 0.1 mmol/L
b-mercaptoethanol, NEAA and 1×106 U/L LIF. Cultures of differentiating ES cells were established by the formation of EB in hanging drop
cultures[2,9,10] with differentiation medium. On d 5, EBs were plated separately onto gelatin-coated 24-well culture plates, and
at that time, icariin was added to the medium at concentrations of
1×10-7, 1×10-8, or
1×10-9 mol/L, on the basis of preliminary
test results. EBs treated with 1×10-8 mol/L RA or with 0.1% dimethyl sulfoxide
(Me2SO) solvent were used as positive or
negative controls, respectively. To explore whether icariin promoted the differentiation of ES cells into cardiomyocytes in
part through regulating the endogenous NO signaling pathway, AG at a concentration of
1×10-3 mol/L was added to differentiation medium together with icariin on d 5 and maintained until d 5+11 (ie, 11 d after EB was plated onto gelatin-coated culture
plates on d 5). EB was observed with a light microscope every day to record the number of spontaneously beating EB.
In the experiment, d 1 corresponds to the day of dissociation of ES cells from MEF cells, and the initiation of differentiation
by the formation of EB. To identify whether the observed cell types derived from ES-D3 cells were cardiomyo-cytes, indirect
immunofluorescence for cardiac-specific protein was carried out according to a method published
elsewhere[2,3].
Detection of cardiac-specific transcripts by semi-quantitative RT-PCR
analysis Total RNA was isolated from EBs
(n=20) induced by 1×10-7,
1×10-8 or 1×10-9 mol/L icariin,
1×10-8 mol/L RA or solvent at different time points by using Trizol reagent.
After extraction, mRNA was precipitated and dissolved in a 0.1% diethylpyrocarbonate solution. To synthesize first strand
cDNA, 7 µL total RNA was incubated in 0.5 µg of oligo
(dT)6 primer and 5 mL deionized water at 65
oC for 15 min. Reverse transcription reactions were performed with 200 U M-MuLV reverse transcriptase in 5× reaction buffer and 1 mmol/L dNTP
mixture for 1 h at 42 oC. Multiplex polymerase chain reaction mixtures of 50 µL contained 1 µL of the RT reaction product,
10×PCR buffer, 25 U Taq polymerase, 1 µL of 10 mmol/L dNTP mixture, and 30 pmol of each primer. The specific primer pairs
designed by using Primer 3.0 software and published sequences were as follows (Table 1):
For the semi-quantitative determination of
Nkx2.5, GATA4, a-MHC,
MLC-2v and b-AR mRNA levels, the products of the
reverse transcription reactions were denatured for 3 min at 94
oC, followed by 35 cycles
(Nkx2.5), 35 cycles (GATA4), 40 cycles
(a-MHC), 45 cycles (MLC-2v), 40 cycles
(b-AR) or 30 cycles (b-actin) of amplification with Ampli
Taq DNA polymerase as follows: 45 s denaturation at 94
oC, 40 s annealing at 54.0 oC
(Nkx2.5 and GATA4), 66.4
oC (a-MHC), 61.1
oC (MLC-2v), 54.0 oC
(b-AR) or 55.0 oC
(b-actin), then 45 s elongation at 72
oC. The PCR products were separated by 1.5% agarose gel electrophoresis,
visualized with ethidium bromide staining, and quantified using a Bio-imaging Analyzer (Bio-Rad,CA,USA), and the density
of the products were quantitated using Quantity One (version 4.2.2) software (Bio-Rad).
Measurement of contraction of cardiomyocytes derived from ES-D3
cells The in vitro function of ES-D3 cell-derived
cardiomyocytes was examined by evaluating the chronotropic effects of cardio-active
drugs[12]. Isoprenaline (a b-AR agonist)
was cumulatively added to the pulsating clusters of EB induced by
1×10-7 mol/L icariin, 1×10-8 mol/L RA or solvent on d 5+5,
d 5+9, and d 5+13, respectively. Then the frequencies were measured in control (ie, the basal level of beating frequency,
before agonist treatment) and agonist-treated variants to create dose_response curves. Measurements were also carried out
in the presence of the antagonist propranolol. Images of the beating cardiomyocytes were monitored with a charge coupled
device (CCD) video camera attached to the microscope observation port, and also displayed on a computer screen. The
frequency of contraction was detected by using a photoelectricity transformation system, which transformed photic signals
into electrical signals[13].
cAMP and cGMP determination assay To clarify the mechanism by which icariin affects the early differentiation of ES
cells before a shift to the cardiomyocyte phenotype, cAMP and cGMP levels in ES cells were measured by using a
125I-cAMP and 125I-cGMP double antibody radioimmunoassay
method[14]. ES-D3 cells were cultivated without MEF
feeders and LIF, and treated with
1×10-7 mol/L icariin or
1×10-8 mol/L RA. After 24_48 h, the cells were harvested, placed into a polypropylene tube
and centrifuged. The supernatant was removed and 1 mL acetic acid buffer (pH 4.5) at 4
oC was added to the cell pellet. Then samples were repeatedly frozen in liquid nitrogen and quickly melted in warm water (5 times). Protein was removed by
centrifugation at 5000×g for 30 min.
Measurement of supernatant NO production
Supernatant NO concentration was measured by using the Griess
reaction[14]. The nitrite concentration was determined by using a curve calibrated with a sodium nitrite standard.
Statistical analysis Data were expressed as Mean±SD. At least 3 independent experiments were carried out to calculate
mean. Statistical significance was evaluated with one-way ANOVA by using SPSS 10.0 for Windows.
P<0.05 was considered statistically significant.
Results
Identification of cardiomyocytes derived from ES-D3 cells
Apparent cell morphological changes during the course of
differentiation were shown in Figure 1. The differentiated beating cardiac cells stained positively with
anti-a-actinin mAb. These results accorded with those of our previous
studies[2,3].
Expression of cardiac-specific genes during the early cardiac developmental
stage Cardiomyocytes from EB expressed the cardiac transcription factors
GATA4 and Nkx2.5, as well as the cardiac-specific genes
a-MHC, MLC-2v and
b-AR. Icariin significantly upregulated the mRNA levels of transcription factors
GATA4 and Nkx2.5 from the onset of cardiac
differentiation (P<0.01). The mRNA levels of
a-MHC, MLC-2v and
b-AR of cells in the presence of icariin increased during
the early cardiac developmental stage in a concentration- and time-dependent manner
(P<0.05). In particular,
1×10_7 mol/L icariin-induced MLC-2v
expression was detected as early as d 5+2, whereas increased expression in control cells was not
observed until d 5+7 (Figure 2).
Chronotropic responses of ES-D3 cell-derived cardio-myocytes
The spontaneously beating capacity of ES cell-derived
cardiomyocytes enables the investigation of the chronotropic effect of cardioactive substances by measuring the beating
frequency of cardiac cells[12]. A positive chronotropic response was observed after administration of the
b-agonist isoprenaline. Furthermore, the effect of isoprenaline on ES-D3 cell derived cardiomyocytes was different in the early and late
developmental stages. The beating EBs early on d 5+5 were insensitive to isoprenaline (Figure 3A). In an intermediate phase (d 5+9), the
beating frequency was significantly increased in the presence of isoprenaline in a concentration-dependent manner
(P<0.01). The frequency of beating EB treated with
1×10-7 mol/L icariin was increased by 20% when
1×10-11 mol/L isoprenaline was added. As the concentration of isoprenaline increased gradually to
1×10-6 mol/L, the frequency was increased by
approximately 80%, whereas the corresponding increase was only 50% for controls (Figure 3B). At the terminal differentiation stage
(d 5+13), the cardiomyocytes were highly sensitive to isoprenaline
(P<0.01). In the presence of isoprenaline at
concentrations ranging from 1×10-11 to
1×10-6 mol/L, the con
traction rate of cardiomyocytes induced by
1×10-7 mol/L icariin increased from approximately 30% to 130% (Figure 3C).
Propranolol (a b-adrenoceptor antagonist) at a concentration of
1×10-6 mol/L caused a reduction in the frequency increase. A
representative trace showing the effect of isoprenaline and propranolol on the beat frequency of the cardiomyocytes is
shown in Figure 4.
Levels of cAMP and cGMP in ES cells The concentrations of cAMP and cGMP in ES cells were 0.233 nmol/L and 0.060
nmol/L, respectively, and the cAMP/cGMP ratio was 3.875. When ES cells were treated with icariin at a concentration of
1×10-7 mol/L for 24 h, the levels of cAMP and cGMP significantly increased to 0.660 nmol/L and 0.083 nmol/L, respectively
(P<0.05). The cAMP/cGMP ratio also increased to 7.870. Treatment of ES cells with icariin for 48 h caused elevation of the
concentration of cAMP and the ratio of cAMP/cGMP
(P<0.01), but the concentration of cGMP was barely increased (Table
2).
Production of supernatant NO In the control, supernatant NO production was low. When ES cells were treated with
icariin at a concentration of 1×10-7 mol/L for 24 h or 48 h, supernatant NO production was markedly elevated
(P<0.05). In particular, the NO-cGMP pathway was involved in the early differentiation of ES cells treated with icariin for 24 h. During the
course of differentiation (from d 5+2 to d 5+11), icariin facilitated the directional differentiation of ES cells into cardiomyocytes,
and supernatant NO generation
increased (P<0.05). In the presence of AG, an effective and selective inhibitor of iNOS, NO generation during differentiation
decreased significantly, but was rescued by icariin
(P<0.05; Figures 5 and 6).
Inducible effect of icariin together with AG on the directional differentiation of ES-D3 cells into
cardiomyocytes To further determine the effects of icariin on modulation of the endogenous NO signaling pathway during the differentiation of
ES cells into cardiomyocytes, cultures were supplemented with AG to partially block NO production. Under these conditions,
whether the percentage cardiac differentiation was influenced, and the number of spontaneously beating EB were recorded.
ES cells were induced into rhythmically beating EB by icariin in a concentration- and time-dependent
manner[3]. The percentage differentiation in cultures containing contracting EB with
1×10-7 mol/L icariin was approximately 7% on d 5+3, and
reached a peak level of 86% on d 5+11 (P<0.05). In the control, only approximately 55% beating EBs were found on d 5+11.
AG significantly delayed and decreased the incidence of contracting EB compared with control cells: beating EB was not
observed until d 5+4 and the percentage was only approximately 35% on d 5+11. When icariin-treated cells were co-cultured
with AG, AG produced a decrease in the percentage of beating EB to 73% on d 5+11 compared with cells treated with icariin
alone. The potential of EB to undergo cardiac differentiation was
significantly enhanced in comparison with that in solvent
together with AG treatment starting from d 5+3
(P<0.05;
Figure 7).
Discussion
Our experiments demonstrated that icariin markedly enhanced the mRNA levels
of GATA4, Nkx2.5,
a-MHC, MLC-2v and b-AR at the early stage of differentiation. It has been reported that the
GATA4 and Nkx2.5 transcription factors appear before
other cardiac genes are
expressed[15]. Myocardial genes such as
a-MHC are expressed relatively early during the course of
cardiogenesis[16]; however, MLC-2v
mRNA is expressed at high levels relatively late, and is spatially restricted to the
ventricular area[17]. In the present study RT-PCR analysis revealed that cardiac differentiation of ES cells induced by icariin
recapitulated this developmental pattern, as GATA4
and Nkx2.5 were detected earlier than cardiac
a-MHC, MLC-2v and
b-AR. Icariin-induced MLC-2v expression upregulation was detected as early as d 5+2, whereas increased expression in control
cells was not observed until d 5+7. These results suggest that one of the mechanisms by which icariin is involved in
differentiation is by facilitating the expression of the cardiac developmental-dependent genes in a developmentally
controlled manner. In particular, icariin could enhance the development of ventricular cardiomyocytes at an early differentiation
stage.
It is important to characterize the responses of cardiomyo-cytes derived from ES cells, and one of the key modulators of
contraction is the b-AR system[18,19], therefore in the present study the responses of the beating rate of cardiomyo-cytes to
b-AR stimulation were determined. We confirmed that
b-AR responses were well developed in cardiomyocytes derived from
ES cells. The cardiomyocytes treated with icariin were more sensitive to isoprenaline than the case of in controls. The
different sensitivities of the cardiomyocytes to isoprenaline may result from the different expression levels and the degree of
maturity of b-AR[20], the hypothesis that is supported by the results of our RT-PCR analysis.
NO is a potential differentiation signal that plays an important role in the differentiation of ES cells into
cardiomyo-cytes[21]. In most instances, NO signaling pathways are typically dependent on cGMP. The cGMP-dependent pathways are
initiated by NO-induced activation of soluble guanylate cyclase by binding to heme iron. This activation results in a
transient increase in the second messenger
cGMP[22]. Another messenger, cAMP, also plays an important role in regulating
cellular proliferation and differentiation. The effects of cAMP and cGMP are opposite. It is crucial to maintain the correct
cAMP/cGMP ratio to provide the correct microenvironment for cell survival and function. Upregulation of cAMP is a key
process for facilitating differentiation and inhibiting proliferation. Icariin induces the differentiation of HL-60 cells, possibly
by elevating the cAMP/cGMP ratio[5]. In the present study, treatment of ES cells with icariin for 24_48 h resulted in an
elevated cAMP concentration and cAMP/cGMP ratio. This indicates that upregulation of cAMP concentration and
cAMP/cGMP ratio is important for the differentiation of ES cells in early differentiation events, before a shift to the cardiomyocyte
phenotype. In the present study, when ES cells were treated with icariin for 24_48 h before a shift to the cardiomyocyte
phenotype, supernatant NO production was markedly elevated. The cGMP level was also elevated with icariin treatment for
only 24 h. On the basis of the results of the radioimmunoassays and the Griess reaction measurements, we suggest that the
NO-cGMP pathway is only involved in the early differentiation of ES cells treated with icariin for 24 h before a shift to the
cardiomyocyte phenotype. However, the fact that the involvement of NO was independent of cGMP in early differentiation
warrants further investigation.
Further studies were aimed at the involvement of icariin in the NO signaling pathway during cardiomyocyte phenotype
formation. NO is generated by the NOS family of proteins. Both NO and NOS isoforms have been shown to induce or
facilitate the differentiation of several cell types, including nerve
cells[23], some tumor cell
types[24], and cardiac
cells[25]. ES cell-derived cardiomyocytes have been found to have an identical pattern of NOS expression as that detected in mouse heart.
In the early developmental stage, ES cell-derived cardiomyocytes displayed strong iNOS and eNOS expression. Conversely,
in the late developmental stage, iNOS was not detected and eNOS expression was found only at low levels. iNOS was
expressed only in the early cardiac differentiation stage and was important for cardiac development. Work with mouse ES
cells has shown that NOS inhibitors arrest differentiation toward the cardiac phenotype, and this effect can be rescued by NO
donors[21]. Orally administered icariin has been shown to increase the mRNA and protein expression of eNOS and iNOS and
regulate the level of NO to enhance erectile
function[8]. However, whether icariin improves cardiovascular function in part
through regulating the level of NO has not been demon-strated. In the present study, an
in vitro model was used to investigate the influence of NOS inhibitors on cardiac development within EB treated with icariin. AG is an effective and
specific inhibitor of iNOS. During the course of differentia-tion, icariin facilitated the directional differentiation of ES cells
into cardiomyocytes and supernatant NO concentration increased. AG decreased the endogenous NO level and arrested
differentiation toward the cardiac phenotype. This effect of AG could be rescued by icariin. These findings suggest that
icariin promotes the differentiation of ES cells into cardiomyocytes in part through upregulating the endogenous NO level,
which may be related to inducing iNOS expression. They also indicate that NO plays an important role in normal cardiac
development as a potential differentiation signaling molecule.
In conclusion, the promoting effects of icariin on cardiac differentiation depended on advancing and increasing the
expression of the GATA4,
Nkx2.5, a-MHC, MLC-2v and
b-AR genes. Icariin could also increase cAMP concentration and the
cAMP/cGMP ratio, and promote endogenous NO generation during early differentiation and cardiac
development.
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