Extract
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Introduction
Mitochondrial biogenesis requires coordinated changes
in the metabolic enzymes of oxidative phosphorylation, the
TCA cycle, and fatty acid oxidation. The expression of
hundreds of nuclear-encoded mitochondrial biogenesis-related
genes is coregulated by a few nuclear transcription factors
and co-activators. Nuclear respiratory factor (NRF)-1 and
NRF-2 regulate many of the genes encoding oxidative
phosphorylation proteins[1, 2]. Peroxisome proliferator-activated
receptors (PPAR) regulate genes encoding enzymes and
transporters of fatty acid
oxidation[3-6]. Peroxisome proliferator-activated receptor
g coactivator-1 alpha (PGC-1α) is a nuclear-encoded transcriptional co-activator which
plays a critical role in the control of mitochondrial
biogenesis based on its ability to interact with transcription factors that
activate nuclear genes encoding mitochondrial
proteins[7-9]. Accumulating evidence supports a major role for NRF-1 in
mediating the effects of PGC-1α on mitochondrial biogenesis.
PGC-1α can interact specifically with NRF-1 through the
NRF-1 DNA binding domain. It can transactivate the
transcription of NRF-1 target genes involved in mitochondrial
respiration and induces NRF-1 mRNA[10]. In addition to its
activation of respiratory subunit genes, PGC-1α can also
upregulate genes involved in the mitochondrial fatty acid
oxidation pathway through co-activating
PPARα[11]. PGC-1α and PPARα are abundantly expressed in tissues with high
oxidative energy demands, such as cardiac and skeletal
muscles[12].
The p38 mitogen-activated protein kinase (MAPK) serves
as upstream events to regulate PGC-1α both at the
transcriptional and post-transcriptional level. For example, the
activation of the p38 MAPK was involved in exercise which
stimulates PGC-1α transcription in skeletal
muscles[13]. The p38 MAPK can phosphorylate
PGC-1α in 3 residues (T262, S265, and
T298)[13] and leads to increased stability and
half-life[14]. PGC-1α could also be regulated through the p38
MAPK-sensitive interaction with the repressor p160
Myb-binding protein[15].
Icariin (Figure 1) is an active ingredient of plant herb
Epimedium, which possesses many kinds of biological
actions, improving cardiovascular function, hormone
regulation, immunological function modulation, and
antitumor activity[16]. Using a model system comprised of
embryonic stem (ES) cells, our previous work demonstrated that
icariin significantly stimulated the cardiac differentiation of
ES cells in vitro and resulted in increased and accelerated
gene expression of α-myosin heavy chain (α-MHC) and
myosin light chain 2 (MLC2v)[17,18]. Since the cardiomyocyte
differentiation of ES cells in vitro faithfully replicates the
process in vivo and ES cell-derived cardiomyocytes display
properties similar to those observed in
vivo or in primary cultures[19, 20], the present study was designed to address
the modulation of the most common factors (PGC-1α,
PPARα, and NRF-1) implicated in the control of mitochondrial
biogenesis by icariin during cardiomyocyte differentiation. In
addition, the activation of the p38 MAPK was evaluated as
it may be partly responsible for the effect of icariin.
Materials and methods
Cell culture and differentiation The permanent ES cell
line D3 (CRL-1934, American Type Culture Collection,
Manassas, VA, USA) was cultivated in an undifferentiated
state on primary cultures of mouse embryonic fibroblasts in
Dulbecco's modified Eagle's minimal essential medium
(DMEM, Gibco BRL, Life Technologies, Germany),
supplemented with 10% fetal calf serum (FCS, Gibco BRL, Germany),
0.1 mmol/L beta-mercaptoethanol (Sigma, St Louis, MO,
USA), non-essential amino acids (NEAA, stock solution
diluted at 1:100, Hyclone, Logan, UT, USA) and
106 units/L recombinant mouse leukemia inhibitory factor (Chemicon,
Temecula, California, USA). For the differentiation of ES
cells, embryoid bodies (EB) were generated using the
hanging drop method with small modifications[21,
22]. On d 0, 30 μL of drops containing approximately 600 ES cells were placed
on the lids of Petri dishes filled with D-Hanks' solution and
cultivated in hanging drops for 3 d followed by another 2 d
in the Petri dishes. On d 5, the EB were plated separately
onto gelatin-coated, 24-well culture plates in differentiation
medium that consisted of DMEM, 20% FCS, 0.1 mmol/L
mercaptoethanol, and 1% NEAA. After incubation for 24 h
(d 6), outgrown EB were subjected to
10-7, 10-8, and
10-9 mol/L icariin, respectively.
10-8 mol/L retinoic acid (RA) was used as the positive control.
Reagents Icariin was purchased from the National
Institute for the Control of Pharmaceutical and Biological
Products (China; Batch No 0737-200011). Icariin was dissolved
in DMSO to prepare a stock solution of 10-4
mol/L and diluted to 2 gradient concentrations
(10-5 and 10-6 mol/L) by DMSO.
For all the experiments, the final concentrations of the test
compound were prepared by diluting the stock with medium.
SB203580 was purchased from Biomol (Plymouth Meeting,
PA, USA). It was dissolved in DMSO at ×1000 immediately
prior to use. The unused inhibitor was aliquoted into Eppendorf
tubes and stored at -20 °C.
Immunofluorescence analysis Differentiated EB that had
been grown on coverslips were fixed for 20 min in methanol
at -20°C, followed by permeabilization in 0.1% Tween 20 in
phosphate-buffered solution (PBS). After washing in PBS 3
times, the EB on coverslips were transferred to PBS
containing 10% goat serum (Sigma, USA) for 30 min at room
temperature. The EB were then placed into PBS containing
mouse monoclonal anti-a-actinin (Sigma, USA; dilution
1:100) and incubated overnight at 4 °C. The EB were washed
in PBS 3 times, followed by incubation in PBS containing the
FITC-conjugated antimouse IgG (Sigma, USA; dilution 1:500).
Fluorescence recordings were performed by means of
confocal laser scanning setup (Leica TCS SP2, Bensheim,
Germany) connected to an inverted microscope.
Semiquantitative RT-PCR Total RNA was isolated from
ES cells and the EB using Trizol reagent (Gibco BRL,
Germany) in accordance with the manufacturer's instructions.
To synthesize first-strand cDNA, 1 µg total RNA was
incubated with 0.5 µg of oligo (dT) 6 primer (Sangon, Shanghai,
China) and 5 µL deionized water at 65 °C for 15 min.
Reverse transcription reactions of 20 µL were performed with 200
units of M-MuLV reverse transcriptase (Gibco BRL), 4
µL of 5×reaction buffer, and 1 mmol/L deoxynucleoside
triphosphate (dNTP) mixture for 1 h at 42 °C. PCR of 50 µL
contained 1 µL of the RT reaction product, 5 µL of 10×PCR buffer,
25 units Taq polymerase (Sangon, China), 1 µL of 10 mmol/L
dNTP mixture, and 30 pmol of each primer.
Primers, annealing temperature, product size, and the
number of PCR cycles are depicted in Table 1. The PCR
products were analyzed by 1.5% agarose gel electrophoresis,
visualized with ethidium bromide staining, and then
quantified using a bio-imaging analyzer (Bio-Rad, USA). The
density of the products was quantified using Quantity One
version 4.2.2 software (Bio-Rad, USA). β-Actin was used as an
internal standard.
Western blot analysis The cells were washed with PBS,
collected in RIPA buffer (containing 0.2% Triton X-100, 5
mmol/L EDTA, 1 mmol/L PMSF, 10 µg/mL leupeptin, and 10
µg/mL aprotinin) and lysed for 30 min on ice. The aliquots
were assayed for protein concentration using the Bio-Rad
protein assay kit and equal amounts of protein were loaded
per well on a 12% SDS_PAGE. Subsequently, the proteins
were transferred onto 0.45 µm pore size nitrocellulose
membranes and blocked with blotto (5% dry milk in PBS, pH 7.4,
with 0.1% Tween 20) at room temperature.
The blots were challenged with primary antibody in blotto
overnight at 4 °C, followed by washing 3 times with
PBST (0.1% Tween 20) at room temperature and challenged with
horseradish peroxidase-conjugated goat anti-rabbit, rabbit
anti-goat, or mouse anti-mouse antibodies (Affinity
Bioreagents, Golden, CO, USA; dilution 1:1000),
respec-tively, followed by detection with an enhanced
chemiluminescent substrate (Pierce, USA). As primary antibodies, the
goat polyclonal anti-actin, the mouse monoclonal
anti-troponin T, rabbit polyclonal anti-PGC-1α, rabbit polyclonal
anti-PPARα, rabbit polyclonal anti-NRF-1 (Santa Cruz
Biotechno-logy, Santa Cruz, CA, USA; dilution
1:500), and the mouse monoclonal anti-α-actinin (Sigma_Aldrich; dilution 1:500) the
rabbit polyclonal anti-p38 MAPK, and anti-p-p38 MAPK (Cell
Signaling, USA; dilution 1:1000) were used.
Statistics Student's t-test and one-way ANOVA were
used to determine the statistical significance of differences
between values for various experimental and control groups.
P<0.05 was considered statistically significant.
Results
In vitro cardiomyocyte differentiation of ES cells
The attached culture was established by plating a single, d 5 EB
culture onto a 24-well plate and allowing continued cellular
proliferation and differentiation. Within this multicellular
arrangement in the EB outgrowths, cardiomyocytes appeared
as spontaneously contracting, round cell clusters within the
EB on average 3 d later (d 8). An increase in size, strength of
contraction, and beat frequency was observed during
further differentiation. Cardiomyocytes derived from ES cells
were positive for the α-actinin antibody, and cross striations
were observed at higher magnification (Figure 2A). A mass
increase in expression of α-actinin and troponin T was
detected on d 10 (Figure 2B).
After incubation for 24 h (d 6), the attached EB were
subjected to 1×10-7,
1×10-8, and 1×10-9 mol/L icariin,
respectively, and cardiac sarcomeric proteins were
evaluated on d 12. It was apparent that the protein level of
α-actinin and troponin T in the EB was dose-dependently
upregulated by icariin exposure (Figure 2C).
Transcription analysis of PGC-1α,
PPARα, and NRF-1 during cardiomyocyte
differentiation Semiquantitative RT-PCR was employed to elucidate the pattern of
PGC-1α, PPARα, and NRF-1 gene expression during the
differentiation course. The data showed that
PGC-1α, PPARα, and NRF-1 mRNA levels increased obviously in early
differentiation and the prominent changes took place between d 7 and
d 9. Compared to the control group, the mRNA levels of
PGC-1α, PPARα, and NRF-1 were markedly upregulated when
icariin was present (Figure 3).
Protein analysis of PGC-1α, PPARα, and NRF-1
during cardiomyocyte differentiation To confirm the result of
the gene expression found by semiquantitative RT-PCR, the
overall level of protein expression in the EB during
cardio-myocyte differentiation was analyzed. Different
concentrations of icariin were applied to confirm the effect of icariin on
the expression of PGC-1α, PPARα, and NRF-1. Showing an
analogy with mRNA expression, the protein level of
PGC-1α, PPARα, and NRF-1 increased in early differentiation (Figure 4).
This prominent expression abated before the mass
expression of α-actinin and troponin T, suggesting that
PGC-1α, PPARα, and NRF-1 may be critical to normal cardiac
develop-ment in vitro. Moreover, elevations in the protein
expression of PGC-1α, PPARα, and NRF-1 were enhanced by icariin
in a dose-dependent-manner (Figures 4, 5).
Involvement of p38 MAPK activation in icariin-induced
cardiomyocyte differentiation We hypothesized that icariin
relayed p38 MAPK activity to the transcription of genes
involved in cardiomyocyte commitment. The
phosphorylation of the p38 MAPK was further activated and prolonged
by icariin in early differentiation. In the absence of icariin
treatment (control), p38 MAPK activity peaked
spontaneously on d 6 and decreased on d 8, while in the presence of
icariin, the p-p38 MAPK was maintained at a high level until
d 8 (Figure 6A). Moreover, the activation of the p38 MAPK
by icariin was in a dose-dependent manner (Figure 6B).
To further investigate the impact of p38 MAPK activity
on icariin-enhanced cardiomyocyte differentiation and the
expression of PGC-1α, PPARα, and NRF-1, the EB were treated
with icariin alone or together with SB203580 (10 µmol/L) from
d 6. Subsequently, cardiomyocyte differentiation was
assessed by calculating the percentage of spontaneously-contracting EB on d 12. It was shown that icariin-stimulated
cardiac differentiation was abolished by SB203580, while
SB203580 alone had a slight effect on differentiation (Figure
7A). Similarly, the increase in the expression of
PGC-1α, PPARα, and NRF-1 following treatment of icariin was
inhibited in the presence of SB203580 (Figure 7B). These results
implied that high p38 MAPK activity was associated with
cardiogenesis and was responsible for icariin-induced
cardio-myocyte differentiation.
Discussion
Cardiomyocyte differentiation can be divided in 2
processes: cardiogenesis and cardiac myofibrillogenesis.
Early cardiogenesis is regulated by 3 families of
transcription factors (ie Nkx2.5, MEF2C, GATA4), which start to be
fully expressed on d 5. While cardiac transcription factors
(ie α-MHC and MLC2v) encoding contracting proteins have
not appeared at d 5. Thus, d 5 is a critical window at which
the cardiac differentiation program becomes fully activated,
but no cardiac cells identified by organized cardiac
sarcomeric proteins or by functional automatic contractions are yet
present[23]. Our previous study showed that there would be
increasing and accelerating gene expression of α-MHC and
MLC2v in EB when treated with icariin from d 5. In present
study, the inducible effect of icariin was further demonstrated
by evaluating cardiac sarcomeric proteins. The expression
of α-actinin and troponin T were upregulated when the EB
were subjected to icariin 24 h after transferring to 24-well
plates, implying that icariin plays a role in myofibril-logenesis.
It has been reported that treatment with high concentrations
of RA (10-7 and 10-8 mol/L) between d 5 and d 7 would
accelerate cardiomyocyte
differentiation[24]. Therefore, RA was
employed in this study as the positive control and its
inducible effect was replicated as we expected.
Most studies on the control of mitochondrial gene
expression implicate PGC-1α as a "master controller" of
mitochondrial biogenesis, co-activating PPARα and
NRF-1[7]. The conditions that provoke mitochondrial biogenesis, such as
contractile activity, could induce the expression of
PGC-1α[25]. Previous studies have demonstrated that the expression of
the PGC-1α gene is greatly increased in the developing
mouse heart, immediately before the large burst of
mitochondrial biogenesis and the oxidative metabolism that precedes
birth. Cardiomyocytes derived from murine ES cells display
properties similar to those observed in cardiomyocyte
in vivo or in primary cultures, expressing cardiac gene
products in a developmentally-controlled manner, showing
characteristic sarcomeric structures and possessing
membrane-bound ion channels. Mitochondrial number and functional
capacity in cardiomyocytes are dynamically regulated in
accordance with energy demands during developmental
stages and in response to diverse physiological
conditions[26]. Here we showed that the protein expression of
PGC-1α was induced in early differentiation, implying that the
cardiomyo-cyte differentiation of ES cells was accompanied with an
organization of mitochondrial biogenesis.
The expression of PGC-1α, PPARα, and NRF-1 was
coincidently induced in early cardiomyocyte differentiation,
representing a highly favorable environment for the onset of
mitochondrial biogenesis in the EB. The parallel increase in
NRF-1 ensured the coordinate induction of mtDNA
transcription and replication, subsequently leading to the enhanced
expression of mitochondrial proteins that are vital for
respiratory chain function. In the transition from fetal to neonatal
and adult life, cardiac metabolism switches from glucose to
fatty acids as a preferred energy substrate to generate
ATP[27]. This transition is accompanied by changes in activity and
expression levels of several enzymes and regulators involved
in fatty acid metabolism. The main regulators of fatty acid
enzymes on the transcriptional level are the so-called PPAR
which are up-regulated at birth[28, 29]. In the present study,
the expression of PPARα was observed to increase in early
differentiation, which correlated to the increase in the
expression of cardiac-specific transcription factors and proteins,
indicating that in addition to the activation of suites of genes
encoding contractile proteins, myofibrillogenesis is
accompanied by an organization of enzymes involved in fatty acid
metabolism. Interestingly, this phenomenon was recently
replicated as the mRNA levels of NRF-1 and PPARα increased
significantly during myogenesis in
vitro[30]. It should be noted that
PPARα relied upon ligands for activation. EB were grown in the presence of 20% fetal bovine serum
during differentiation, so there would have been abundant
natural ligands (eg fatty acids) for the activation of
PPARα.
Mitochondrial biogenesis and the activation of both
oxidative phosphorylation as well as the transcription and
replication of the mitochondrial genome are key regulatory
events in cell differentiation. The aim of this study is to
analyze the mediation of mitochondrial biogenesis-related
transcription factors by icariin, as final ES cell commitment
may be influenced by mitochondrial proliferation and mtDNA
transcription. The expression of PGC-1α, PPARα, and
NRF-1 were upregulated by icariin in a dose-dependent manner
during cardiac differentiation in vitro, suggesting that icariin
facilitated mitochondrial adaptation to increase energy
demand. Mitochondrial has proven to be important in
maintaining the proper function of cardiomyocytes. During
development of the heart, mitochondrial enzyme activities and
proteins increase in the bovine and human
heart[31]. The blockade of mitochondrial activity by the inhibition of
mitochondrial protein synthesis, the uncoupling of the inner
membrane potential from ATP synthesis and the inhibition of
mitochondrial ATP production (oligomycin) inhibits
differentiation of skeletal muscle cells from myoblast
precursors[32].
The p38 MAPK was proposed to be a link between cardiogenesis and mitochondrial biogenesis. P38 MAPK
activity stimulates PGC-1α gene transcription in
cardio-myocytes, and the activation of this pathway is sufficient to
induce, and is necessary for, cardiac muscle adaptation. In
addition, the p38 MAPK could directly phosphorylate
PPARα and then drive its own transcription in a ligand-dependent
manner[33]. Interestingly, accumulating evidence indicates
that the p38 MAPK plays a critical role in cardiomyocyte
differentiation of murine carcinoma stem cells and ES
cells in vitro[34, 35]. The control of p38 MAPK activity constitutes an
early switch, committing ES cells into either neurogenesis
(p38 off) or cardiomyogenesis (p38
on)[36]. Consistent with previous studies, the p-p38 MAPK was found to be at a high
level from d 5 and followed with a decrease on d 8. Because
PGC-1α is a direct downstream target of the p38 MAPK, the
resulting phosphorylation and protein stabilization could be
important in mediating the increase in the PGC-1α protein
observed here. In this study, we demonstrated that the
phosphorylation of the p38 MAPK was enhanced and prolonged
by icariin, suggesting that icariin triggered the activation of
the p38 MAPK and thus mediated cardiac differentiation.
Taken together, icariin treatment stimulated the
phosphorylation of the p38 MAPK and upregulated the expression of
PGC-1α, PPARα, and NRF-1 (Figure 8) which may act as part
mechanisms for the inducible effect of icariin on the cardiac
differentiation of murine ES cells.
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