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Chromatin remodeling,
particularly histone
acetylation, plays a
critical role in the
progression of
pathological
cardiac hypertrophy and
heart failure. We
hypothesized that
curcumin, a natural
polyphenolic compound
abundant in the spice
turmeric and a known
suppressor of histone
acetylation, would
suppress cardiac
hypertrophy
through the disruption
of p300 histone
acetyltransferase–dependent
(p300-HAT–dependent)
transcriptional
activation. We tested
this hypothesis using
primary cultured rat
cardiac myocytes and
fibroblasts as well
as two well-established
mouse models of cardiac
hypertrophy. Curcumin
blocked phenylephrin-induced
(PEinduced)
cardiac hypertrophy in
vitro in a
dose-dependent manner.
Furthermore, curcumin
both prevented
and reversed mouse
cardiac hypertrophy
induced by aortic
banding (AB) and PE
infusion, as assessed by
heart
weight/BW and lung
weight/BW ratios,
echocardiographic
parameters, and gene
expression of
hypertrophic
markers. Further
investigation
demonstrated that
curcumin abrogated
histone acetylation,
GATA4 acetylation,
and DNA-binding activity
through blocking
p300-HAT activity.
Curcumin also blocked
AB-induced
inflammation and
fibrosis through
disrupting
p300-HAT–dependent
signaling pathways. Our
results indicate
that curcumin has the
potential to protect
against cardiac
hypertrophy,
inflammation, and
fibrosis through
suppression of p300-HAT
activity and downstream
GATA4, NF-κB, and TGF-β–Smad
signaling pathways.
Introduction
Cardiac hypertrophy is
an adaptive enlargement
of the myocardium
in response to increased
workload, characterized
by an increase
in the size of
individual cardiac
myocytes and whole-organ
enlargement.
Although cardiac
hypertrophy may
initially be
compensatory,
sustained pathologic
hypertrophy is
deleterious and may lead
to
heart failure, sudden
death, and stroke (1–3).
Histone acetylation
is one of the key
control points for gene
regulation in the
hypertrophic
myocardium (4).
Acetylation of histone
tails, mediated by
histone
acetyltransferases (HATs),
confers accessibility of
the DNA
template to the
transcriptional
machinery and is
associated with
activation of gene
expression (5). Histone
deacetylases (HDACs),
on the other hand,
catalyze removal of
acetyl groups on
aminoterminal
lysine residues of
histones and, by
promoting nucleosomal
condensation, act as
transcriptional
repressors or silencers
of
genes (5). The status of
histone acetylation is
therefore determined
by the balanced action
of HATs and HDACs. p300
is a critical HAT
in muscle that modifies
chromatin and associated
transcription
factors and promotes
gene activation (6, 7).
Recent studies have
demonstrated that p300
transcriptional activity
is enhanced during
agonist-induced cardiac
hypertrophy and that
subsequent
blocking of p300-HAT
activity inhibits
agonist-mediated cardiac
growth (6, 8). Moreover,
transgenic mice that
overexpress p300 in
the heart develop
cardiac hypertrophy and
eventual heart failure
(9). Therefore, p300-HAT
is an attractive target
to treat or prevent
cardiac hypertrophy and
subsequent heart
failure.
Curcumin is a natural
polyphenolic compound
abundant in the
rhizome of the perennial
herb turmeric, Curcuma
longa (10). It is
commonly used as a
dietary spice and
coloring agent in
cooking
and is used anecdotally
as an herb in
traditional Indian and
Chinese
medicine (11). However,
to our knowledge no
study to date
has addressed the effect
of curcumin on cardiac
hypertrophy and
related signaling
pathways. Although
evidence demonstrates
that
curcumin is an inhibitor
of p300-HAT (12, 13),
very little is known
about whether this
regulatory effect is
related to a protective
role
cardiac dysfunction.
Therefore, we aimed to
determine whether
curcumin attenuates
cardiac hypertrophy in
vitro and in vivo by
impairing p300-HAT
activity.
Results
Pretreatment with
curcumin inhibits
cardiac hypertrophy in
vitro. Neonatal
rat ventricular myocytes
were cultured to greater
than
97% purity, as confirmed
by phase-contrast
microscopy and
immunocytochemical
staining. To rule out
the possibility of
cytotoxicity, we
determined the number of
viable cells in all
wells
using trypan blue
exclusion analysis and
lactate dehydrogenase
(LDH) release assay.
When 5–50 μM curcumin
was applied to cultured
neonatal cardiomyocytes,
cells were observed to
be healthy
even in the presence of
50 μM curcumin at the
end of 48 hours
(Supplemental Figure 1;
supplemental material
available online
with this article;
doi:10.1172/JCI32865DS1).
At 100 μM curcumin,
however, we observed
decreased cell
viability, leading us to
choose
a lower dose for the in
vitro experiments. There
were no observable
adverse effects by the
administration of DMSO
and phenylephrin
(PE) or infection with a
wild-type human p300
cDNA adenoviral
(Ad-p300) and an AT2
mutant p300 adenoviral
lacking HAT activity
(Ad-DN-p300) (data not
shown). In this study,
curcumin was
dissolved in DMSO medium
for the in vitro
studies. DMSO alone
without curcumin served
as a control and did not
show any effect
on cell viability,
cardiac hypertrophy,
collagen synthesis, and
related
molecular mechanisms
(data not shown).
Cardiac hypertrophy can
be monitored by
increased protein
synthesis,
myocyte cross-sectional
area, and induction of
fetal gene
expression (14). Cardiac
myocytes were incubated
with curcumin
for 60 minutes and
subsequently treated
with 100 μM PE for 48
hours. Pretreatment with
curcumin demonstrated a
dose-dependent
reduction in PE-induced
increases of [3H]leucine
incorporation
that showed maximal
effects at 50 μM (Figure
1A). Additionally,
the increase in cardiac
myocyte size seen after
48 hours
of culture in the
presence of PE was also
markedly attenuated by
curcumin (Figure 1, B
and C). Curcumin
markedly reduced atrial
natriuretic peptide
(ANP) and brain
natriuretic peptide
(BNP)
mRNA expression levels
induced by PE (Figure
1D). The inhibition
of cardiac hypertrophy
in vitro by curcumin was
sustained
for all tested times.
However, curcumin alone
had no effect on
[3H]leucine
incorporation, cardiac
myocyte size, or
expression of
ANP and BNP. These data
demonstrate that
curcumin attenuates
cardiac hypertrophy in
vitro.
Pretreatment with
curcumin inhibits
cardiac hypertrophy in
vivo. To
determine the
physiological relevance
of our in vitro
findings, we
investigated the effects
of curcumin in a murine
pressure-overload
model of cardiac
hypertrophy. To evaluate
the dose-response
relationship, we
administered 3 different
doses of curcumin (50,
75, and 100 mg/kg/d) for
1 week and then
subjected the mice to
either chronic pressure
overload generated by
aortic banding (AB)
or sham surgery
(control). We found that
maximal efficacy was
achieved at a curcumin
dose of 75 mg/kg/d
(Supplemental Table 1).
Moreover, no apparent
effect on cell toxicity
was observed with
any dose of curcumin,
and 75 mg/kg/d was
therefore chosen as the
experimental dose. These
findings are in
agreement with previous
publications in which
curcumin showed no
toxicity in the liver or
kidney at doses of 100
or 200 mg/kg/d (15, 16).
In order to further
evaluate the effects of
curcumin on cardiac
hypertrophy, mice were
randomly assigned to 4
groups: pretreatment
with either vehicle or
75 mg/kg/d curcumin for
1 week prior
to either AB surgery or
sham operation. Curcumin
treatment of
the AB mice resulted in
significant attenuation
of hypertrophy, as
measured by heart
weight/BW (HW/BW) ratio,
lung weight/BW
(LW/BW) ratio, and
cardiomyocyte
cross-sectional area
(Figure
2A). No significant
changes were observed in
the sham-operated
mice treated with
curcumin or vehicle.
Gross heart and wheat
germ
agglutinin (WGA)
staining further
confirmed the inhibitory
effect
of curcumin on cardiac
remodeling in AB hearts
(Figure 2B). As
shown in Table 1,
curcumin pretreatment
prevented adverse
cardiac
remodeling and
ventricular dysfunction,
as evidenced by
improvements
in LV end-systolic
diameter (LVESD), LV
end-diastolic diameter
(LVEDD), and percent
fractional shortening
(FS).
We next examined the
potential effect of
curcumin on hypertrophy
mediated by PE infusion.
Mice were randomly
allocated into 4
groups: pretreatment
with either vehicle or
75 mg/kg/d curcumin
for 1 week prior to
either PE or saline
infusion. Osmotic
minipumps
were implanted
subcutaneously for a
3-week administration
period
followed by cardiac
functional assessment.
As shown in Table 2,
curcumin
abrogated PE-induced
cardiac chamber dilation
in both systole
and diastole. The
PE-induced increase in
HW/BW and LW/BW
ratios as well as
cardiomyocyte
cross-sectional area
were also attenuated
by 4 weeks of curcumin
administration (Figure
2C). These findings
were confirmed by
morphological assessment
(Figure 2D).
ANP, BNP, and myosin
heavy chain β (β-MHC)
are markers for
cardiac hypertrophy
(14). To determine
whether curcumin
affected
the mRNA expression
levels of these markers,
we performed Northern
blot analysis. Curcumin
attenuated the observed
increase in
hypertrophic marker
expression caused by AB
or PE infusion (Figure
2, E and F). These
findings suggest that
curcumin prevents the
development of cardiac
hypertrophy in vivo.
Pretreatment with
curcumin inhibits
histone acetylation in
response to
hypertrophic stimuli. To
explore the molecular
mechanisms through
which curcumin impairs
the hypertrophic
response, we examined
the state of acetylation
of histones by assaying
the incorporation
of [3H]acetate into
histones. We exposed
cultured neonatal rat
cardiomyocytes
to 100 μM PE with or
without curcumin. As
expected,
PE induced a significant
increase in histone
acetylation that was
dose-dependently blocked
and sustained for all
tested time points
by curcumin (Figure 3, A
and B). These findings
were confirmed
by concomitant
attenuation of histone
H3, histone H4, and
tubulin
acetylation (Figure 3C).
To test its efficacy in
vivo, Western blot
analysis was performed
using samples from the 2
differing hypertrophic
animal models. Similar
findings confirmed that
vehicle-treated
AB or PE-infused mice
markedly induced the
acetylation of histone
H3, histone H4, and
tubulin and that
curcumin-treated mice
significantly
attenuated the
acetylation of these
targets (Figure 3, D and
E). Our findings suggest
that curcumin inhibits
histone acetylation
in vitro and in vivo in
response to hypertrophic
stimuli.
Pretreatment with
curcumin blocks p300-HAT
activity, but not HDAC
activity. Histone
acetylation is regulated
by p300-HAT and plays an
important role in heart
disease (9). To
elucidate the role of
p300 in
the inhibitory effect of
curcumin on cardiac
hypertrophy, we analyzed
the effects of curcumin
on p300-HAT activity
induced by PE.
As shown in Figure 4A,
at concentrations of 50
and 100 μM PE,
p300-HAT activity
increased 11.3- and
17.9-fold, respectively,
in
cardiac myocytes. The
linear increase of HAT
activity by increased
concentrations of PE
indicated that
PE-induced p300-HAT
activity
was dose dependent and
maximally achieved at
100 μM. p300-HAT
activity reached its
maximum value at 6 hours
after the addition
of 100 μM PE (Figure
4B). We also found that
curcumin dosedependently
blocked PE-induced
p300-HAT activity and
sustained
this inhibition for all
tested time points
(Figure 4, C and D). We
further demonstrated
that blocking p300-HAT
activity inhibited,
whereas increased
p300-HAT activity
promoted, histone
acetylation
induced by PE, as
estimated by the global
acetylation of histones
and the acetylation of
histone H3, histone H4,
and tubulin (Figure
4, E and F). This
suggests that the
hyperacetylation induced
by PE
in cardiac myocytes is
dependent on p300-HAT
activity. To define the
role of HDAC activity in
curcumin-induced histone
hypoacetylation,
the effect of curcumin
on HDAC activity was
determined.
Curcumin did not alter
its activity at each of
the tested
concentrations
(Supplemental Figure 2).
Collectively, these
results indicate
that curcumin inhibits
histone acetylation
through direct
inhibition
of p300-HAT activity
rather than HDAC
activity.
Pretreatment with
curcumin blocks GATA4
acetylation and
DNAbinding
activity. p300 protein
serves as an adaptor for
hypertrophy-
responsive transcription
factors including GATA4,
which is
required for the
activation of cardiac
genes that are
upregulated
during cardiac
hypertrophy (17, 18). As
expected, both PE
stimulation
and AB markedly induced,
and curcumin
pretreatment completely
abolished, GATA4
acetylation and
DNA-binding activity
(Figure 5, A and B).
Based on these observed
inhibitory effects, we
investigated whether
PE-mediated GATA4
activation is dependent
on p300-HAT activity.
Our results demonstrated
that PE-induced
GATA4 acetylation and
DNA-binding activity
were almost completely
inhibited by infection
with Ad-DN-p300 but were
augmented
by infection with
Ad-p300 (Figure 5C). In
addition, the
inhibitory effects of
curcumin on GATA4
acetylation and
DNAbinding
activity were reversed
by infection with
Ad-p300 (Figure
5D). These data suggest
that GATA4 acetylation
and activation by
PE require p300-HAT
activity in cardiac
myocytes.
Pretreatment with
curcumin blunts
inflammation and
fibrosis. Inflammation
is known to play an
important role in the
development and
progression to
hypertrophy and heart
failure (19–21). To
determine
whether curcumin
suppresses inflammation
in the heart, we
first examined the
cellular infiltrates by
immunostaining analyses.
As shown in Figure 6A,
the population of cells
that stained positive
for myeloperoxidase
(MPO), Mac-1, and Mac-3
significantly
increased at 8 weeks
after AB; these
increases were
dramatically
reduced by curcumin
treatment. Furthermore,
curcumin decreased
NF-κB activation and
MCP-1, IL-6, IL-1β, and
TNF-α mRNA and
protein expression
induced by AB
(Supplemental Figure 3,
A and B,
and Figure 6B). Curcumin
treatment also impaired
IκBα phosphorylation
and degradation, IκB
kinase (IKK) activation,
and
p65 translocation
mediated by AB
(Supplemental Figure 3,
C–F).
A previous report
describing the
regulation of NF-κB
activation
by direct
protein-protein
interaction with p300
(22) prompted
us to investigate the
functional significance
of p300 on NF-κB
signaling. Infection
with Ad-p300 promoted,
and infection with
Ad-DN-p300
downregulated, NF-κB
transcription and
cytokine
expression after PE
treatment in myocytes
(Supplemental Figure 3,
G and H). Furthermore,
p300 overexpression
significantly reversed
the curcumin-induced
inhibitory effects on
inflammation
(Supplemental
Figure 3I). These
results indicate that
curcumin blocks
NF-κB signaling and
NF-κB–dependent
inflammatory responses
through the disruption
of p300-HAT activity.
Pathological cardiac
hypertrophy is
associated with
increased
fibrosis in the
myocardium (1), and, as
expected, marked
perivascular
and interstitial
fibrosis were detected
in the vehicle-treated
AB mice by picrosirius
red (PSR) staining.
Curcumin treatment,
however, remarkably
reduced the extent of
cardiac fibrosis in vivo
(Figure 6, C and D).
Subsequent analysis of
mRNA and protein
expression levels of
known mediators of
fibrosis including
TGF-β1,
collagen I, and
connective tissue growth
factor (CTGF)
demonstrated
a blunted response by
curcumin treatment
(Figure 6, E and F).
In addition, curcumin
effectively blocked
collagen synthesis and
COL1A2, PAI-1, and CTGF
protein expression
levels and promoter
activities induced by
TGF-β1 in cultured
cardiac fibroblasts
(Supplemental
Figure 4, A–C).
To further elucidate the
cellular mechanisms
underlying the
antifibrotic
effects of curcumin, we
then assessed the
regulatory role
of curcumin in Smad
cascade activation in
hearts subjected to AB.
Smad-2 phosphorylation
and Smad-2/3/4 nuclear
translocation
were markedly blocked by
curcumin (Supplemental
Figure 4D).
Our in vitro studies
using neonatal rat
cardiac fibroblasts
yielded
identical results
(Supplemental Figure
4E). To our knowledge,
no
previous study has shown
the physiologic link
between TGF-β
and p300 with respect to
collagen synthesis in
the heart. Using
confluent cardiac
fibroblasts infected
with Ad-GFP, Ad-p300,
or Ad-DN-p300 along with
COL1A2-, PAI-1–, or
CTGF-luc reporter
constructs, we incubated
the cells with TGF-β1.
Forced expression
of ectopic p300 revealed
a significant increase
in, whereas inhibiting
p300-HAT activity almost
completely abrogated,
collagen
synthesis, promoter
activities, and protein
levels of markers of
fibrosis (Supplemental
Figure 4F). Immunoblot
analysis further
demonstrated that
Ad-DN-p300 almost
completely abrogated
the phosphorylation of
Smad-2 and nuclear
translocation of
Smad-2/3/4 in response
to TGF-β1 (Supplemental
Figure 4G).
Ad-p300 infection
partially rescued the
curcumin-induced
inhibitory
effects on collagen
synthesis, markers of
fibrosis,
phosphorylation
of Smad-2, and
translocation of
Smad-2/3/4 (Supplemental
Figure 4, H and I).
These findings suggest
that curcumin blocks
collagen synthesis by
disrupting p300-HAT
activity–dependent
TGF-β–Smad signaling.
Curcumin ameliorates
established cardiac
hypertrophy in vivo. For
greater clinical
relevance, we next
assessed whether
curcumin can
reverse established
cardiac hypertrophy. For
these studies, we
subjected
mice to AB surgery and
sham operation
(control). Cardiac
hypertrophy was
confirmed by the
increase in HW/BW ratio,
by the
gross morphology of the
heart, and from
echocardiographic
analyses
after a 2-week period.
Continuation of AB for a
subsequent 6
weeks resulted in the
transition to heart
failure, as evidenced by
a
further decline in
percent FS, increase in
LVEDD and LVESD, and
increase in HW/BW and
LW/BW ratios (Table 3
and Figure 7, A
and B). Interestingly,
curcumin treatment for a
period of 6 weeks
after the initial 2
weeks of AB reversed the
remodeling, contractile
dysfunction, and cardiac
ANP, BNP, and β-MHC mRNA
expression
levels toward normal
control values,
ultimately preventing
the transition to heart
failure (Figure 7, A, B,
and E). To verify this
observation, we tested
the impact of curcumin
on established PEinduced
cardiac hypertrophy.
Curcumin was
administered to mice
at a dose of 75 mg/kg/d
starting after 1 week of
PE infusion and
continuing for 2 weeks.
Cardiac hypertrophy was
markedly reversed
by curcumin as assessed
by HW/BW and LW/BW
ratios,
echocardiographic
measurements,
cardiomyocyte area, and
expression levels
of hypertrophic markers
compared with
vehicle-treated controls
(Table 2 and Figure 7,
C, D and F). These data
confirm our findings
from the AB model and
suggest that curcumin is
able to reverse
established cardiac
hypertrophy and heart
failure.
p300 partially reverses
the inhibitory effects
of curcumin in vivo. The
above experimental
results suggested that
curcumin inhibits
cardiac
hypertrophy,
inflammation, and
fibrosis through
blocking
p300-HAT–dependent
signaling pathways. To
confirm these findings,
we evaluated whether the
inhibitory effects of
curcumin could
be reversed through
ectopic expression of
p300 in vivo. Therefore,
we established a
protocol to locally
increase p300 expression
via
direct
adenovirus-mediated gene
transfer into the heart.
Western
blot analysis showed
that p300 protein levels
were significantly
increased in our
Ad-p300–infected samples
when compared with
the Ad-GFP controls,
which reached peak
levels after 2 days and
tapered off by 21 days
(Figure 8A). Next, we
studied the functional
consequences of
increased p300
expression. The
Ad-GFP–infected
mice demonstrated
significant ventricular
dilatation and decreased
percent FS compared with
the sham-operated mice
after 2 weeks
of AB (data not shown).
Hypertrophied hearts
treated with p300
gene transfer showed
significant functional
deterioration compared
with those treated with
GFP gene transfer after
2 weeks of
AB (Figure 8B).
Interestingly, curcumin
treatment markedly
attenuated
the functional
deterioration observed
in Ad-GFP–infected
mice but had no
alleviating effect on
Ad-p300–treated mice
(Figure
8B). Furthermore,
Ad-p300 infection also
partially but obviously
reversed the inhibitory
effects of curcumin on
the HW/BW ratio,
cardiomyocyte
cross-sectional area,
and cardiac morphology
after
2 weeks of AB (Figure 8,
C and D). Northern blot
analysis further
revealed that Ad-p300
infection significantly
reversed the attenuated
mRNA levels of ANF and
BNP at 2 weeks after AB
compared
with those
Ad-GFP–infected groups
after treatment with
curcumin
(Figure 8E). These
results suggest that
p300 overexpression
partially
reverses the inhibitory
effects of curcumin on
cardiac hypertrophy.
As inflammation and
pathological fibrosis
have been shown to be
inhibited by curcumin,
we determined whether
overexpression of
p300 might annul the
inhibitory effects of
curcumin on inflammation
and fibrosis. Ad-p300
infection significantly
reversed the
inhibitory effect of
curcumin on NF-κB
activation and NF-κB–
dependent TNF-α and IL-6
expression (Supplemental
Figure 5A
and Figure 8F).
Additionally,
overexpression of p300
significantly
reversed the inhibitory
effect of
curcumin on AB-mediated
fibrosis
and collagen I and III
protein
expression (Figure 8, G
and H, and
Supplemental Figure 5B).
Discussion
Our study demonstrates,
for the
first time to our
knowledge, that
curcumin protects
against cardiac
hypertrophy both in
vitro and in
vivo. The
cardioprotection of
curcumin
is mediated by
interruption
of p300-HAT
activity–dependent
signaling pathways,
resulting in
protection of the host
from the
combined deleterious
effects of cardiac
hypertrophy,
inflammation,
and fibrosis. Curcumin
also reversed
established cardiac
hypertrophy and
dysfunction induced by
sustained
pressure overload or PE
infusion.
These findings support
the concept
that curcumin could be
an effective
preventive and
therapeutic candidate
against cardiac
hypertrophy
and heart failure.
Curcumin is a
low–molecular
weight polyphenol and
has been
used as a natural
compound in
the treatment of many
conditions,
including cardiovascular
diseases
(10, 11). However, to
our knowledge there was
no evidence to date
regarding the effects of
curcumin on cardiac
hypertrophy. Despite
significantly increased
blood pressure in our 2
cardiac hypertrophy
models, curcumin
treatment did not affect
blood pressure. This
indicates that the
primary target of
curcumin action is
cardiac protection,
rather than lowering
blood pressure. Of
particular clinical
relevance is the finding
that curcumin can
reverse preestablished
cardiac hypertrophy and
dysfunction induced by
different animal
models. The raw
ingredient for curcumin
is abundant and
inexpensive,
and the amount of
curcumin used is within
the physiologic
range and well below the
maximum tolerable
pharmacological level
(equivalent to 0.4 g/d
for humans).
The mechanism by which
curcumin mediates its
antihypertrophic
effects remains unclear.
There is increasing
evidence for the
involvement of chromatin
remodeling, especially
histone acetylation
in pathological cardiac
hypertrophy and heart
failure (23–25).
HATs are believed to
acetylate histone
proteins, relax
chromatin,
and expose
prohypertrophic genes
for activation by
cardiogenic
transcription factors.
Several lines of
evidence have shown that
a
critical HAT in the
heart is p300, which
plays a key role in the
physiological
growth and
differentiation of
cardiac myocytes during
development (9). p300
knockout mice die
between days 9 and 11.5
of gestation, exhibiting
defects of cardiac
muscle differentiation
and trabeculation,
indicating the
importance of p300 for
early cardiac
morphogenesis and heart
development (26).
However, p300
is also involved in the
pathological process of
cardiac hypertrophy
(8). The results of the
present study indicate
that inhibition of
histone acetylation is a
key mechanism for the
antihypertrophic
activity of curcumin and
that p300-HAT serves as
its molecular
target. We found that
treatment with curcumin
had a negligible
effect on the normal
heart, in contrast to
its effects on the heart
subjected to hemodynamic
stress. In addition, we
did not find
any appreciable increase
in cell death with
curcumin treatment in
the adult heart,
suggesting that the role
of p300 in the postnatal
heart is different from
its role during early
cardiac morphogenesis.
Consistent with our
findings, Miyamoto et
al. demonstrated that
cardiac-specific
overexpression of
wild-type p300 protein
in
the normal heart had no
effect on cardiac
morphology or function,
in contrast to the
resulting deteriorated
function observed
after myocardial
infarction (9). We
hypothesize that in the
context
of the postnatal
myocardium, p300 may
maintain basal function
in the normal heart but
promote cardiac growth
under chronic
loading stress or PE
infusion. Importantly,
in contrast to the
complete
depletion of p300 in
knockout mice, elevated
levels of p300
in cardiac hypertrophy
were reduced (but not
eliminated) with a
therapeutic dose of
curcumin in this study.
The lack of
physiological
levels of p300-HAT
activity during
embryonic cardiac
development
or other organogenesis
is not comparable with
the suppression
of elevated p300-HAT
activity in adult
animals with cardiac
hypertrophy. Based on
our present results, we
believe curcumin
to correct the
disease-related increase
in p300-HAT activity.
Our
data also indicate that
curcumin inhibits
p300-HAT activity but
does not affect the
activity of HDAC.
Blocking p300-HAT
activity
completely blocks the
inhibition by curcumin
of histone acetylation,
suggesting that the
balance of HATs and
HDACs is primarily
modulated by curcumin
through its effects on
HAT activity. p300
acetylates GATA4 and
increases its
DNA-binding ability,
leading
to nuclear
hyperacetylation of
cardiac myocytes and
cardiac hypertrophy
(27–29). Consistent with
recent reports (29), we
observed
GATA4 hyperacetylation
and increased
DNA-binding activity in
response to hypertrophic
stimuli. Curcumin
treatment abolished
GATA4 acetylation and
attenuated DNA-binding
activity in our
hypertrophic models, and
these effects were
markedly reversed by
p300 overexpression in
vitro and in vivo. These
findings suggest
that curcumin blunts
cardiac hypertrophy by
inhibiting p300-
HAT–dependent GATA4
activation.
Inflammation plays an
important role in the
progression to cardiac
hypertrophy (20), and
perivascular
infiltration by
neutrophils
and macrophages has been
observed in the hearts
of several animal
species subjected to AB
(30, 31). After curcumin
administration, we
observed marked
attenuation of leukocyte
infiltration and
cytokine
production, suggesting
potential cross-talk
between leukocyte
infiltration
and inflammation in
modulating the
microenvironment
for the development of
cardiac hypertrophy. By
blocking NF-κB
signaling, curcumin may
inhibit the early steps
of inflammation
and modulate the
amplification of
multiple cytokine
signaling
cascades (32–34).
Acetylation is also an
important modification
step for the regulation
of the nuclear function
of NF-κB (22, 35),
and p300 appears to play
a major role in the
acetylation of p65 and
subsequent regulation
NF-κB activity (22). Our
data suggest that
suppression of p300-HAT
activity results in
impaired NF-κB activity,
while upregulation of
p300-HAT activity
promotes NF-κB
activation,
indicating that p300-HAT
plays an important role
in the
activation of NF-κB
signaling. We have shown
that curcumin can
inhibit these signaling
pathways in the heart.
Cardiac fibrosis is a
classical feature of
pathological hypertrophy
and is characterized by
the expansion of the
extracellular
matrix due to the
accumulation of collagen
(1). The present study
demonstrated that
curcumin blocks cardiac
fibrosis in vivo and
inhibits collagen
synthesis in vitro. Our
study is the first to
our
knowledge to report
inhibition of
TGF-β1–induced collagen
synthesis
in cardiac fibroblasts
by curcumin.
Furthermore, our data
suggest that curcumin
abrogates Smad-2
phosphorylation and
Smad-2/3/4 translocation
in both cardiac
fibroblast culture and
hypertrophied hearts,
important downstream
components of
TGF-β1 signaling (36,
37). Recent studies
indicate that TGF-β1–
Smad signaling can be
regulated by p300 (37,
38). Our findings
demonstrated that
blocking of p300-HAT
activity led to complete
inhibition, while forced
expression of p300 led
to upregulation,
of collagen synthesis
and Smad-2/3 activation.
p300 gene transfer
also markedly reversed
the inhibitory effects
of curcumin on
fibrosis in vivo,
indicating that the
inhibitory effects of
curcumin
on fibrosis and collagen
synthesis are mediated
through blocking
p300-HAT activity. In
conclusion, our present
work demonstrates that
curcumin
inhibits cardiac
hypertrophy in response
to pathological stimuli
both in vitro and in
vivo. Curcumin prevented
the development of
cardiac hypertrophy by
blocking
p300-HAT–dependent
hypertrophy,
inflammation, and
fibrosis. Our data
confirm that p300-HAT
is the main target of
curcumin’s inhibitory
actions, although we
cannot determine whether
p300-HAT is its only
target. This study is
relevant to the
understanding of the
inhibitory effect of
curcumin
on cardiac hypertrophy
and related molecular
mechanisms. It also
serves to elucidate the
dominant signaling
pathways leading to
cardiac
hypertrophy,
inflammation, and
fibrosis in response to
hypertrophic
stimuli. Curcumin is a
natural polyphenolic
compound
that has already been
used clinically and is
approved by the FDA as
a safe food additive.
Future studies should
examine the hypothesis
that curcumin may be a
safe and effective
approach to preventing
and treating cardiac
hypertrophy and the
transition to failure.
Methods
Materials.
Anti–phospho-Smad2,
anti–phospho-p65,
anti-IKKα, anti-IKKβ,
anti–phospho-IκBα, and
anti-IκBα antibodies
were purchased from Cell
Signaling Technology.
The antibodies used to
recognize histones H3
and
H4 at their N-terminal
lysine residues and
GATA4 were purchased
from
Upstate Biotechnology.
The anti-acetylated
tubulin was obtained
from
Sigma-Aldrich.
[3H]leucine,
[3H]proline,
[3H]acetate, and
[3H]acetyl-CoA
were purchased from
Amersham. The BCA
protein assay kit was
purchased
from Pierce, and the IKK
activity kit was
obtained from BD
Biosciences. All
other antibodies were
purchased from Santa
Cruz Biotechnology.
TGF-β1
was purchased from R&D
Systems. FCS was
obtained from Hyclone.
CTGF-luc and COL1A2-luc
report constructs were
provided by M.
Trojanowska
(Medical University of
South Carolina,
Charleston, South
Carolina,
USA), and PAI-1–luc
report construct was
provided by F.M. Stanley
(New York University
School of Medicine, New
York, New York, USA).
Ad-p300 and Ad-DN-p300
were provided by L. Hua
(Oregon Health and
Sciences University,
Portland, Oregon, USA;
ref. 39). Cell culture
reagents,
curcumin, and all other
reagents were obtained
from Sigma-Aldrich.
Cultured neonatal rat
cardiac myocytes and
fibroblasts. Primary
cultures of
cardiac myocytes were
prepared as described
previously (33). Cells
from the
hearts of 1- to
2-day-old Sprague-Dawley
rats (Charles River
Laboratories)
were seeded at a density
of 1 × 106 cells/well
onto 6-well culture
plates
coated with fibronectin
(BD) in plating medium
consisting of F10 medium
supplemented with 10%
FCS and
penicillin/streptomycin.
After 48 hours,
the culture medium was
replaced with F10 medium
containing 0.1% FCS
and BrdU (0.1 mM). After
12 hours of serum
starvation, curcumin
alone or
curcumin followed by 100
μM PE was added to the
medium, and cultures
were incubated for the
indicated times.
Viability was determined
by cell
number, frequency of
contractions, cellular
morphology, and trypan
blue
exclusion. Cultures of
neonatal rat ventricular
nonmyocytes, which have
been shown to be
predominantly
fibroblasts, were
prepared as described
previously by Sadoshima
and Izumo (40). All
experiments were
performed
on cells from the first
or second passages,
which were placed in
DMEM
containing 0.1% FCS for
24 hours before the
experiment. The purity
of
these cultures was
greater than 95% cardiac
fibroblasts, as
determined by
positive staining for
vimentin and negative
staining for smooth
muscle
actin and von Willebrand
factor. For cell
infection, cardiac
myocytes or
cardiac fibroblasts were
cultured at 1 × 106
cells/well in 6-well
plates and
exposed to 2 × 108 pfu
of each virus in 1 ml
serum-free medium for 24
hours. The cells were
then washed and
incubated in
serum-containing
medium for 24 hours. All
additional treatments to
which cells were
subjected
are described in the
figure legends.
LDH release. Cardiac
myocytes or cardiac
fibroblasts in 6-well
plates
were preincubated with
different concentrations
of curcumin for 48
hours or 25 μM curcumin
for various time periods
and then incubated
with PE or TGF-β1 for
the indicated times. LDH
activity was measured
using an LDH assay kit
according to the
manufacturer’s
instructions
(Cayman). The absorbance
was determined at 492 nm
in an ELISA reader.
The percentage of LDH
released from the cells
was determined as the
LDH activity in
supernatant divided by
the combined LDH
activity in
supernatant and cell
lysate.
[3H]Leucine
incorporation and
surface area.
[3H]Leucine
incorporation was
measured as described
previously (33).
Briefly, cardiac
myocytes were pretreated
with curcumin for 60
minutes and subsequently
stimulated with
PE (100 μM) and
coincubated with
[3H]leucine (2 μCi/ml)
for the indicated
time. At the end of the
experiment, the cells
were washed with Hanks’
solution,
scraped off the well,
and then treated with
10% trichloroacetic acid
at
4°C for 60 minutes. The
precipitates were then
dissolved in NaOH (1 N)
and subsequently counted
with a scintillation
counter. For surface
areas,
the cells were fixed
with 3.7% formaldehyde
in PBS, permeabilized in
0.1%
Triton X-100 in PBS, and
stained with α-actinin
(Sigma-Aldrich) at a
dilution
of 1:100 by standard
immunocytochemical
techniques.
Reporter assays. Cardiac
myocytes or cardiac
fibroblasts were seeded
in
triplicate in 6-well
plates. Cells were
transfected with 0.5 μg
luciferase
reporter constructs, and
internal control plasmid
DNA using 10 μl of
LipofectAMINE reagent
(Invitrogen), according
to the manufacturer’s
instructions. After 6
hours of exposure to the
DNA-LipofectAMINE
complex,
cells were cultured in
medium containing 10%
serum for 18 hours
and then incubated with
serum-free medium for 12
hours. Cells were
pretreated
with curcumin for 60
minutes and then treated
with PE for cardiac
myocytes or TGF-β1 for
fibroblasts. Cells were
harvested using passive
lysis
buffer (Promega)
according to the
manufacturer’s protocol.
The luciferase
activity was normalized
by control plasmid. All
experiments were done in
triplicate and repeated
at least 3 times.
Collagen synthesis
assay. Collagen
synthesis was evaluated
by measuring
[3H]proline
incorporation as
described previously
(41). In brief, cardiac
fibroblasts were made
quiescent by culturing
in 0.1% FCS DMEM for 24
hours, pretreating with
curcumin for 60 minutes
and subsequently
incubating
with TGF-β1 and 5 μCi/ml
[3H]proline for the
indicated time. Cells
were washed with PBS
twice, treated with
ice-cold 5%
trichloroacetic acid
for 1 hour, and washed
with distilled water
twice. Cells were then
lysed with
1 N NaOH solutions and
counted in a liquid
scintillation counter.
The
count representing the
amount Quantitative
real-time RT-PCR. Total
RNA was extracted from
frozen, pulverized
mouse tissues using
TRIzol (Invitrogen) and
synthesized cDNA using
oligo(dT) primers with
the Advantage RT-for-PCR
kit (BD Biosciences). We
quantified PCR
amplifications using
SYBR Green PCR Master
Mix (Applied
Biosystems) and
normalized results
against GAPDH gene
expression.
Western blotting and
Northern blot. Cardiac
tissue and cultured
cardiac
myocytes or fibroblasts
were lysed in RIPA lysis
buffer. Nuclear protein
extracts were isolated
as described previously
(21). Cell lysate (50
μg) was
used for SDS-PAGE, and
proteins were then
transferred to an
Immobilon-P
membrane (Millipore).
Specific protein
expression levels were
normalized
to either the GAPDH
protein for total cell
lysate and cytosolic
protein or
the lamin-B1 protein for
nuclear protein signal
on the same
nitrocellulose
membrane. For mRNA
analysis, RNA was
separated by 1%
formaldehydeagarose
gel electrophoresis and
transferred to a NYTRAN
SuPerCharge
nylon membrane.
Hypertrophy markers,
fibrosis markers, and
GAPDH
probes were prepared by
PCR as described
previously (21) and
labeled with
α-[32P]dCTP using
Prime-a-Gene labeling
system. The
radioactivity was
detected by
Phosphoimager using a
scanner.
EMSA and IKK. Nuclear
proteins were isolated
as described previously
(33). EMSA was performed
according to the
manufacturer’s
instructions
(Gel Shift Assay System
E3300; Promega).
Synthetic, double-strand
oligonucleotides
containing NF-κB and
GATA4 binding domains
were labeled
with [γ-32P] ATP using
T4 polynucleotide kinase
and separated from
unincorporated
[γ-32P] ATP by gel
filtration using a Nick
column (Pharmacia).
IKK complexes were
isolated from mouse
heart lysates and
cultured cardiac
myocytes using anti-IKKγ
antibodies (BD
Biosciences —
Pharmingen)
coupled to protein A
sepharose, and analyzed
using in vitro kinase
assays,
performed at 30°C with
[γ-32P]ATP and 3 μg
GST-IκBα.
Histone acetylation
assay and histone
deacetylation activity.
For the global
acetylation of histones,
cells were plated at a
density of 1 × 106
cells/ml
and exposed to the
indicated amounts of
curcumin in the presence
of 10
μCi/ml [3H]acetate (5.0
Ci/mmol) for the
indicated times. Before
collecting
the cells, TSA was used
(or not) to stimulate
the cells for 6 hours.
Histones
were then isolated, and
3H-labeled histones were
determined by liquid
scintillation
counting. To analyze the
acetylation of histone
H3, histone H4, or
tubulin, histones from
cardiac tissue and
cardiac myocytes were
prepared
as described previously
(42). The prepared
histones were suspended
in 4M
urea and stored at –20°C
until used. Equal
amounts of histones (10
μg)
were subjected to
SDS-PAGE on 15%
polyacrylamide gels and
were electrophoretically
transferred to a
nitrocellulose membrane.
Nitrocellulose blots
were blocked with 5%
milk in TTBS
(Tris-buffered saline
plus 0.05% Tween
20, pH 7.5) and
incubated overnight at
4°C with an antibody
against acetyl-
histone H3, histone H4,
or tubulin in TTBS
containing 5% milk.
After
incubation with
horseradish
peroxidase–conjugated
secondary antibody,
immunoreactivity was
visualized by means of
enhanced
chemiluminescence.
GATA4 acetylation was
detected as described
previously (43). HDAC
activity was measured by
colorimetric HDAC assay
kit (ab1438; Abcam) as
described previously
(25). The absorbance of
samples is expressed as
arbitrary
units equivalent to the
absorbance obtained with
specific concentrations
of deacetylated
standard.
Immunoprecipitation HAT
assay. HAT assays were
performed using antip300
antibody following the
manufacturer’s protocol
(Upstate) as described
previously (42).
Briefly, cardiac
myocytes were collected,
pelleted, and
resuspended
in RIPA lysis buffer
after various
treatments. Protein
concentrations
were determined using
BCA Protein Assay. For
immunoprecipitation, 500
μg lysate proteins were
precipitated with
anti-p300 antibody
(Upstate) and
incubated at 4°C
overnight. Protein G
sepharose beads (30 μl)
were added
and incubated at 4°C for
1 hour. HAT assay
cocktail (50 μl)
containing
10 μl biotinylated
histone H4 peptide was
added to the
immunoprecipitated
p300. The mixture was
then incubated at 30°C
for 1 hour in a shaking
incubator. [3H]acetyl
incorporation into the
substrates was
determined by
counting in a liquid
scintillation counter.
Animal models. All
protocols were approved
by the Animal Care and
Use
Committee of University
Health Network (Toronto,
Ontario, Canada). All
surgeries and subsequent
analyses were performed
in a blinded fashion
for all groups. The
adult male C57BL/6 mice
(8–10 weeks old) used in
the
current study were
purchased from the
Jackson Laboratory and
acclimatized
for 1 week prior to
experimental use. AB was
performed as described
previously (44). Doppler
analysis was performed
to ensure that
physiologic
constriction of the
aorta was induced. To
further detect the
effects of curcumin
on cardiac hypertrophy,
PE infusion models were
performed. PE
(65 mg/kg/d dissolved in
0.9% NaCl) was
subcutaneously infused
for 3
weeks using an osmotic
minipump (Alzet model
2004; Alza Corp.)
implanted
in each mouse.
Saline-infused animals
served as infusion
controls and
were subjected to the
same procedures as the
experimental animals
with
the exception of PE
infusion. Cardiac
adenoviral delivery was
performed
according to the method
of Lebeche et al. (45).
With preliminary
experiments
demonstrating maximal
p300 expression at 2
days after cardiac gene
transfer, all subsequent
experiments were carried
out with the transfer of
Ad-GFP or Ad-p300 2 days
prior to AB. Curcumin
suspension was prepared
using 0.5% carboxy
methylcellulose solution
for animal experiments.
Suspensions
were freshly prepared
and administered at a
constant volume of
1 ml/100 g BW by oral
gavage 3 times a day.
The control group of
these
animal experiments was
given the same volume of
liquid but composed
solely of the vehicle
solution (0.5% carboxy
methylcellulose). The
internal
diameter and wall
thickness of LV were
assessed by
echocardiography at
the indicated times
after surgery or
infusion. Hearts and
lungs of the sacrificed
mice were dissected and
weighed to compare HW/BW
(mg/g) and
LW/BW (mg/g) ratios in
curcumin- and
vehicle-treated mice.
Blood pressure and
echocardiography. A
microtip catheter
transducer
(SPR-839; Millar
Instruments) was
inserted into the right
carotid artery
and advanced into the LV
under pressure control.
After stabilization for
15 minutes, the pressure
signals and heart rate
were recorded
continuously
with an ARIA
pressure-volume
conductance system
coupled with a
Powerlab/4SP A/D
converter, stored, and
displayed on a personal
computer
as described previously
(44). Echocardiography
was performed by SONOS
5500 ultrasound (Philips
Electronics) with a
15-MHz linear array
ultrasound
transducer. The LV was
assessed in both
parasternal long-axis
and
short-axis views at a
frame rate of 120 Hz.
End-systole or
end-diastole was
defined as the phase in
which the smallest or
largest LV area,
respectively,
was obtained. LVEDD and
LVESD were measured from
the LV M-mode
tracing with a sweep
speed of 50 mm/s at the
midpapillary muscle
level.
Histological analysis.
Hearts were excised,
washed with saline
solution, and
placed in 10% formalin.
Hearts were cut
transversely close to
the apex to
visualize the LV and RV.
Several sections of
heart (4–5 μm thick)
were prepared
and stained with H&E for
histopathology or PSR
for collagen deposition
and then visualized by
light microscopy. For
myocyte cross-sectional
area, sections were
stained for membranes
with FITC-conjugated WGA
(Invitrogen) and for
nuclei with DAPI. A
single myocyte was
measured with
an image quantitative
digital analysis system
(NIH Image version 1.6).
The
outline of 100–200
myocytes was traced in
each section. To
identify infiltrating
mononuclear cell
populations, sections
were incubated with
anti–Mac-1
and anti–Mac-3 (BD)
antibody for macrophages
and anti-MPO (Abcam)
antibody for
neutrophils.
Quantitative assessments
for MPO-, Mac-1–, and
Mac-3–positive cells
were performed, and the
number of positive cells
in 5
randomly selected fields
of view was calculated
for each animal.
Statistics. Data are
expressed as means ±
SEM. Differences among
groups were tested by
1-way ANOVA. Comparisons
between 2 groups
were performed by
unpaired Student’s t
test. A P value less
than 0.05 was
considered significant.
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