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The dietary compound
curcumin inhibits
p300 histone
acetyltransferase
activity
and prevents heart
failure in rats.
Hemodynamic overload in
the heart can trigger
maladaptive hypertrophy
of cardiomyocytes. A key
signaling
event in this process is
nuclear acetylation by
histone deacetylases and
p300, an intrinsic
histone
acetyltransferase(HAT). It has been
previously shown that curcumin, a polyphenol
responsible for the
yellow color of
the spice turmeric,
possesses HAT inhibitory
activity with
specificity for the
p300/CREB-binding
protein. We
found that curcumin
inhibited the
hypertrophy-induced
acetylation and
DNA-binding abilities of
GATA4, a
hypertrophy-responsive
transcription factor, in
rat cardiomyocytes.
Curcumin also disrupted
the p300/GATA4
complex and repressed
agonist- and
p300-induced
hypertrophic responses
in these cells. Both the
acetylated
form of GATA4 and the
relative levels of the
p300/GATA4 complex
markedly increased in
rat hypertensive
hearts in vivo. The
effects of curcumin were
examined in vivo in 2
different heart failure
models: hypertensive
heart disease in
salt-sensitive Dahl rats
and surgically induced
myocardial infarction in
rats. In both models,
curcumin prevented
deterioration of
systolic function and
heart failure–induced
increases in both
myocardial
wall thickness and
diameter. From these
results, we conclude
that inhibition of p300
HAT activity by the
nontoxic
dietary compound
curcumin may provide a
novel therapeutic
strategy for heart
failure in humans.
Introduction
Heart failure remains
one of the leading
causes of death in
industrialized
countries (1, 2). In
response to an increase
in pressure or
volume overload,
cardiomyocytes undergo
hypertrophy, a
compensatory
response to increased
wall stress required to
maintain
normal cardiac output
(3, 4). However,
hypertrophy is
associated
with the diastolic
dysfunction of the heart
and with a significant
increase in the risk for
sudden death, and it
eventually leads to
systolic dysfunction or
decompensated heart
failure (3–5). This
process is accompanied
by activation of various
neurohormonal
factors, such as
angiotensin II,
endothelin-1 (ET-1), and
catecholamines
(6–7). While present
pharmacological therapy
for heart
failure targets such
extracellular molecules,
mortality due to heart
failure is still high
(2, 6). To establish a
more effective
therapeutic
strategy for heart
failure, it will be
necessary to target a
common
downstream pathway
within cardiomyocytes.
Neurohormonal factors
bind to myocardial
cell-surface receptors
and activate a number of
subcellular signaling
pathways.
These signals finally
reach nuclei of
cardiomyocytes and
activate
a subset of
hypertrophy-responsive
transcription factors
(8). These include serum
response factors;
myocyte enhancer
factor–2 (9); and a zinc
finger protein, GATA4
(10, 11). Activation
of these transcription
factors is mediated, in
part, through
acetylation control by
histone deacetylases and
an intrinsic
histone
acetyltransferase (HAT),
p300 (12, 13). p300
serves as a
coactivator of GATA4 and
induces expression of
genes encoding
atrial natriuretic
factor (ANF), ET-1, and
β–myosin heavy chain
(β-MHC) (14–16). By its
HAT activity, p300 not
only acetylates
histone to promote an
active chromatin
configuration but also
is able to acetylate
GATA4 and to increase
its DNA-binding and
transcriptional
activities (16). Cardiac
overexpression of intact
p300 in transgenic mice
induces acetylation of
GATA4 and myocardial
cell hypertrophy and
promotes LV remodeling
after myocardial
infarction (MI) in vivo
(16, 17). However,
overexpression
of mutant p300 lacking
HAT activity is unable
to achieve such
effects (17). These
findings suggest that
nuclear acetylation by
p300 is a critical event
during myocardial cell
hypertrophy. However,
a pharmacological heart
failure therapy that
targets p300
HAT activity has yet to
be established.
Although a number of
studies regarding such a
strategy have
been performed, very few
HAT inhibitors are known
so far. Lys-
CoA and H3-CoA-20 are
synthetic HAT inhibitors
that are specific
for p300 and for PCAF,
respectively. However,
these agents are not
easily able to permeate
cells (18, 19).
Anacardic acid and
garcinol
are cell-permeable
natural HAT inhibitors
without specificity for
p300/CBP and PCAF (20,
21). It has been
reported that a
cell-permeable
natural compound,
curcumin, possesses HAT
inhibitory
activity with
specificity for p300/CBP
(22). Curcumin is a
polyphenol
that is responsible for
the yellow color of the
spice turmeric
and is commonly used for
its perceived health
benefits.
The goal of this study
was to determine whether
a natural p300-
specific HAT inhibitor,
curcumin, can be used as
a therapeutic
agent for heart failure.
First, we tested the
effects of curcumin on
nuclear acetylation and
hypertrophic responses
in cultured neonatal
rat cardiomyocytes. Then
we examined the effects
of curcumin
in 2 different heart
failure models in vivo:
one model was
hypertensive heart
disease in
salt-sensitive Dahl (DS)
rats, and
the other model was MI
in rats. We demonstrated
that inhibition
of p300 HAT activity by
curcumin prevented the
development of
heart failure in both
models. Thus, we believe
that the present
study will provide a
novel therapeutic
strategy for heart
failure
that targets a common
nuclear pathway in
cardiomyocytes.
Results
Curcumin represses
hypertrophic responses
in cardiomyocytes. We
first
examined the effects of
curcumin on hypertrophic
responses in
primary cardiomyocytes
prepared from neonatal
rats. These cells
were stimulated with
saline or 30 μM
phenylephrine (PE), an
α1-adrenergic agonist,
in the presence of
curcumin (5 or 10 μM) or
a corresponding amount
of its solvent, DMSO.
After 48 hours, we
stained these cells with
an antibody against
cardiac MHC (Figure 1A).
Treatment with curcumin
inhibited the PE-induced
increase in
cell size (myocardial
cell-surface area) but
did not affect the basal
cell size (Figure 1B).
We then examined the
effects of curcumin
by transient
transfection experiments
on PE-induced activities
of
promoters for ANF and
β-MHC, upregulated
expression of which
are well-established
markers of myocardial
cell hypertrophy.
Curcumin
significantly inhibited
the induction of ANF
(Figure 1C)
and β-MHC (Figure 1D)
promoters. However,
curcumin alone did
not affect the basal
levels of these
activities.
We then examined the
effects of curcumin on
myocardial cell
hypertrophy induced by
overexpression of p300.
Transfection of an
expression vector
encoding p300 resulted
in a marked increase in
the amount of p300
protein (Figure 2A) and
a significant increase
in the myocardial
cell-surface area
compared with
transfection of
β-gal (Figure 2B).
Treatment with curcumin
significantly inhibited
the p300-induced
increase in cell size
but did not affect the
amount of p300.
Furthermore, treatment
with curcumin
significantly
inhibited the
p300-induced activities
of the ANF (Figure 2C)
and β-MHC (Figure 2D)
promoters. Taken
together, these data
demonstrate that
curcumin can selectively
suppress the PE- and
p300-induced
hypertrophic responses
in cardiomyocytes.
We also used an HDAC
inhibitor, trichostatin
A (TSA), to induce
nuclear
hyperacetylation, and
mutant p300
(HATmutp300), which
loses its HAT activity
but retains the ability
to interact with
transcription
factors. Treatment with
TSA and transfection of
an expression
vector encoding
HATmutp300 induced a
significant increase in
cell size. However, the
increase by TSA or
HATmutp300 was smaller
than that caused by
intact p300 (Figure 2E,
bars 1, 2, 4, and 6).
Curcumin
inhibited both TSA- and
HATmutp300-induced
hypertrophy
as well as intact
p300-induced hypertrophy
(Figure 2E, bars 2–6).
Curcumin inhibits
PE-induced acetylation
and DNA binding of
GATA4.
To assess whether
curcumin can suppress
the acetylation of
histones
during myocardial cell
hypertrophy, protein
extracts from
cardiomyocytes treated
with PE/curcumin or
their vehicles were
subjected to
immunoblotting with the
antibody against
acetylated
forms of histone-3 or
histone-4 (Figure 3A).
Curcumin repressed
the PE-induced
acetylation of histone-3
and histone-4 in
cardiomyocytes.
Then we evaluated the
nuclear hyperacetylation
in cardiomyocytes
by immunostaining with
an anti–acetylated
lysine
antibody (Figure 3B). PE
stimulation markedly
induced nuclear
staining, indicating
hyperacetylation.
Curcumin almost
completely
blocked such nuclear
hyperacetylation.
We then evaluated
effects of curcumin on
acetylation and DNA
binding of GATA4 during
myocardial cell
hypertrophy. The
expression levels of
GATA4 and β-actin were
similar in saline- and
PE-stimulated
cardiomyocytes. In
contrast, p300 levels
were markedly
increased by stimulation
with PE. Curcumin did
not affect
the protein levels of
p300, GATA4, or β-actin
(Figure 3C). Then,
nuclear extracts from
cardiomyocytes were
immunoprecipitated
with anti-GATA4
antibody, followed by
Western blotting with
anti–acetylated lysine
antibody and anti-p300
antibody. Curcumin
repressed the PE-induced
increase in the level of
the acetylated
form of GATA4 (Figure 3,
D and G, middle lanes).
Curcumin also
repressed the PE-induced
increase in the binding
of GATA4 with
p300 (Figure 3, D and F,
top lanes). To control
for differences in
protein loading after
immunoprecipitation, the
same membrane
was reblotted with
anti-GATA4 antibody. The
amounts of total
GATA4 in lysates after
immunoprecipitation were
similar among
these 3 groups (Figure
3D, bottom lanes). For
the reverse experiments,
the same extracts were
immunoprecipitated with
antip300
antibody, followed by
Western blotting with
anti-GATA4
antibody. Curcumin
inhibited the PE-induced
increase in the
binding of p300 with
GATA4 (Figure 3, E and
H). Next, the same
extracts were subjected
to EMSAs using the GATA4
site of the
ET-1 promoter as a probe
(Figure 4A). Competition
and supershift
experiments demonstrated
that the retarded band
(indicated
by arrows) represents an
interaction of the probe
with GATA4
(lanes 2, 4–7). Notably,
curcumin significantly
repressed the PE induced
increase in GATA4/DNA
binding (Figure 4, A and
C,
bars 1–3). Sp-1/DNA
binding was similar
among these 3 groups
(Figure 4B). These
findings demonstrate
that curcumin inhibits
binding of p300 to GATA4
and represses PE-induced
acetylation
and DNA binding of
GATA4.
Curcumin prevents the
development of
hypertension-induced
heart failure.
To determine whether
curcumin can prevent the
development
of heart failure in
vivo, we utilized a
salt-sensitive Dahl (DS)
rat
model of hypertension.
In this model, LV
concentric hypertrophy
with preserved systolic
function at the age of
11 weeks is followed
by a decrease in
systolic function at the
age of 17–18 weeks. We
performed physiological
studies on 11-week-old
DS rats (n = 39)
and control normotensive
salt-resistant Dahl rats
(DR rats, n = 6).
Then these rats were
randomly assigned to
daily oral treatment
with curcumin or vehicle
(1% gum arabic). Before
treatment, there
were no differences
between the curcumin and
vehicle groups
in pretreatment data,
including body weight,
blood pressure,
heart rate, LV
dimensions, fractional
shortening (FS), and
wall
thickness. Preliminary
experiments suggested
that 50 mg/kg/d
of curcumin is
sufficient to exert its
beneficial effect. Then,
we
evaluated whether
administering 50 mg/kg/d
of curcumin can
preserve LV function. At
the age of 18 weeks (7
weeks after curcumin
or vehicle treatment),
we performed
physiological studies
in all of the surviving
rats (3 rats in each DR
group, 8 rats in the
vehicle-treated DS
group, and 6 rats in the
curcumin-treated DS
group). LV systolic
function represented by
FS was much higher
in the curcumin-treated
group than in the
vehicle-treated group
(Figure 5, A, C, and H).
Wall thickness was
significantly smaller
in the curcumin-treated
group than in the
vehicle-treated group
(Figure 5, D and I).
Meanwhile, curcumin did
not alter FS or wall
thickness in DR rats
(Figure 5, H and I). LV
end-diastolic dimension
(LVEDD), body weight,
blood pressure, and
heart rate did
not differ between the
curcumin- and
vehicle-treated groups
of
DS rats (Figure 5, B, E,
F, and G).
After physiological
studies, we measured
plasma and LV mRNA
levels of B-type
natriuretic peptide
(BNP), a biochemical
marker of
heart failure. LV mRNA
levels of BNP were
higher in DS rats than
DR rats in
vehicle-treated groups
(Figure 6A). In DS rats,
these levels were
significantly higher in
the vehicle-treated
group than the
curcumin-treated group.
Consistent with these
results, treatment
with curcumin
significantly inhibited
increase in plasma BNP
levels
in DS rats (Figure 6B).
Histological analysis
demonstrated that
cross-sectional
myocardial cell diameter
was larger in DS rats
than
in DR rats in
vehicle-treated groups.
Curcumin almost
completely
inhibited
hypertension-induced
increase in myocardial
cell diameter
(Figure 6, C and D). In
vehicle-treated groups,
the amount of
fibrosis in perivascular
areas was markedly
increased in DS compared
with DR rats. Curcumin
significantly but not
completely
inhibited the increase
in perivascular fibrosis
(Figure 6, E and F).
Curcumin inhibits
hypertension-induced
acetylation of GATA4 in
Dahl rats. To determine
whether treatment with
curcumin affects
hypertension-induced
acetylation of GATA4 in
Dahl rats, we performed
immunoprecipitation/Western
blotting in LV nuclear
extracts from
18-week-old DS and
control normotensive DR
rats.
Before
immunoprecipitation,
Western blotting
demonstrated
that p300 levels were
increased in DS compared
with DR rats,
while GATA4 levels were
similar in DR and DS
rats (Figure 7A).
Then, these extracts
were subjected to
immunoprecipitation with
anti-GATA4 antibody,
followed by sequential
Western blotting
with anti–acetylated
lysine antibody and
anti-p300 antibody.
LV complex containing
p300 and GATA4 was
increased in DS
compared with DR rats
(Figure 7B, bottom
lanes). In accord with
this, the acetylated
form of GATA4 increased
in DS compared
with DR rats (Figure 7B,
top lanes). Notably, in
DS rats, administration
of curcumin decreased
the acetylated form of
GATA4
(Figure 7D, top lanes)
and p300/GATA4 complex
(Figure 7D,
bottom lanes), while it
did not change the
expression of GATA4
or p300 themselves
(Figure 7C).
Quantitative analysis
revealed
that curcumin decreased
by 60% the ratio of the
acetylated form
of GATA4 to total GATA4
(Figure 7E). Thus,
curcumin inhibited
the hypertension-induced
acetylation of GATA4 as
well as p300/
GATA4 complex in the LV
of Dahl rats.
Curcumin prevents the
deterioration of LV
systolic function after
MI.
To determine whether the
beneficial effect of
curcumin is limited
to a model of
hypertension-induced
heart failure or
generalized
to other types of heart
failure, we examined the
effect
of curcumin on LV
remodeling after MI.
Fifty-four rats were
subjected to MI (n = 40)
or sham operation (n =
14). One week
later, we performed
physiological studies in
all surviving rats.
We excluded from this
study 2 MI rats whose FS
was more than
40% before
randomization. Then, the
remaining rats (MI: n =
29,
sham operation: n = 14)
were randomly assigned
to oral chronic
treatment with curcumin
(50 mg/kg/d) or vehicle
(1% gum arabic).
Before treatment, there
were no differences
between the curcumin
and vehicle groups for
any of the data
examined, including
body weight, blood
pressure, heart rate, LV
dimensions,
FS, and wall thickness.
There was no death
during the treatment
with curcumin or vehicle
for 6 weeks. At 7 weeks
after MI
(6 weeks after the
treatment), LV systolic
function (represented
by FS) was much higher
in the curcumin-treated
group than in
the vehicle-treated
group (Figure 8, A, C,
and H). Wall thickness
was significantly lower
in the curcumin-treated
group than in
the vehicle-treated
group (Figure 8, D and
I). However, curcumin
did not alter FS or wall
thickness in
sham-operated rats
(Figure 8,
H and I). LVEDD, body
weight, blood pressure,
and heart rate
did not differ between
the curcumin- and
vehicle-treated groups
(Figure 8, B, E, F, and
G). Histological
analysis demonstrated
that cross-sectional
myocardial cell diameter
was larger in MI
rats than in
sham-operated rats in
the vehicle-treated
group.
Curcumin almost
completely inhibited the
MI-induced increase
in myocardial cell
diameter (Figure 8J).
Discussion
Accumulating evidence
suggests that nuclear
acetylation controlled
by HAT and histone
deacetylases is a
critical event during
pathological
cardiomyocyte
hypertrophy (12, 13). An
intrinsic
HAT, p300, is able to
acetylate histone and
hypertrophy-responsive
transcription factors
such as serum response
factor, myocyte
enhancer factor–2, and
GATA4. Activation by
p300 is required
not only for
pathological myocyte
growth but also for
normal
myocardial development
and differentiation.
p300-knockout
mice die between days 9
and 11.5 of gestation
and show reduced
expression of muscle
structural proteins as
well as cardiac
structural
defects and reduced
trabeculation (23). A
gene knock-in
approach demonstrated
that the HAT domain of
p300 is essential
for heart formation
(24). In contrast to
myocyte overgrowth,
antitumor agent
doxorubicin markedly
reduces p300 protein
levels in cardiomyocytes
and induces their
apoptosis (25). However,
the present study
demonstrated that the
acetylated form
of GATA4 and p300/GATA4
complex were markedly
increased
in hypertensive hearts
in vivo as well as
hypertrophied
cardiomyocytes in
culture. These findings
suggest that cardiac
p300
activity is increased in
common types of heart
failure in which
pathological
cardiomyocyte overgrowth
occurs in response to
hemodynamic overload.
A natural compound,
curcumin, repressed
agonist- and p300-
induced hypertrophic
responses in cultured
neonatal cardiomyocytes.
Curcumin inhibited
acetylation of histones
and GATA4
nearly to the control
level. These findings
suggest that
curcuminmediated
inhibition of nuclear
acetylation mainly
contributes to
the repression of
myocardial cell
hypertrophy.
Interestingly, curcumin
also disrupted the
p300/GATA4 complex
formation in
cardiomyocytes.
It has been reported
that p300, by its HAT
activity,
is able to induce
autoacetylation of p300
(26) and its significant
conformational change
and dissociate p300 from
mediator complexes
(27). Paradoxically,
curcumin strongly
inhibits binding of
p300 to mediator
complexes (27). This
effect of curcumin
appears
to be independent of its
inhibitory action on
p300 HAT activity,
since curcumin also
inhibits binding of
catalytically inactive
p300 mutant to
mediators. Our finding
that curcumin disrupts
p300/GATA4 complex
formation is compatible
with this report.
At present, the precise
mechanisms by which
curcumin inhibits
binding of p300 to GATA4
in cardiomyocytes are
unclear. One
possible explanation
would be that curcumin
induces a p300
allosteric
transition that prevents
its binding to GATA4. At
any event,
curcumin may repress
hypertrophic responses
in cardiomyocytes
through at least 2
mechanisms: (a)
inhibiting acetylation
of histones
and
hypertrophy-responsive
transcription factors;
and (b)
disrupting the
p300/GATA4 complex by a
mechanism distinct
from inhibiting p300 HAT
activity.
We showed that curcumin
inhibited HATmutp300-
and TSAas
well as intact
p300-induced
hypertrophy. The smaller
extent
of hypertrophy brought
about by TSA or
HATmutp300 than
that by intact p300
suggests that both the
bridging function and
HAT activity of p300 are
required for maximal
hypertrophy. Further
studies are needed to
clarify to what extent
the inhibition of
p300 HAT activity and/or
disruption of the
p300/GATA4 complex
contribute to the
inhibitory effects of
curcumin on hypertrophy
induced by PE in culture
or via hypertension or
MI in animal
models. However, our
data suggest that both
of the 2 mechanisms
are significantly
involved in the
curcumin-mediated
inhibition of
hypertrophic responses
in cardiomyocytes.
Myocardial cell
hypertrophy is
associated with systolic
and
diastolic dysfunction of
the heart. The present
study demonstrated
that in 2 different
models of heart failure,
hypertension
and MI, curcumin
prevented deterioration
of systolic function
without affecting
systemic hemodynamics.
In both models,
curcumin significantly
decreased LV wall
thickness and the
diameter of each
myocyte.
Curcumin-mediated
inhibition of
cardiomyocyte
hypertrophy was also
documented by reduced
gene expression of BNP,
one of the markers of
myocardial cell
hypertrophy (28). While
curcumin significantly
reduced perivascular
fibrosis as well, the
reduction was minimal.
Strikingly,
curcumin reduced the
wall thickness and
myocyte diameter
of MI rats nearly to the
level of those of
sham-operated rats.
Furthermore, curcumin
treatment inhibited the
hypertensioninduced
acetylation of GATA4 and
formation of p300/GATA4
complex in DS rats, in
accord with the data in
cultured neonatal
cardiomyocytes. These
findings suggest that
curcumin acts directly
on the heart and that
reversal of myocyte
hypertrophy
is the main mechanism by
which curcumin improves
LV systolic
function in these rat
heart failure models.
As a HAT inhibitor,
curcumin is able to
inhibit the
p300-mediated
acetylation of p53 in
vitro and in vivo (22).
Furthermore, curcumin
can disrupt the
conformation of the p53
protein required
for its serine
phosphorylation.
Recently, Sano et al.
reported that
sustained pressure
overload induces an
accumulation of p53 that
inhibits Hif-1 activity
(29). This inhibition
results in the
impairment
of cardiac angiogenesis
and in the development
of heart
failure. Therefore,
another possible
explanation for the
beneficial
effect by curcumin might
be induction of
angiogenesis by
inhibiting
cardiac p53 activity. In
addition to its
inhibitory action on
p300 HAT activity,
curcumin inhibits
transcriptional
activation
by NF-κB (30), a pivotal
intracellular mediator
of the inflammatory
response. NF-κB
signaling is also
involved in
cardiomyocyte
hypertrophy (31). The
contribution of this
action of curcumin
to the improvement of
systolic function in
hemodynamic overload–
induced heart failure
should be further
investigated. However,
inhibition of NF-κB may
be important in such
human disease
states as myocarditis,
in which inflammatory
processes play a key
role in the development
of heart failure (32).
Curcumin also acts as
a free radical scavenger
and antioxidant,
inhibiting lipid
peroxidation
and oxidative DNA
damage. Puvanakrishnan
and colleagues
reported that curcumin
played a protective role
against isoproterenol-
induced myocardial
necrosis in rats and
that this protective
effect is attributed to
its antioxidant
properties (33). These
multiple
actions of curcumin may
be beneficial when
clinically applying
this agent to patients
with heart failure.
As curcumin has potent
antiproliferative
effects against a
variety
of cancer cells in vitro
and in vivo, 7 clinical
trials regarding
neoplasms
are now ongoing, mainly
targeting colon cancer
(34). Other
clinical trials
utilizing curcumin are
also underway, including
trials
for chronic psoriasis
vulgaris and Alzheimer
disease. The phase
I–II human clinical
trials have revealed
that oral administration
of
curcumin at doses up to
10 g/d is safe. This
dosage exceeds the
dosage
used in this study (50
mg/kg/d). Hypertension
and MI-induced
heart failure are common
diseases in industrial
countries and predispose
patients to circulatory
failure and sudden
death. Conventional
pharmacological therapy
such as angiotensin
blockers (converting
enzyme inhibitors and/or
receptor blockers)
targets extracellular
neurohormonal factors.
We believe that the use
of curcumin, which
targets nuclear
signaling pathways in
cardiomyocytes, will
provide a
novel therapeutic
strategy against heart
failure. Future
application
of this nontoxic dietary
natural compound as a
therapeutic agent
for heart failure in
humans would be
particularly
interesting.
Methods
Immunocytochemistry and
measurement of
cell-surface area.
Primary neonatal rat
ventricular
cardiomyocytes were
prepared as previously
described (16, 37).
The cardiomyocytes were
grown in flask-style
chambers with glass
slides
(Nalgen Nunc) and
stained for cardiac MHC
using the indirect
immunoperoxidase
method, as previously
described (35). A total
of 40 myocardial fibers
were selected randomly
from cardiomyocytes
stained with anti–β-MHC
antibody that reacts
with both α- and cardiac
β-MHC. Then, the surface
area of these cells was
measured
semiautomatically with
the aid of an image
analyzer (Image
Pro-Plus;
MediaCybernetics) as
described previously
(35).
Plasmid constructs. The
expression vectors
pCMVβ-gal, pCMVwtp300,
and
pCMVHATmutp300 contain
the cytomegalovirus
promoter/enhancer fused
to cDNA encoding β-gal,
a full-length human
p300, and a mutant p300,
respectively, in which
double amino acid
substitutions were
introduced in the
HAT domain. The plasmids
pCMVwtp300 and
pCMVHATmutp300 were a
gift from Richard
Eckner, University of
Medicine and Dentistry
of New Jersey,
Newark, New Jersey, USA.
The plasmid constructs
pANF-luc and pβ-MHCluc
directly on the heart
and that reversal of
myocyte hypertrophy
is the main mechanism by
which curcumin improves
LV systolic
function in these rat
heart failure models.
As a HAT inhibitor,
curcumin is able to
inhibit the
p300-mediated
acetylation of p53 in
vitro and in vivo (22).
Furthermore, curcumin
can disrupt the
conformation of the p53
protein required
for its serine
phosphorylation.
Recently, Sano et al.
reported that
sustained pressure
overload induces an
accumulation of p53 that
inhibits Hif-1 activity
(29). This inhibition
results in the
impairment
of cardiac angiogenesis
and in the development
of heart
failure. Therefore,
another possible
explanation for the
beneficial
effect by curcumin might
be induction of
angiogenesis by
inhibiting
cardiac p53 activity. In
addition to its
inhibitory action on
p300 HAT activity,
curcumin inhibits
transcriptional
activation
by NF-κB (30), a pivotal
intracellular mediator
of the inflammatory
response. NF-κB
signaling is also
involved in
cardiomyocyte
hypertrophy (31). The
contribution of this
action of curcumin
to the improvement of
systolic function in
hemodynamic overload–
induced heart failure
should be further
investigated. However,
inhibition of NF-κB may
be important in such
human disease
states as myocarditis,
in which inflammatory
processes play a key
role in the development
of heart failure (32).
Curcumin also acts as
a free radical scavenger
and antioxidant,
inhibiting lipid
peroxidation
and oxidative DNA
damage. Puvanakrishnan
and colleagues
reported that curcumin
played a protective role
against isoproterenol-
induced myocardial
necrosis in rats and
that this protective
effect is attributed to
its antioxidant
properties (33). These
multiple
actions of curcumin may
be beneficial when
clinically applying
this agent to patients
with heart failure.
As curcumin has potent
antiproliferative
effects against a
variety
of cancer cells in vitro
and in vivo, 7 clinical
trials regarding
neoplasms
are now ongoing, mainly
targeting colon cancer
(34). Other
clinical trials
utilizing curcumin are
also underway, including
trials
for chronic psoriasis
vulgaris and Alzheimer
disease. The phase
I–II human clinical
trials have revealed
that oral administration
of
curcumin at doses up to
10 g/d is safe. This
dosage exceeds the
dosage
used in this study (50
mg/kg/d). Hypertension
and MI-induced
heart failure are common
diseases in industrial
countries and predispose
patients to circulatory
failure and sudden
death. Conventional
pharmacological therapy
such as angiotensin
blockers (converting
enzyme inhibitors and/or
receptor blockers)
targets extracellular
neurohormonal factors.
We believe that the use
of curcumin, which
targets nuclear
signaling pathways in
cardiomyocytes, will
provide a
novel therapeutic
strategy against heart
failure. Future
application
of this nontoxic dietary
natural compound as a
therapeutic agent
for heart failure in
humans would be
particularly
interesting.
Methods
Immunocytochemistry and
measurement of
cell-surface area.
Primary neonatal rat
ventricular
cardiomyocytes were
prepared as previously
described (16, 37).
The cardiomyocytes were
grown in flask-style
chambers with glass
slides
(Nalgen Nunc) and
stained for cardiac MHC
using the indirect
immunoperoxidase
method, as previously
described (35). A total
of 40 myocardial fibers
were selected randomly
from cardiomyocytes
stained with anti–β-MHC
antibody that reacts
with both α- and cardiac
β-MHC. Then, the surface
area of these cells was
measured
semiautomatically with
the aid of an image
analyzer (Image
Pro-Plus;
MediaCybernetics) as
described previously
(35).
Plasmid constructs. The
expression vectors
pCMVβ-gal, pCMVwtp300,
and
pCMVHATmutp300 contain
the cytomegalovirus
promoter/enhancer fused
to cDNA encoding β-gal,
a full-length human
p300, and a mutant p300,
respectively, in which
double amino acid
substitutions were
introduced in the
HAT domain. The plasmids
pCMVwtp300 and
pCMVHATmutp300 were a
gift from Richard
Eckner, University of
Medicine and Dentistry
of New Jersey,
Newark, New Jersey, USA.
The plasmid constructs
pANF-luc and pβ-MHCluc
monoclonal antibody
(Molecular Probes;
Invitrogen) for Western
blotting.
The levels of signals
were estimated by
photographing them using
LAS-1000
Plus (FUJIFILM) and
quantified using Multi
Gauge V3.0 (FUJIFILM).
EMSAs. EMSAs were
carried out as
previously described
using the GATA4
site in the rat ET-1
promoter as a probe (16,
17, 37). We also used a
doublestranded
oligonucleotide
containing the Sp-1
binding site (Santa Cruz
Biotechnology Inc.) as a
control probe. The
levels of the signals
were quantified
by densitometry using
NIH Image 1.61
(http://rsb.info.nih.gov).
Animals. All procedures
were performed in
conformity with the
Guide
for the Care and Use of
Laboratory Animals by
the Institute of
Laboratory
Animals, Graduate School
of Medicine, Kyoto
University, and the
protocol was approved by
the ethics committee of
the Graduate School of
Medicine, Kyoto
University.
DS rats. Male DS and DR
rats were purchased from
Japan SLC Inc. After
weaning, the rats were
fed a 0.3% NaCl
(low-salt) diet until
the age of
6 weeks, after which
they were fed an 8% NaCl
(high-salt) diet.
Eleven-weekold
DS and DR rats were
randomly assigned to
treatment with either
curcumin
(Wako Japan) or vehicle
(1% gum arabic). All
drugs were given to
these rats orally by
gastric gavage once a
day. Animals were
monitored, and
body weights and in vivo
blood pressures were
measured weekly.
MI rats. MI was induced
in male rats weighing
250–290 g by proximal
ligation of the left
anterior descending
coronary artery through
left thoracotomy,
as previously described
(38). Rats were
anesthetized with
1.0%–1.5%
isoflurane. After
coronary ligation, ST
elevation on
electrocardiography and
a color change in the LV
myocardium were observed
in all rats. The same
surgical procedure was
performed in a
sham-operated group of
rats, except
that the suture around
the coronary artery was
not tied. Animals were
monitored,
and body weights were
measured weekly. Serial
echocardiography
and measurement of in
vivo blood pressures
were performed biweekly.
Transthoracic
echocardiography.
Cardiac function was
noninvasively evaluated
by echocardiography
according to a method
previously described
(38). In brief, images
were recorded using a
10- to 12-MHz
phased-array
transducer (model 21380A
with HP SONOS 5500
imaging system; Agilent
Technologies). LVEDD and
end-systolic dimension
(LVESD) were measured
with M-mode tracings
from the short-axis view
of the LV at the
papillary
muscle level. Percent FS
was calculated as
follows: %FS = [(LVEDD
– LVESD) / LVEDD] × 100.
All measurements were
performed in a blinded
fashion according to the
guidelines of the
American Society for
Echocardiology
and averaged over 3
consecutive cardiac
cycles.
Histological analysis.
After physiological
studies, all surviving
rats were
euthanized, and their
hearts were removed. The
excised hearts were cut
into 2 transverse slices
at the mid-level of the
papillary muscles; the
specimens
were fixed in 10%
buffered formalin and
embedded in paraffin,
after
which 4-μm-thick
sections were stained
with H&E and Masson
trichrome.
Quantitative assessments
of cross-sectional
myocardial cell diameter
and
perivascular fibrotic
area were performed on
20 randomly chosen
highpower
fields in each section
with the use of an
Axioskop 2 FS plus
(Zeiss).
Measurement of plasma
BNP and LV BNP mRNA.
Blood samples were
obtained from all
surviving rats, and
plasma BNP
concentrations were
determined using a
radioimmunoassay kit
(Peninsula Lab.).
Measurement
of LV BNP mRNA was
performed by real-time
RT-PCR analysis. Total
RNA
was isolated from left
ventricles using TRIzol
reagent (Invitrogen).
RNA
samples were treated
with DNaseI (Invitrogen)
to eliminate genomic DNA
contamination, and cDNA
was synthesized using
SuperScriptTMII reverse
transcriptase
(Invitrogen). For
real-time PCR, the
reaction was performed
with SYBR Green PCR
master mix (Applied
Biosystems), and the
products
were analyzed with a
thermal cycler (ABI
Prism 7900HT sequence
detection
system). Levels of GAPDH
transcript were used to
normalize cDNA levels.
Rat BNP-specific PCR
primers were: sense,
5′-TTCCGGATCCAGGAGAGACTT-
3′; antisense,
5′-CCTAAAACAACCTCAGCCCGT-3′.
Statistics. Data are
presented as mean ± SEM.
Statistical comparisons
were
performed with the use
of unpaired 2-tailed
Student t tests or ANOVA
with
Scheffé’s test where
appropriate, with a
probability value less
than 0.05
taken to indicate
significance.
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