Curcumin prevents and reverses murine cardiac hypertrophy

Research article
Curcumin prevents and reverses murine
cardiac hypertrophy
Hong-Liang Li,1 Chen Liu,1 Geoffrey de Couto,1 Maral Ouzounian,1 Mei Sun,1 Ai-Bing Wang,2
Yue Huang,3 Cheng-Wei He,4 Yu Shi,1 Xin Chen,1 Mai P. Nghiem,1 Youan Liu,1 Manyin Chen,1
Fayez Dawood,1 Masahiro Fukuoka,1 Yuichiro Maekawa,1 Liyong Zhang,1 Andrew Leask,5
Asish K. Ghosh,6 Lorrie A. Kirshenbaum,7 and Peter P. Liu1,8
1Division of Cardiology, Heart and Stroke/Richard Lewar Centre of Excellence, University Health Network, University of Toronto, Toronto,
Ontario, Canada. 2Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland, USA.
3Wellcome Trust Genome Campus and Sanger Institute, Hinxton, Cambridge, United Kingdom. 4Department of Medicine, Massachusetts General Hospital,
and Harvard Medical School, Boston, Massachusetts, USA. 5Division of Oral Biology and Department of Physiology and Pharmacology,
Canadian Institutes of Health Research (CIHR) Group in Skeletal Development and Remodeling, Schulich School of Medicine and Dentistry,
University of Western Ontario, London, Ontario, Canada. 6Division of Rheumatology, Northwestern University Feinberg School of Medicine,
Chicago, Illinois, USA. 7Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Winnipeg, Manitoba, Canada.
8Institute of Circulatory and Respiratory Health, Canadian Institutes of Health Research, Ottawa, Ontario, Canada.
Chromatin remodeling, particularly histone acetylation, plays a critical role in the progression of pathologi-
cal 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 hyper-
trophy through the disruption of p300 histone acetyltransferase–dependent (p300-HAT–dependent) transcrip-
tional 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 (PE-
induced) 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 acetyla-
tion, 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
in muscle that modifies chromatin and associated transcription 
Cardiac hypertrophy is an adaptive enlargement of the myocardi-
factors and promotes gene activation (6, 7). Recent studies have 
um in response to increased workload, characterized by an increase  demonstrated that p300 transcriptional activity is enhanced dur-
in the size of individual cardiac myocytes and whole-organ enlarge-
ing agonist-induced cardiac hypertrophy and that subsequent 
ment. Although cardiac hypertrophy may initially be compensato-
blocking of p300-HAT activity inhibits agonist-mediated cardiac 
ry, sustained pathologic hypertrophy is deleterious and may lead to  growth (6, 8). Moreover, transgenic mice that overexpress p300 in 
heart failure, sudden death, and stroke (1–3). Histone acetylation  the heart develop cardiac hypertrophy and eventual heart failure 
is one of the key control points for gene regulation in the hyper-
(9). Therefore, p300-HAT is an attractive target to treat or prevent 
trophic myocardium (4). Acetylation of histone tails, mediated by  cardiac hypertrophy and subsequent heart failure.
histone acetyltransferases (HATs), confers accessibility of the DNA 
Curcumin is a natural polyphenolic compound abundant in the 
template to the transcriptional machinery and is associated with  rhizome of the perennial herb turmeric, Curcuma longa (10). It is 
activation of gene expression (5). Histone deacetylases (HDACs),  commonly used as a dietary spice and coloring agent in cooking 
on the other hand, catalyze removal of acetyl groups on amino-
and is used anecdotally as an herb in traditional Indian and Chi-
terminal lysine residues of histones and, by promoting nucleoso-
nese medicine (11). However, to our knowledge no study to date 
mal condensation, act as transcriptional repressors or silencers of  has addressed the effect of curcumin on cardiac hypertrophy and 
genes (5). The status of histone acetylation is therefore determined  related signaling pathways. Although evidence demonstrates that 
by the balanced action of HATs and HDACs. p300 is a critical HAT  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 
Nonstandard abbreviations used: AB, aortic banding; ANP, atrial natriuretic 
peptide; BNP, brain natriuretic peptide; CTGF, connective tissue growth factor; FS, 
curcumin attenuates cardiac hypertrophy in vitro and in vivo by 
fractional shortening; HAT, histone acetyltransferase; HDAC, histone deacetylase; 
impairing p300-HAT activity.
HW, heart weight; IKK, IκB kinase; LDH, lactate dehydrogenase; LVEDD, LV end-
diastolic diameter; LVESD, LV end-systolic diameter; LW, lung weight; MHC, myosin 
Results
heavy chain; MPO, myeloperoxidase; PE, phenylephrin; PSR, picrosirius red; WGA, 
wheat germ agglutinin.
Pretreatment with curcumin inhibits cardiac hypertrophy in vitro. Neo-
Conflict of interest: The authors have declared that no conflict of interest exists.
natal  rat  ventricular  myocytes  were  cultured  to  greater  than 
Citation for this article: J. Clin. Invest. doi:10.1172/JCI32865.
97%  purity,  as  confirmed  by  phase-contrast  microscopy  and 

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research article
Figure 
Pretreatment with curcumin inhibits cardiac hypertrophy in vitro. (A) Curcumin inhibited PE-induced [3H]leucine incorporation. (B) Repre-
sentative fields of cardiac myocytes stained with α-actinin. Original magnification, ×200. (C) Quantification of cell cross-sectional area from
experiments shown in B by measuring 50 random cells. (D) Curcumin blunted PE-induced ANP and BNP mRNA expression levels by North-
ern blot. [3H]leucine incorporation and Northern blot were measured as described in Methods. The results were reproducible in 3 separate
experiments. *P < 0.05 versus control.

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research article
immunocytochemical staining. To rule out the possibility of  shown in Table 1, curcumin pretreatment prevented adverse cardiac 
cytotoxicity, we determined the number of viable cells in all wells  remodeling and ventricular dysfunction, as evidenced by improve-
using trypan blue exclusion analysis and lactate dehydrogenase  ments in LV end-systolic diameter (LVESD), LV end-diastolic diam-
(LDH) release assay. When 5–50 μM curcumin was applied to cul-
eter (LVEDD), and percent fractional shortening (FS).
tured neonatal cardiomyocytes, cells were observed to be healthy 
We next examined the potential effect of curcumin on hypertro-
even in the presence of 50 μM curcumin at the end of 48 hours  phy mediated by PE infusion. Mice were randomly allocated into 4 
(Supplemental Figure 1; supplemental material available online  groups: pretreatment with either vehicle or 75 mg/kg/d curcumin 
with this article; doi:10.1172/JCI32865DS1). At 100 μM curcumin,  for 1 week prior to either PE or saline infusion. Osmotic minipumps 
however, we observed decreased cell viability, leading us to choose  were implanted subcutaneously for a 3-week administration period 
a lower dose for the in vitro experiments. There were no observable  followed by cardiac functional assessment. As shown in Table 2, cur-
adverse effects by the administration of DMSO and phenylephrin  cumin abrogated PE-induced cardiac chamber dilation in both sys-
(PE) or infection with a wild-type human p300 cDNA adenoviral  tole and diastole. The PE-induced increase in HW/BW and LW/BW 
(Ad-p300) and an AT2 mutant p300 adenoviral lacking HAT activ-
ratios as well as cardiomyocyte cross-sectional area were also attenu-
ity (Ad-DN-p300) (data not shown). In this study, curcumin was  ated by 4 weeks of curcumin administration (Figure 2C). These find-
dissolved in DMSO medium for the in vitro studies. DMSO alone  ings were confirmed by morphological assessment (Figure 2D).
without curcumin served as a control and did not show any effect 
ANP, BNP, and myosin heavy chain β (β-MHC) are markers for 
on cell viability, cardiac hypertrophy, collagen synthesis, and related  cardiac hypertrophy (14). To determine whether curcumin affected 
molecular mechanisms (data not shown).
the mRNA expression levels of these markers, we performed North-
Cardiac hypertrophy can be monitored by increased protein syn-
ern blot analysis. Curcumin attenuated the observed increase in 
thesis, myocyte cross-sectional area, and induction of fetal gene  hypertrophic marker expression caused by AB or PE infusion (Fig-
expression (14). Cardiac myocytes were incubated with curcumin  ure 2, E and F). These findings suggest that curcumin prevents the 
for 60 minutes and subsequently treated with 100 μM PE for 48  development of cardiac hypertrophy in vivo.
hours. Pretreatment with curcumin demonstrated a dose-depen-
Pretreatment with curcumin inhibits histone acetylation in response to
dent reduction in PE-induced increases of [3H]leucine incorpo-
hypertrophic stimuli. To explore the molecular mechanisms through 
ration that showed maximal effects at 50 μM (Figure 1A). Addi-
which curcumin impairs the hypertrophic response, we examined 
tionally, the increase in cardiac myocyte size seen after 48 hours  the state of acetylation of histones by assaying the incorporation 
of culture in the presence of PE was also markedly attenuated by  of [3H]acetate into histones. We exposed cultured neonatal rat car-
curcumin (Figure 1, B and C). Curcumin markedly reduced atrial  diomyocytes to 100 μM PE with or without curcumin. As expected, 
natriuretic peptide (ANP) and brain natriuretic peptide (BNP)  PE induced a significant increase in histone acetylation that was 
mRNA expression levels induced by PE (Figure 1D). The inhibi-
dose-dependently blocked and sustained for all tested time points 
tion of cardiac hypertrophy in vitro by curcumin was sustained  by curcumin (Figure 3, A and B). These findings were confirmed 
for all tested times. However, curcumin alone had no effect on  by concomitant attenuation of histone H3, histone H4, and tubu-
[3H]leucine incorporation, cardiac myocyte size, or expression of  lin acetylation (Figure 3C). To test its efficacy in vivo, Western blot 
ANP and BNP. These data demonstrate that curcumin attenuates  analysis was performed using samples from the 2 differing hypertro-
cardiac hypertrophy in vitro.
phic animal models. Similar findings confirmed that vehicle-treated 
Pretreatment with curcumin inhibits cardiac hypertrophy in vivo. To  AB or PE-infused mice markedly induced the acetylation of histone 
determine the physiological relevance of our in vitro findings, we  H3, histone H4, and tubulin and that curcumin-treated mice signif-
investigated the effects of curcumin in a murine pressure-over-
icantly attenuated the acetylation of these targets (Figure 3, D and 
load model of cardiac hypertrophy. To evaluate the dose-response  E). Our findings suggest that curcumin inhibits histone acetylation 
relationship, we administered 3 different doses of curcumin (50,  in vitro and in vivo in response to hypertrophic stimuli.
75, and 100 mg/kg/d) for 1 week and then subjected the mice to 
Pretreatment with curcumin blocks p300-HAT activity, but not HDAC
either chronic pressure overload generated by aortic banding (AB)  activity. Histone acetylation is regulated by p300-HAT and plays an 
or sham surgery (control). We found that maximal efficacy was  important role in heart disease (9). To elucidate the role of p300 in 
achieved at a curcumin dose of 75 mg/kg/d (Supplemental Table 1).  the inhibitory effect of curcumin on cardiac hypertrophy, we ana-
Moreover, no apparent effect on cell toxicity was observed with  lyzed the effects of curcumin on p300-HAT activity induced by PE. 
any dose of curcumin, and 75 mg/kg/d was therefore chosen as the  As shown in Figure 4A, at concentrations of 50 and 100 μM PE, 
experimental dose. These findings are in agreement with previous  p300-HAT activity increased 11.3- and 17.9-fold, respectively, in 
publications in which curcumin showed no toxicity in the liver or  cardiac myocytes. The linear increase of HAT activity by increased 
kidney at doses of 100 or 200 mg/kg/d (15, 16).
concentrations of PE indicated that PE-induced p300-HAT activity 
In order to further evaluate the effects of curcumin on cardiac  was dose dependent and maximally achieved at 100 μM. p300-HAT 
hypertrophy, mice were randomly assigned to 4 groups: pretreat-
activity reached its maximum value at 6 hours after the addition 
ment with either vehicle or 75 mg/kg/d curcumin for 1 week prior  of 100 μM PE (Figure 4B). We also found that curcumin dose-
to either AB surgery or sham operation. Curcumin treatment of  dependently blocked PE-induced p300-HAT activity and sustained 
the AB mice resulted in significant attenuation of hypertrophy, as  this inhibition for all tested time points (Figure 4, C and D). We 
measured by heart weight/BW (HW/BW) ratio, lung weight/BW  further demonstrated that blocking p300-HAT activity inhibited, 
(LW/BW) ratio, and cardiomyocyte cross-sectional area (Figure  whereas increased p300-HAT activity promoted, histone acetylation 
2A). No significant changes were observed in the sham-operated  induced by PE, as estimated by the global acetylation of histones 
mice treated with curcumin or vehicle. Gross heart and wheat germ  and the acetylation of histone H3, histone H4, and tubulin (Figure 
agglutinin (WGA) staining further confirmed the inhibitory effect  4, E and F). This suggests that the hyperacetylation induced by PE 
of curcumin on cardiac remodeling in AB hearts (Figure 2B). As  in cardiac myocytes is dependent on p300-HAT activity. To define 

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Figure 
tive for myeloperoxidase (MPO), Mac-1, and Mac-3 significantly 
Pretreatment with curcumin blunts cardiac hypertrophy in vivo. (A and increased at 8 weeks after AB; these increases were dramatically 
C) Statistical results of HW/BW ratio, LW/BW ratio, and myocyte cross-
reduced by curcumin treatment. Furthermore, curcumin decreased 
sectional areas (n = 200 cells per section) at 8 weeks after AB surgery NF-κB activation and MCP-1, IL-6, IL-1β, and TNF-α mRNA and 
(A; n = 6) or 3 weeks after PE infusion (C; n = 7). (B and D) Gross protein expression induced by AB (Supplemental Figure 3, A and B, 
heart and WGA-FITC staining of sham and AB mice at 8 weeks after and Figure 6B). Curcumin treatment also impaired IκBα phos-
surgery (B) or 3 weeks of saline- and PE-infused mice (D) treated with
or without curcumin. Scale bars: 20 mm (gross heart); 50 μm (WGA phorylation and degradation, IκB kinase (IKK) activation, and 
stain). (E and F) Analysis of hypertrophic markers (n = 4). Total RNA p65 translocation mediated by AB (Supplemental Figure 3, C–F). 
was isolated from hearts of mice of the indicated groups, and expres-
A previous report describing the regulation of NF-κB activation 
sion of transcripts for ANP, BNP, β-MHC, and α-MHC induced by by direct protein-protein interaction with p300 (22) prompted 
AB (E) or PE infusion (F) was determined by Northern blot analysis. us to investigate the functional significance of p300 on NF-κB 
*P < 0.05 versus respective vehicle control.
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, 
the role of HDAC activity in curcumin-induced histone hypoacety-
G and H). Furthermore, p300 overexpression significantly reversed 
lation, the effect of curcumin on HDAC activity was determined.  the curcumin-induced inhibitory effects on inflammation (Sup-
Curcumin did not alter its activity at each of the tested concentra-
plemental Figure 3I). These results indicate that curcumin blocks 
tions (Supplemental Figure 2). Collectively, these results indicate  NF-κB signaling and NF-κB–dependent inflammatory responses 
that curcumin inhibits histone acetylation through direct inhibi-
through the disruption of p300-HAT activity.
tion of p300-HAT activity rather than HDAC activity.
Pathological cardiac hypertrophy is associated with increased 
Pretreatment with curcumin blocks GATA4 acetylation and DNA-
fibrosis in the myocardium (1), and, as expected, marked perivas-
binding activity. p300 protein serves as an adaptor for hypertro-
cular and interstitial fibrosis were detected in the vehicle-treated 
phy-responsive transcription factors including GATA4, which is  AB mice by picrosirius red (PSR) staining. Curcumin treatment, 
required for the activation of cardiac genes that are upregulated  however, remarkably reduced the extent of cardiac fibrosis in vivo 
during cardiac hypertrophy (17, 18). As expected, both PE stimula-
(Figure 6, C and D). Subsequent analysis of mRNA and protein 
tion and AB markedly induced, and curcumin pretreatment com-
expression levels of known mediators of fibrosis including TGF-β1, 
pletely abolished, GATA4 acetylation and DNA-binding activity  collagen I, and connective tissue growth factor (CTGF) demonstrat-
(Figure 5, A and B). Based on these observed inhibitory effects, we  ed a blunted response by curcumin treatment (Figure 6, E and F). 
investigated whether PE-mediated GATA4 activation is dependent  In addition, curcumin effectively blocked collagen synthesis and 
on p300-HAT activity. Our results demonstrated that PE-induced  COL1A2, PAI-1, and CTGF protein expression levels and promoter 
GATA4 acetylation and DNA-binding activity were almost com-
activities induced by TGF-β1 in cultured cardiac fibroblasts (Sup-
pletely inhibited by infection with Ad-DN-p300 but were aug-
plemental Figure 4, A–C).
mented by infection with Ad-p300 (Figure 5C). In addition, the 
To further elucidate the cellular mechanisms underlying the anti-
inhibitory effects of curcumin on GATA4 acetylation and DNA-
fibrotic effects of curcumin, we then assessed the regulatory role 
binding activity were reversed by infection with Ad-p300 (Figure  of curcumin in Smad cascade activation in hearts subjected to AB. 
5D). These data suggest that GATA4 acetylation and activation by  Smad-2 phosphorylation and Smad-2/3/4 nuclear translocation 
PE require p300-HAT activity in cardiac myocytes.
were markedly blocked by curcumin (Supplemental Figure 4D). 
Pretreatment with curcumin blunts inflammation and fibrosis. Inflam-
Our in vitro studies using neonatal rat cardiac fibroblasts yielded 
mation is known to play an important role in the development and  identical results (Supplemental Figure 4E). To our knowledge, no 
progression to hypertrophy and heart failure (19–21). To deter-
previous study has shown the physiologic link between TGF-β 
mine whether curcumin suppresses inflammation in the heart, we  and p300 with respect to collagen synthesis in the heart. Using 
first examined the cellular infiltrates by immunostaining analyses.  confluent cardiac fibroblasts infected with Ad-GFP, Ad-p300, 
As shown in Figure 6A, the population of cells that stained posi-
or Ad-DN-p300 along with COL1A2-, PAI-1–, or CTGF-luc reporter 
Table 
Effects of curcumin on cardiac hypertrophy induced by AB over time

Vehicle
Curcumin, 75 mg/kg/d
Group (n)
Basal (12)
AB, 4 wk (10) AB, 6 wk (8)
AB, 8 wk (8)
Basal (12) AB, 4 wk (11) AB, 6 wk (7) AB, 8 wk (8)
BW (g)
24.2 ± 1.3
25.0 ± 2.1
26.3 ± 1.4
28.0 ± 1.2
24.6 ± 2.0
26.4 ± 2.3
26.8 ± 1.7
28.3 ± 1.3
SBP (mmHg)
105.0 ± 3.7
137.6 ± 5.7A
158.0 ± 9.6A
163.0 ± 3.9A
108.0 ± 3.8 136.6 ± 6.5A
155.7 ± 7.1A 161.4 ± 6.2A
HR (bpm)
445 ± 3
446 ± 22
452 ± 24
454 ± 23
441 ± 36
445 ± 26
459 ± 17
436 ± 25
LVEDD (mm)
3.65 ± 0.03
4.12 ± 0.02A
4.89 ± 0.05A
5.22 ± 0.08A
3.69 ± 0.06 3.72 ± 0.05B
3.81 ± 0.06B 4.02 ± 0.03B
LVESD (mm)
1.79 ± 0.02
2.52 ± 0.04A
3.22 ± 0.06A
3.81 ± 0.04A
1.70 ± 0.03 1.78 ± 0.03B
1.96 ± 0.06B 2.09 ± 0.04B
IVSd (mm)
0.64 ± 0.06
0.92 ± 0.08
1.25 ± 0.07A
1.62 ± 0.04A
0.67 ± 0.02 0.73 ± 0.02
0.81 ± 0.07B 0.88 ± 0.07B
LVPWd (mm)
0.67 ± 0.07
0.78 ± 0.04
1.19 ± 0.05A
1.36 ± 0.08A
0.60 ± 0.03 0.71 ± 0.05
0.78 ± 0.03B 0.82 ± 0.02B
FS (%)
55.4 ± 3.1
43.0 ± 2.3A
30.0 ± 1.7A
18.2 ± 4.2A
56.8 ± 4.3
50.7 ± 2.9A,B
47.9 ± 1.5A,B 40.7 ± 2.5A,B
All values are mean ± SEM. SBP, systolic blood pressure; HR, heart rate; IVSd, LV septum, diastolic; LVPWd, LV posterior wall, diastolic. AP < 0.05 versus
sham. BP < 0.05 versus vehicle after AB.

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Table 
Effects of curcumin on cardiac hypertrophy induced by PE infusion

Vehicle
Curcumin, 75 mg/kg/d
Group (n)
Basal (15)
Saline (13)
PE, 3 wk (8)
Saline (14)
PE, 3 wk +
PE, 3 wk +





Cur, 4 wk (10)
Cur, 2 wk (8)
BW (g)
26.2 ± 2.2
27.8 ± 2.3
26.4 ± 1.8
27.5 ± 1.4
27.4 ± 2.1
27.0 ± 1.7
SBP (mmHg)
110.5 ± 2.1
113.0 ± 4.2
159.5 ± 3.6A
104.2 ± 3.2
153.0 ± 4.1A
155.7 ± 3.9A
HR (bpm)
488 ± 18
465 ± 15
481 ± 21
475 ± 34
467 ± 49
464 ± 26
LVEDD (mm)
3.81 ± 0.03
3.82 ± 0.05
5.16 ± 0.02A
3.74 ± 0.05
3.98 ± 0.01B
4.20 ± 0.02A,B
LVESD(mm)
1.67 ± 0.02
1.73 ± 0.03
3.78 ± 0.02A
1.59 ± 0.01
1.88 ± 0.01B
2.14 ± 0.01A,B
IVSd (mm)
0.64 ± 0.02
0.73 ± 0.04
1.35 ± 0.01A
0.58 ± 0.03
0.83 ± 0.02B
0.87 ± 0.02B
LVPWd (mm)
0.61 ± 0.01
0.62 ± 0.02
1.21 ± 0.02A
0.63 ± 0.01
0.66 ± 0.03B
0.72 ± 0.03B
FS (%)
56.0 ± 1.3
55.0 ± 2.2
25.3 ± 2.1A
54.3 ± 1.6
45.0 ± 1.3A,B
39.7 ± 1.7A,B
All values are mean ± SEM. Cur, curcumin; SBP, systolic blood pressure; HR, heart rate; IVSd, LV septum, diastolic; LVPWd, LV posterior wall, diastolic.
AP < 0.05 versus saline. BP < 0.05 versus vehicle after PE infusion.
constructs, we incubated the cells with TGF-β1. Forced expression  the phosphorylation of Smad-2 and nuclear translocation of 
of ectopic p300 revealed a significant increase in, whereas inhib-
Smad-2/3/4 in response to TGF-β1 (Supplemental Figure 4G). 
iting p300-HAT activity almost completely abrogated, collagen  Ad-p300 infection partially rescued the curcumin-induced inhibi-
synthesis, promoter activities, and protein levels of markers of  tory effects on collagen synthesis, markers of fibrosis, phosphory-
fibrosis (Supplemental Figure 4F). Immunoblot analysis further  lation of Smad-2, and translocation of Smad-2/3/4 (Supplemental 
demonstrated that Ad-DN-p300 almost completely abrogated  Figure 4, H and I). These findings suggest that curcumin blocks 
Figure 
Pretreatment with curcumin inhibits histone acetylation in vitro and in vivo. (A and B) The dose and time courses of curcumin on the global
acetylation of histones induced by PE. The results of 4 paral el experiments are shown. (C) Curcumin inhibited the acetylation of histone H3,
histone H4, and tubulin. Experiments were performed in triplicate. The levels of histone H3 acetylation, histone H4 acetylation, and tubulin were
quantified and normalized relative to GAPDH. *P < 0.05 versus control. (D and E) Curcumin blocked AB- and PE infusion–mediated acetylation of
histone H3, histone H4, and tubulin in mice (n = 5). Densitometric quantification of acetyl-H3, acetyl-H4, and tubulin was normalized to GAPDH.
*P < 0.05 versus respective vehicle control.

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Figure 
Pretreatment with curcumin blocks
p300-HAT activity. (A and B) The
dose and time course of PE on HAT
activity of p300. Cells were treated
with different doses of PE (A) or with
100 μM PE for the indicated times (B)
and then harvested and subjected to
analysis of HAT activity as described
in Methods. (C and D) Curcumin
inhibited PE-induced p300-HAT activ-
ity. Cel s were either pretreated for 60
minutes with different doses of cur-
cumin and then incubated with 100
μM PE for 6 hours (C) or pretreated
for 60 minutes with 25 μM curcumin
and then incubated with 100 μM PE
for different times up to 48 hours (D).
(E and F) Effect of p300 on histone
acetylation. Cells were infected with
Ad-p300, Ad-DN-p300, or Ad-GFP
for 24 hours and then treated with
100 μM PE for 6 hours. The global
acetylation of histones (E) and the
acetylation of histone H3, histone
H4, and tubulin (F) were determined.
Each assay was performed at least 3
times. *P < 0.05 versus control.
collagen synthesis by disrupting p300-HAT activity–dependent  induced cardiac hypertrophy. Curcumin was administered to mice 
TGF-β–Smad signaling.
at a dose of 75 mg/kg/d starting after 1 week of PE infusion and 
Curcumin ameliorates established cardiac hypertrophy in vivo. For  continuing for 2 weeks. Cardiac hypertrophy was markedly reversed 
greater clinical relevance, we next assessed whether curcumin can  by curcumin as assessed by HW/BW and LW/BW ratios, echocar-
reverse established cardiac hypertrophy. For these studies, we sub-
diographic measurements, cardiomyocyte area, and expression lev-
jected mice to AB surgery and sham operation (control). Cardiac  els of hypertrophic markers compared with vehicle-treated controls 
hypertrophy was confirmed by the increase in HW/BW ratio, by the  (Table 2 and Figure 7, C, D and F). These data confirm our findings 
gross morphology of the heart, and from echocardiographic analy-
from the AB model and suggest that curcumin is able to reverse 
ses after a 2-week period. Continuation of AB for a subsequent 6  established cardiac hypertrophy and heart failure.
weeks resulted in the transition to heart failure, as evidenced by a 
p300 partially reverses the inhibitory effects of curcumin in vivo. The 
further decline in percent FS, increase in LVEDD and LVESD, and  above experimental results suggested that curcumin inhibits car-
increase in HW/BW and LW/BW ratios (Table 3 and Figure 7, A  diac hypertrophy, inflammation, and fibrosis through blocking 
and B). Interestingly, curcumin treatment for a period of 6 weeks  p300-HAT–dependent signaling pathways. To confirm these find-
after the initial 2 weeks of AB reversed the remodeling, contractile  ings, we evaluated whether the inhibitory effects of curcumin could 
dysfunction, and cardiac ANP, BNP, and β-MHC mRNA expres-
be reversed through ectopic expression of p300 in vivo. Therefore, 
sion levels toward normal control values, ultimately preventing  we established a protocol to locally increase p300 expression via 
the transition to heart failure (Figure 7, A, B, and E). To verify this  direct adenovirus-mediated gene transfer into the heart. Western 
observation, we tested the impact of curcumin on established PE-
blot analysis showed that p300 protein levels were significantly 

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reversed  the  inhibitory  effect  of 
curcumin on AB-mediated fibro-
sis 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 cur-
cumin is mediated by interruption 
of  p300-HAT  activity–dependent 
signaling  pathways,  resulting  in 
protection  of  the  host  from  the 
combined deleterious effects of car-
diac  hypertrophy,  inflammation, 
and fibrosis. Curcumin also reversed 
established cardiac hypertrophy and 
dysfunction induced by sustained 
Figure 
pressure overload or PE infusion. 
Pretreatment with curcumin blocks GATA4 activation. (A and B) Curcumin blocked the acetylation and These findings support the concept 
DNA-binding activity of GATA4 induced by PE infusion (A) or AB (B). n = 4. Oct-1 DNA-binding activity that curcumin could be an effective 
was used as a control. (C) Effect of p300 on the acetylation and DNA-binding activity of GATA4 induced
by PE. Cel s were infected with Ad-p300, Ad-DN-p300, or Ad-GFP for 24 hours and then treated with
preventive and therapeutic candi-
100 μM PE for 24 hours. Extracts were assayed for GATA4 acetylation and DNA-binding activity. (D) date  against  cardiac  hypertrophy 
p300 partial y reversed the inhibitory effect of curcumin on the acetylation and DNA-binding activity of and heart failure.
GATA4 induced by PE. Cel s were infected with Ad-p300 or Ad-GFP for 24 hours, treated with 25 μM
Curcumin  is  a  low–molecular 
curcumin for 60 minutes, and then incubated with 100 μM PE for 24 hours. The results were reproduc-
weight  polyphenol  and  has  been 
ible in 3 separate experiments.
used  as  a  natural  compound  in 
the treatment of many conditions, 
including cardiovascular diseases 
increased in our Ad-p300–infected samples when compared with  (10, 11). However, to our knowledge there was no evidence to date 
the Ad-GFP controls, which reached peak levels after 2 days and  regarding the effects of curcumin on cardiac hypertrophy. Despite 
tapered off by 21 days (Figure 8A). Next, we studied the functional  significantly increased blood pressure in our 2 cardiac hypertrophy 
consequences of increased p300 expression. The Ad-GFP–infected  models, curcumin treatment did not affect blood pressure. This 
mice demonstrated significant ventricular dilatation and decreased  indicates that the primary target of curcumin action is cardiac pro-
percent FS compared with the sham-operated mice after 2 weeks  tection, rather than lowering blood pressure. Of particular clinical 
of AB (data not shown). Hypertrophied hearts treated with p300  relevance is the finding that curcumin can reverse preestablished 
gene transfer showed significant functional deterioration com-
cardiac hypertrophy and dysfunction induced by different animal 
pared with those treated with GFP gene transfer after 2 weeks of  models. The raw ingredient for curcumin is abundant and inexpen-
AB (Figure 8B). Interestingly, curcumin treatment markedly atten-
sive, and the amount of curcumin used is within the physiologic 
uated the functional deterioration observed in Ad-GFP–infected  range and well below the maximum tolerable pharmacological level 
mice but had no alleviating effect on Ad-p300–treated mice (Figure  (equivalent to 0.4 g/d for humans).
8B). Furthermore, Ad-p300 infection also partially but obviously 
The mechanism by which curcumin mediates its antihypertro-
reversed the inhibitory effects of curcumin on the HW/BW ratio,  phic effects remains unclear. There is increasing evidence for the 
cardiomyocyte cross-sectional area, and cardiac morphology after  involvement of chromatin remodeling, especially histone acetyla-
2 weeks of AB (Figure 8, C and D). Northern blot analysis further  tion in pathological cardiac hypertrophy and heart failure (23–25). 
revealed that Ad-p300 infection significantly reversed the attenu-
HATs are believed to acetylate histone proteins, relax chromatin, 
ated mRNA levels of ANF and BNP at 2 weeks after AB compared  and expose prohypertrophic genes for activation by cardiogenic 
with those Ad-GFP–infected groups after treatment with curcumin  transcription factors. Several lines of evidence have shown that a 
(Figure 8E). These results suggest that p300 overexpression partially  critical HAT in the heart is p300, which plays a key role in the phys-
reverses the inhibitory effects of curcumin on cardiac hypertrophy.  iological growth and differentiation of cardiac myocytes during 
As inflammation and pathological fibrosis have been shown to be  development (9). p300 knockout mice die between days 9 and 11.5 
inhibited by curcumin, we determined whether overexpression of  of gestation, exhibiting defects of cardiac muscle differentiation 
p300 might annul the inhibitory effects of curcumin on inflam-
and trabeculation, indicating the importance of p300 for early car-
mation and fibrosis. Ad-p300 infection significantly reversed the  diac morphogenesis and heart development (26). However, p300 
inhibitory effect of curcumin on NF-κB activation and NF-κB–
is also involved in the pathological process of cardiac hypertrophy 
dependent TNF-α and IL-6 expression (Supplemental Figure 5A  (8). The results of the present study indicate that inhibition of 
and Figure 8F). Additionally, overexpression of p300 significantly  histone acetylation is a key mechanism for the antihypertrophic 

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Figure 
Pretreatment with curcumin inhibits inflammation and fibrosis induced by AB. (A) Quantitative analysis revealed the population of MPO-, Mac-1–,
and Mac-3–positive cells in the hearts of the indicated mice (n = 6). (B) Western blot analysis of TNF-α, IL-1β, IL-6, and MCP-1 protein expres-
sion in the myocardia obtained from the indicated mice (n = 6). Each assay was performed in triplicate. (C) PSR staining on histological sections
of the LV was performed on each group 8 weeks after AB. Scale bars: 10 μm. (D) Fibrotic areas from histological sections were quantified using
an image-analyzing system (n = 6). (E and F) Northern blot and Western blot analyses of TGF-β1, col agen I, and CTGF were performed to
determine mRNA (E) and protein (F) expression levels in each group 8 weeks after AB (n = 3). GAPDH was used as the sample loading control.
*P < 0.05 versus vehicle-treated sham control.
activity of curcumin and that p300-HAT serves as its molecular  any appreciable increase in cell death with curcumin treatment in 
target. We found that treatment with curcumin had a negligible  the adult heart, suggesting that the role of p300 in the postnatal 
effect on the normal heart, in contrast to its effects on the heart  heart is different from its role during early cardiac morphogen-
subjected to hemodynamic stress. In addition, we did not find  esis. Consistent with our findings, Miyamoto et al. demonstrated 

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