Inhibition of histone acetylation by curcumin reduces alcohol-induced fetal cardiac apoptosis
Yan et al. Journal of Biomedical Science
Inhibition of histone acetylation by curcumin reduces alcohol-induced fetal cardiac apoptosis
Xiaochen Yan 0 2 3 4 5
Bo Pan 0 2 3 4 5
Tiewei Lv 0 4
Lingjuan Liu 0 4
Jing Zhu 3
Wen Shen 1
Xupei Huang 1
Jie Tian 0 4
0 Department of Cardiology, Heart Centre, The Children's Hospital of Chongqing Medical University , 136 Zhongshan Er Rold, Yu Zhong District, Chongqing 400014 , China
1 Department of Biomedical Science, Charlie E. Schmidt College of Medicine, Florida Atlantic University , 777 Glades Road, Boca Raton, FL 33431 , USA
2 China International Science and Technology Cooperation base of Child Development and Critical Disorders , Chongqing , China
3 Children's Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders , Chongqing , China
4 Department of Cardiology, Heart Centre, The Children's Hospital of Chongqing Medical University , 136 Zhongshan Er Rold, Yu Zhong District, Chongqing 400014 , China
5 Chongqing Key Laboratory of Pediatrics , Chongqing , China
Background: Prenatal alcohol exposure may cause cardiac development defects, however, the underlying mechanisms are not yet clear. In the present study we have investigated the roles of histone modification by curcumin on alcohol induced fetal cardiac abnormalities during the development. Methods and results: Q-PCR and Western blot results showed that alcohol exposure increased gene and active forms of caspase-3 and caspase-8, while decreased gene and protein of bcl-2. ChIP assay results showed that, alcohol exposure increased the acetylation of histone H3K9 near the promoter region of caspase-3 and caspase-8, and decreased the acetylation of histone H3K9 near the promoter region of bcl-2. TUNEL assay data revealed that alcohol exposure increased the apoptosis levels in the embryonic hearts. In vitro experiments demonstrated that curcumin treatment could reverse the up-regulation of active forms of caspase-3 and caspase-8, and down-regulation of bcl-2 induced by alcohol treatment. In addition, curcumin also corrected the high level of histone H3K9 acetylation induced by alcohol. Moreover, the high apoptosis level induced by alcohol was reversed after curcumin treatment in cardiac cells. Conclusions: These findings indicate that histone modification may play an important role in mediating alcohol induced fetal cardiac apoptosis, possibly through the up-regulation of H3K9 acetylation near the promoter regions of apoptotic genes. Curcumin treatment may correct alcohol-mediated fetal cardiac apoptosis, suggesting that curcumin may play a protective role against alcohol abuse caused cardiac damage during pregnancy.
Alcohol; Apoptosis; Histone acetylation; Fetal cardiac development; Caspase
During the past two decades, about 1.35 million children
were born annually having congenital heart disease
(CHD). The incidence of CHD is very high (9.3%) and
accounts to one third of the total number of birth
defects . Thus, it has been a major concern in relation
to public health. The causes of CHD are complex, only
about 15% of CHD are known to stem from clear
genetic reasons, while vast majority are caused by the
interaction of environmental and genetic factors. One of the
common environmental teratogenic factors is maternal
alcohol abuse during pregnancy. Epidemiological data
have shown that mothers consume alcohol during
pregnancy and the rate of their offspring suffering from
CHD would be doubled . Animal studies have also
shown that alcohol exposure can lead to congenital
cardiac defects such as ventricular septal defects [3–5] and
several types of cardiomyopathy . Both in vivo and in
vitro studies have shown that alcohol exposure can
induce apoptosis of cardiomyocytes [6, 7].
Epigenetics modification plays a key role in the
regulation of embryonic development and cardiogenesis
[8–12]. Acetylation of histone generated by histone
acetyltransferases (HATs) can neutralize the positive
charges between histone and DNA allowing the
binding of transcription factors to DNA, and consequently
advances gene transcription [13, 14]. In addition,
studies have also revealed that alcohol exposure can lead to
hyperacetylation of histone H3K9 [15–17], an event
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believed to be associated with abnormal heart
development [14, 18–21]. H3K9 plays a critical role in cell
cycle progression and apoptosis [22, 23]. Pregnant
mice expose to alcohol results in elevated levels of
acetylated histone H3K9 followed by apoptosis in fetal
lung , suggesting that increased acetylation of
H3K9 could alter the expression of genes that induce
apoptosis. Caspase-3 is a caspase protein that encoded
by the caspase-3 gene, the protein itself can be cleaved
and activated during apoptosis. And cleaved caspase-3
plays a central role in the execution-phase of cell
apoptosis. Acetylation of histone H3 also regulate the
cleaved caspase-3 level. Exposure of N27 dopaminergic
cells to paraquat induced histone H3 acetylation and
cleaved caspase-3 upregulation, while inhibition of
HAT activity by anacardic acid significantly reduced
paraquat-induced caspase-3 proteolytic cleavage .
Curcumin, the main constituent of the spice turmeric, is
reported as the first naturel HAT inhibitor . Several
investigators have provided evidences suggesting that
curcumin has protective effects against abnormal heart
development . In this study, we have measured the mRNA
of caspase-3, caspase-8 and bcl-2, and the protein of
cleaved caspase-3, cleaved caspase-8 and bcl-2. The
purpose of the study is to understand whether histone H3K9
acetylation can regulate the cardiomyocytes apoptosis
induced by alcohol. And, whether inhibition of histone
H3K9 acetylation by curcumin, can reverse the
overexpressed apoptosis genes.
Prenatal ethanol exposure
Fifty healthy adult C57BL6 mice (weight 21–28 g,
specific pathogen free (SPF) grade) were purchased from
the Experimental Animal Center of Chongqing Medical
University (Chongqing, China). All experimentation
procedures were conducted in accordance with the ethical
guidelines provided by Chongqing Medical University on
laboratory animals. Mice were raised in a stable
environment (22 ± 1 °C, 55 ± 5% humidity) with 12 h’ light: 12 h’
dark cycle (light 19:00–07:00) and provided food ad
libitum. After mating in the evening (5 pm), female mice
were examined for the vaginal plug at 8:00 AM in the
following morning. The day on which the vaginal plug
was detected was considered as embryonic day 0.5
(E0.5). Between the embryonic day 7.5 (E7.5) and
embryonic day 15.5 (E15.5), every morning at 8 am, the study
mice were given ethanol (56% v/v in saline) using oral
gavage at dosage of 10 μl/g while the controls were given
isovolumetric, isocaloric glucose saline solution. The
mice were monitored after treatment and it was seen
that the study mice immediately appeared intoxicated
and lethargic following ethanol exposure; however,
they began moving around normally 35 mins after the
exposure. At E 17.5, all pregnant mice were
euthanized using carbon dioxide asphyxia and the
embryonic hearts were promptly retrieved from the mouse
Culture and treatment of cardiac progenitor cells
The cardiac progenitor cells were purchased from
Molecular Oncology Laboratory at the University of
Chicago Medical Center . The cells were cultured
in Dulbecco’s modified Eagle medium (DMEM)/high
glucose (Thermo, USA) supplemented with 10% fetal
bovine serum (FBS) (Hyclone, USA), 100U/ml penicillin
and 100 μg/ml streptomycin (Thermo, Waltham, MA,
USA) in 37 °C humid air with 5% CO2. The cells were
divided into five groups: alcohol; curcumin; alcohol +
curcumin; DMSO; and control. From our previous
studies [14, 18], we found that 200 mM alcohol was an
effective concentration resulting in hyperacetylation of
cardiac progenitor cells, and 25 μM curcumin could
completely reversed the hyperacetylation induced by
alcohol (200 mM). So we used 200 mM alcohol and
25 μM curcumin (SIGMA, USA) to intervene our cells
for 24 h.
Total RNA extraction and quantitative real-time PCR
Total RNA was extracted via RNA extraction kit (Bioteck,
Beijing, China), then reverse transcribed into cDNA with
oligo dT-adaptor primer and AMV reverse transcriptase
kit (TaKaRa, Ostu, Japan). Quantitative real-time
polymerase chain reaction (RT-PCR) was performed using SYBR
Green RealMasterMix kit (Tiangen, Beijing, China) with
the following parameters: pre-denaturation at 95 °C for
3 min, followed by 40 cycles of denaturation at 95 °C for
10s, annealing at proper temperature for 30s, elongation
at 72 °C for 20s. The primers used in the study are listed
in Table 1. The annealing temperatures were 56 °C for
caspase-3, caspase-8, bcl-2 and β-actin. The relative
mRNA levels for each gene were normalized to β-actin
using the 2-ΔΔCt (Ct is cycle threshold) method .
Western blotting assays were performed as previously
reported [19, 21, 30]. The primary and secondary
antibodies used are listed in Table 2. Specific target protein
bands were revealed with enhanced chemiluminescence
(Millipore, Billerica, USA), and analyzed using Quantity
One Version 4.62 software (Bio-Rad, Richmond, CA).
The β-actin was used as the internal control.
Chromatin immunoprecipitation (ChIP) assay
CHIP assays were performed as previously reported
[21, 30, 31]. The primers used in the study are listed
in Table 1. The annealing temperature was 56 °C for
caspase-3, caspase-8 and bcl-2.
Forward 5'-GGAGATTACTGCCCTGGCTCCTA-3' 174 bp
Caspase-3 Forward 5'-AGTCCCTTACATCCAACG-3'
Caspase-8 Forward 5'-CAGAGGGATCAGGTTGGG-3'
TUNEL (Terminal-deoxynucleoitidyl Transferase
Mediated Nick End Labeling) assay (KeyGEN, Nanjing, China)
was used to assess the mode of apoptosis of alcohol
treatment. Fifteen μm thick frozen section was fixed in 10%
formalin for 20 min, then incubated in permeabilizing
solution (0.1% Triton X in 0.1% phosphate-buffered saline)
for 5 min at room temperature. After blocking in 0.3%
H2O2 for 30 min, the frozen section was incubated with
TdT (Terminal Deoxynucleotidyl Transferase) enzyme
reaction mixture for 60 min at 37 °C, and then conjugated
with streptavidin-HRP for 30 min at 37 °C. This was
followed by incubation with 3’-diaminobenzidine (DAB)
solution. Then counterstained the section with hematoxylin
and the positively stained cells were photographed on a
Nikon 800 photomicroscope.
Table 2 The antibodies used in Western blotting assays
Table 1 The primers used in Q-PCR or CHIP assays
Caspase-3 Forward 5'-TGACTGGAAAGCCGAAACTC-3'
Caspase-8 Forward 5'-GCCCTCAAGTTCCTGTGCT-3'
Cardiac progenitor cells (1 × 106/well), which had been
treated with alcohol, curcumin, alcohol + curcumin or
DMSO, were incubated with Annexin-V for 30 min
followed by propidium iodide for 10 min. Subsequently,
the apoptosis was subject to analysis using flow cytometer
(BD FACSCalibur, USA).
Statistical analysis was performed using mean ± SD or
one-way analysis of variance (ANOVA). Value of P < 0.05
was considered to be statistically significant.
Effect of alcohol exposure on pregnant mice
Pregnant C57 mice were exposed to alcohol (56% v/v in
saline) in a dosage of 10 μl/g by gavage between E7.5
and E15.5. The peak blood-alcohol levels was 212.43 ±
56.53 mg/100 ml 40 min after alcohol exposure.
Effect of alcohol exposure on H3K9 acetylation of
embryonic hearts in E17.5
In our previous study, we has discovered that alcohol
exposure increased the levels of H3K9 acetylation in
embryonic hearts between E11.5 and E18.5, and it reached
its highest point in E17.5 . In this study, using
Western blotting assays, we found that H3K9 acetylation was
increased significantly in embryonic hearts at E17.5 after
alcohol exposure (Fig. 1).
Effect of alcohol exposure on the expression of apoptosis
gene of embryonic hearts in E17.5
In order to detect the apoptosis inducted by alcohol
treatment, we monitored caspase-3, caspase-8, and bcl-2 using
Quantitative Real-Time PCR assays and Western blotting
assays. Q-PCR data showed that mRNA of caspase-3 and
caspase-8 in alcohol group was higher than that in control
group (P < 0.05) (Fig. 2a, b). Whereas bcl-2 was decreased
in the alcohol group compared to that in the control
group (P < 0.05) (Fig. 2c). Furthermore, we examined
cleaved and pro caspase-3, caspase-8 and bcl-2 using
Western blotting assays. Cleaved caspase are small
fragments extracted from the caspase, which play a key role in
caspase-dependent pathway for apoptosis. The expression
of caspase-3 (Fig. 2d, h), caspase-8 (Fig. 2e, j) and bcl-2
(Fig. 2f, k) was decreased in the alcohol group compared
to that in the control group (P < 0.05). Whereas the
cleaved caspase-8 was increased in alcohol group
compared to that in the control group (P < 0.05) (Fig. 2e, i).
However, the cleaved caspase-3 was only observed in
alcohol group (Fig. 2d, g).
Fig. 1 Effect of Alcohol Exposure on H3K9 Acetylation of Fetal Hearts in E17.5. a Western blot bands. b Prenatal alcohol exposure increased the
acetylation of histone H3K9. *p < 0.05
Fig. 2 Effect of Alcohol Exposure on the Expression of Apoptosis Gene of Embryonic Hearts in E17.5. a, b The mRNA levels of caspase-3 and caspase-8
increased more with alcohol exposure, compared with the control group. c The mRNA levels of bcl-2 decreased more in the alcohol group than in the
control group. d, e, f Western blot band. g, h Western blotting showed that prenatal alcohol exposure increased the expression of cleaved caspase-3 and
decreased the expression of caspase-3. i, j Western blotting showed that pregnant alcohol exposure increased the expression of cleaved caspase-8 and
decreased the expression of caspase-8. k Western blotting showed that bcl-2 significantly decreased in alcohol group than in control group. *p < 0.05
Effect of alcohol exposure on myocardial apoptosis
TUNEL assay was used to assess the mode of apoptosis
with alcohol treatment. As shown in Fig. 3, the positively
stained cells in the alcohol group were significantly
higher than those in the control group.
Effect of alcohol exposure on acetylation of histone H3K9
near the promoter regions of apoptosis genes
In order to determine whether the change of caspase-3,
caspase-8 and bcl-2 expressions following alcohol
treatment were associated with hyperacetylation of histone
H3K9 near the promoter regions of these genes, we
assessed the levels of the histone H3K9 acetylation near
the promoter regions of caspase-3, caspase-8 and bcl-2
using CHIP assays. We observed that alcohol could
increase the acetylation level of histone H3K9 near the
promoter regions of caspase-3 and caspase-8 (p < 0.05)
(Fig. 4a, b). Meanwhile, alcohol significantly decreased
Fig. 3 Effect of Alcohol Exposure on Myocardial Apoptosis. TUNEL
assay results showed that alcohol exposure increased the apoptosis
cells in the fetal heart. a Control group. b Alcohol group.
TUNELpositive cells are shown in brown (arrow)
Fig. 4 Effect of Alcohol Exposure on Acetylation of Histone H3K9
near the Promoter Regions of Apoptosis Genes. a, b ChIP-Q-PCR
data showed that alcohol increased the acetylation of histone H3K9
near the promoter regions of caspase-3 and caspase-8. c ChIP-Q-PCR
data showed that alcohol decreased the acetylation of histone H3K9
near the promoter region of bcl-2. *p < 0.05
the acetylation level of histone H3K9 near the promoter
region of bcl-2 (p < 0.05) (Fig. 4c).
Effect of curcumin on acetylation of histone H3K9 in
cardiac progenitor cells treated by alcohol
We analyzed whether the hyperacetylation of H3K9
could be reversed by curcumin. Alcohol increased
acetylation of histone H3K9 but when treated with
curcumin and alcohol simultaneously, the hyperacetylation
was reversed (Fig. 5). However, the expression of
acetylation of histone H3K9 had no effect when treated only
Effect of curcumin on expression levels of apoptosis
genes in cardiac progenitor cells treated by alcohol
We then analyzed whether the change of apoptosis genes
could be reversed by curcumin. The results demonstrated
that cleaved caspase-3 (Fig. 6a, d) and cleaved caspase-8
(Fig. 6b, f ) levels increased significantly when treated with
alcohol. At the same time, the expression of caspase-3
(Fig. 6a, e) and caspase-8 (Fig. 6b, g) along with the
expression of bcl-2 (Fig. 6c, h) decreased. After the
treatment with both curcumin and alcohol simultaneously, the
over-expression of cleaved caspase-3 and cleaved caspse-8
were corrected. Moreover, the decrease in caspase-3,
caspase-8 and bcl-2 were also reversed. However,
curcumin did not affect the baseline level of cleaved caspase-3,
cleaved caspase-8, caspase-3, caspase-8 and bcl-2.
Effect of curcumin on apoptosis levels in cardiac
progenitor cells treated by alcohol
Flow cytometry (FCM) assay was used to detect the
apoptosis levels. As shown in Fig. 7, alcohol treatment
increased the level of cardiac progenitor cells apoptosis
Fig. 5 Effect of Curcumin on Acetylation of Histone H3K9 in Cardiac
Progenitor Cells Treated by Alcohol. a Western blot bands. b Alcohol
treatment lead to the hyper-acetylation of histone H3K9 in cardiac
progenitor cells, and curcumin pretreatment could prevent this
change. *p < 0.05
rate to 10%, whereas curcumin intervention at an early
stage could prevent this change.
Alcohol is a common environmental teratogenic factor
and excessive use may generate excessive oxidative stress
 and apoptosis in cardiomyocytes . Prenatal
alcohol exposure exerts a clear teratogenic effect on the
developing heart, such as ventricular septal defect 
and several types of cardiomyopathy . Our previous
study has also revealed that prenatal alcohol exposure
could result in hypertrophy . However, the
underlying mechanisms are still unclear. It has been clearly
established that cardiomyocytes are highly differentiated
cells which rarely replicate after birth. Cardiomyocytes
death (either necrosis or apoptosis) may be associated
with abnormal heart development [33, 34]. Some studies
have focused on the roles of apoptosis in heart
development after prenatal alcohol exposure. Ren and co-workers
 found that prenatal ethanol exposure increases
apoptosis and alters myocardial contractile function in
newborns. Goh et al.  found that, ethanol exposure during
late gestation period may increase the expression of
apoptosis genes, then accelerates the maturation of
cardiomyocytes and increases cardiomyocyte and left ventricular
(LV) tissue volume in the fetal heart. In this study, we find
that prenatal alcohol exposure can increase the protein of
cleaved caspase-3, cleaved caspase-8 and decrease the
expression of bcl-2, consequently leading to cardiomyocytes
apoptosis. Our data reveal that prenatal ethanol exposure
promotes fetal myocyte apoptosis. This is an adverse event
during heart development.
A body of evidence have shown that, histone
acetylation is a key regulator during gene transcription
[11, 36–38]. Histone acetylation regulates the expression
of heart development related genes and plays a critical
role in abnormal heart development when exposed to
alcohol [30, 39, 40]. Although some researches show that
HDACs can repress the growth of myocytes [41, 42], our
previous study have demonstrated that alcohol exposure
increased histone acetylation by enhancing HATs
activities instead of altering HDACs . Recent observations
have further shown that cell death and survival are
determined by the balance of histone
acetylation/deacetylation [43–45]. For instance, the HDAC inhibitor induces
histone hyperacetylation, which results in cancer cell
death [46, 47]. On the contrary, anacardic acid, a HATs
inhibitor, also has a neuroprotective effect against
dieldrin-induced nigral dopaminergic neuronal
degeneration . So we detected the acetylation of histone
H3K9 near the promoter regions of apoptosis genes. We
find prenatal alcohol exposure can increase histone
H3K9 acetylation near the promoter regions of
caspase3, caspase-8, while decrease histone H3K9 acetylation
Fig. 6 Effect of Curcumin on Expression Levels of Apoptosis Genes in Cardiac Progenitor Cells Treated with Alcohol. a, b, c Western blot band. d, e
Western blotting showed that alcohol treatment increased the expression of cleaved caspase-3 and decreased the expression of caspase-3, and curcumin
pretreatment could reverse this change. f, g Western blotting showed that alcohol treatment increased the expression of cleaved caspase-8 and decreased
the expression of caspase-8, and curcumin pretreatment could reverse this change. h Alcohol treatment could decrease the expression of bcl-2 in cardiac
progenitor cells, while curcumin pretreatment could prevent this change. *p < 0.05
near the promoter region of bcl-2. And q-PCR data
reveal that, alcohol can increase the mRNA of caspase-3,
caspase-8 and decrease the mRNA of bcl-2. This data
demonstrate that alcohol exposure can alter the balance
of histone acetylation of apoptosis genes, which may
cause the dysregulated expression of apoptosis genes. So,
we emphasize that any alterations in the balance of
histone acetylation in apoptosis genes may contribute to
programmed cell death.
Curcumin is a curcuminoid found in turmeric, is
reported as the first natural HATs inhibitor . In
our previous studies, we have confirmed that alcohol
can impair the homeostasis of histone acetylation in
cardiomyocytes, while this hyperacetylation can be
reversed by curcumin. In this study, we found curcumin
could down-regulate the hyper-acetylation of histone
H3K9 induced by alcohol, but not back to the
baseline level. This suggests that some factors such as
the instability of curcumin and the turn-over rate of
histone acetylation on those sites should be taken
into account in the study. It is concluded that
curcumin can indeed down-regulate partially the acetylation
of histone H3K9 and correct the up-regulation of
cleaved caspase-3, −8 as well as the down-regulation
of bcl-2 induced by alcohol, which result in a
correction of the high level of apoptosis in cardiac cells.
These findings highlight the role of hyper-acetylation
in the programmed cell death. We provide the
implication that the increased histone acetylation could be
seen as an early sign of cardiomyocytes apoptosis. To
our knowledge, this is the first study of its kind that
has investigated histone modification during the
programmed cell death (apoptosis) in cardiomyocytes
after alcohol exposure.
Fig. 7 Effect of Curcumin on Apoptosis Levels in Cardiac Progenitor Cells Treated by Alcohol. Flow cytometry (FCM) assay was used to detect the
apoptosis levels. a Control group. b Alcohol group. c Curcumin group. d Alcohol + Curcumin group. e DMSO group. f Quantitative analysis (percentage of
apoptosis cells versus total cells) of five group showed that alcohol treatment increased the apoptosis rate and curcumin pretreatment could correct
Herein we giving some novel findings that alcohol induces
hyperacetylation in histone H3K9 as an early event before
cardiomyocytes apoptosis. It’s also unveiled that the HAT
inhibitor curcumin had a protective effect against alcohol
exposure, highlighting the translational potential of the
drug while curing congenital heart disease. These results
provide progression towards the understanding of alcohol
abuse and epigenetic mechanisms in the pathogenesis of
congenital heart disease. This surely will promote the
development of new medicine for preventing congenital
CHD: Congenital heart disease; ChIP: Chromatin immunoprecipitation; FCM: Flow
cytometry; HAT: Histone acetyltransferase; HDAC: Histone deacetylase inhibitors;
TUNEL: Terminal deoxynucleoitidyl Transferase Mediated Nick End Labeling
XY carried out the Western blot and ChIP assays and data analyses. BP and TL
carried out the TUNEL Staining and apoptosis assays. LL and JZ participated in
the result discussion and data analyses. XY, JT, WS and XH participated in
experimental design and manuscript drafting. All authors read and approved
the final manuscript.
1. van der Linde D , et al. Birth prevalence of congenital heart disease worldwide: a systematic review and meta-analysis . J Am Coll Cardiol . 2011 ; 58 ( 21 ): 2241 - 7 .
2. Grewal J , et al. Maternal periconceptional smoking and alcohol consumption and risk for select congenital anomalies . Birth Defects Res A Clin Mol Teratol . 2008 ; 82 ( 7 ): 519 - 26 .
3. Webster WS , et al. Alcohol and congenital heart defects: an experimental study in mice . Cardiovasc Res . 1984 ; 18 ( 6 ): 335 - 8 .
4. Kvigne VL , et al. Characteristics of children who have full or incomplete fetal alcohol syndrome . J Pediatr . 2004 ; 145 ( 5 ): 635 - 40 .
5. May PA , et al. The epidemiology of fetal alcohol syndrome and partial FAS in a South African community . Drug Alcohol Depend . 2007 ; 88 ( 2-3 ): 259 - 71 .
6. Chen DB , Wang L , Wang PH . Insulin-like growth factor I retards apoptotic signaling induced by ethanol in cardiomyocytes . Life Sci . 2000 ; 67 ( 14 ): 1683 - 93 .
7. Ren J , et al. Influence of prenatal alcohol exposure on myocardial contractile function in adult rat hearts: role of intracellular calcium and apoptosis . Alcohol Alcohol . 2002 ; 37 ( 1 ): 30 - 7 .
8. Ohtani K , Dimmeler S. Epigenetic regulation of cardiovascular differentiation . Cardiovasc Res . 2011 ; 90 ( 3 ): 404 - 12 .
9. Connelly JJ , et al. Epigenetic regulation of COL15A1 in smooth muscle cell replicative aging and atherosclerosis . Hum Mol Genet . 2013 ; 22 ( 25 ): 5107 - 20 .
10. Hon GC , et al. Epigenetic memory at embryonic enhancers identified in DNA methylation maps from adult mouse tissues . Nat Genet . 2013 ; 45 ( 10 ): 1198 - 206 .
11. Yu CE , et al. Epigenetic signature and enhancer activity of the human APOE gene . Hum Mol Genet . 2013 ; 22 ( 24 ): 5036 - 47 .
12. Mathiyalagan P , et al. Interplay of chromatin modifications and non-coding RNAs in the heart . Epigenetics . 2014 ; 9 ( 1 ): 101 - 12 .
13. Hasan S , Hottiger MO. Histone acetyl transferases: a role in DNA repair and DNA replication . J Mol Med (Berl) . 2002 ; 80 ( 8 ): 463 - 74 .
14. Wang L , et al. Inhibition of histone acetylation by curcumin reduces alcohol-induced expression of heart development-related transcription factors in cardiac progenitor cells . Biochem Biophys Res Commun . 2012 ; 424 ( 3 ): 593 - 6 .
15. Park PH , Miller R , Shukla SD . Acetylation of histone H3 at lysine 9 by ethanol in rat hepatocytes . Biochem Biophys Res Commun . 2003 ; 306 ( 2 ): 501 - 4 .
16. Kim JS , Shukla SD . Acute in vivo effect of ethanol (binge drinking) on histone H3 modifications in rat tissues . Alcohol Alcohol . 2006 ; 41 ( 2 ): 126 - 32 .
17. Choudhury M , Shukla SD . Surrogate alcohols and their metabolites modify histone H3 acetylation: involvement of histone acetyl transferase and histone deacetylase . Alcohol Clin Exp Res . 2008 ; 32 ( 5 ): 829 - 39 .
18. Zhong L , et al. Ethanol and its metabolites induce histone lysine 9 acetylation and an alteration of the expression of heart development-related genes in cardiac progenitor cells . Cardiovasc Toxicol . 2010 ; 10 ( 4 ): 268 - 74 .
19. Pan B , et al. Alcohol consumption during gestation causes histone3 lysine9 hyperacetylation and an alternation of expression of heart developmentrelated genes in mice . Alcohol Clin Exp Res . 2014 ; 38 ( 9 ): 2396 - 402 .
20. Peng C , et al. Inhibition of histone H3K9 acetylation by anacardic acid can correct the over-expression of Gata4 in the hearts of fetal mice exposed to alcohol during pregnancy . PLoS One . 2014 ; 9 ( 8 ): e104135 .
21. Peng C , et al. Alcohol-induced histone H3K9 hyperacetylation and cardiac hypertrophy are reversed by a histone acetylases inhibitor anacardic acid in developing murine hearts . Biochimie . 2015 ; 113 : 1 - 9 .
22. Chen H , Tini M , Evans RM . HATs on and beyond chromatin . Curr Opin Cell Biol . 2001 ; 13 ( 2 ): 218 - 24 .
23. Li J , et al. Arsenic trioxide promotes histone H3 phosphoacetylation at the chromatin of CASPASE-10 in acute promyelocytic leukemia cells . J Biol Chem . 2002 ; 277 ( 51 ): 49504 - 10 .
24. Wang X , et al. Acute alcohol exposure induces apoptosis and increases histone H3K9/18 acetylation in the mid-gestation mouse lung . Reprod Sci . 2010 ; 17 ( 4 ): 384 - 90 .
25. Song C , et al. Paraquat induces epigenetic changes by promoting histone acetylation in cell culture models of dopaminergic degeneration . Neurotoxicology . 2011 ; 32 ( 5 ): 586 - 95 .
26. Devipriya B , Kumaradhas P. Molecular flexibility and the electrostatic moments of curcumin and its derivatives in the active site of p300: a theoretical charge density study . Chem Biol Interact . 2013 ; 204 ( 3 ): 153 - 65 .
27. Sun H , et al. Curcumin-mediated cardiac defects in mouse is associated with a reduced histone H3 acetylation and reduced expression of cardiac transcription factors . Cardiovasc Toxicol . 2014 ; 14 ( 2 ): 162 - 9 .
28. Zhu GH , et al. Activation of RXR and RAR signaling promotes myogenic differentiation of myoblastic C2C12 cells . Differentiation . 2009 ; 78 ( 4 ): 195 - 204 .
29. Livak KJ , Schmittgen TD . Analysis of relative gene expression data using real-time quantitative PCR and the 2(− Delta Delta C(T)) Method. Methods . 2001 ; 25 ( 4 ): 402 - 8 .
30. Gao W , et al. Alcohol exposure increases the expression of cardiac transcription factors through ERK1/2-mediated histone3 hyperacetylation in H9c2 cells . Biochem Biophys Res Commun . 2015 ; 466 ( 4 ): 670 - 5 .
31. Sun H , et al. Inhibition of p300-HAT results in a reduced histone acetylation and down-regulation of gene expression in cardiac myocytes . Life Sci . 2010 ; 87 ( 23 - 26 ): 707 - 14 .
32. Choudhury M , et al. Evidence for the role of oxidative stress in the acetylation of histone H3 by ethanol in rat hepatocytes . Alcohol . 2010 ; 44 ( 6 ): 531 - 40 .
33. Hallaq H , et al. A null mutation of Hhex results in abnormal cardiac development, defective vasculogenesis and elevated Vegfa levels . Development . 2004 ; 131 ( 20 ): 5197 - 209 .
34. Wang X , et al. Targeted deletion of mek5 causes early embryonic death and defects in the extracellular signal-regulated kinase 5/myocyte enhancer factor 2 cell survival pathway . Mol Cell Biol . 2005 ; 25 ( 1 ): 336 - 45 .
35. Goh JM , et al. Alcohol exposure during late gestation adversely affects myocardial development with implications for postnatal cardiac function . Am J Physiol Heart Circ Physiol . 2011 ; 300 ( 2 ): H645 - 51 .
36. Yin W , et al. Histone acetylation at the human beta-globin locus changes with developmental age . Blood . 2007 ; 110 ( 12 ): 4101 - 7 .
37. Gupta A , et al. The mammalian ortholog of Drosophila MOF that acetylates histone H4 lysine 16 is essential for embryogenesis and oncogenesis . Mol Cell Biol . 2008 ; 28 ( 1 ): 397 - 409 .
38. Chaturvedi P , Tyagi SC . Epigenetic mechanisms underlying cardiac degeneration and regeneration . Int J Cardiol . 2014 ; 173 ( 1 ): 1 - 11 .
39. Si L , et al. Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells . Biochem Biophys Res Commun . 2014 ; 450 ( 1 ): 81 - 6 .
40. Zhang W , et al. Prenatal alcohol exposure causes the over-expression of DHAND and EHAND by increasing histone H3K14 acetylation in C57 BL/6 mice . Toxicol Lett . 2014 ; 228 ( 3 ): 140 - 6 .
41. McKinsey TA , Zhang CL , Olson EN . Signaling chromatin to make muscle . Curr Opin Cell Biol . 2002 ; 14 ( 6 ): 763 - 72 .
42. Zhang CL , et al. Class II histone deacetylases act as signal-responsive repressors of cardiac hypertrophy . Cell . 2002 ; 110 ( 4 ): 479 - 88 .
43. Somech R , Izraeli S , J Simon A. Histone deacetylase inhibitors-a new tool to treat cancer . Cancer Treat Rev . 2004 ; 30 ( 5 ): 461 - 72 .
44. Marchion D , Munster P. Development of histone deacetylase inhibitors for cancer treatment . Expert Rev Anticancer Ther . 2007 ; 7 ( 4 ): 583 - 98 .
45. Soriano FX , et al. Role of histone acetylation in the activity-dependent regulation of sulfiredoxin and sestrin 2 . Epigenetics . 2009 ; 4 ( 3 ): 152 - 8 .
46. Aron JL , et al. Depsipeptide (FR901228) induces histone acetylation and inhibition of histone deacetylase in chronic lymphocytic leukemia cells concurrent with activation of caspase 8-mediated apoptosis and down-regulation of c-FLIP protein . Blood . 2003 ; 102 ( 2 ): 652 - 8 .
47. Condorelli F , et al. Inhibitors of histone deacetylase (HDAC) restore the p53 pathway in neuroblastoma cells . Br J Pharmacol . 2008 ; 153 ( 4 ): 657 - 68 .
48. Song C , et al. Environmental neurotoxic pesticide increases histone acetylation to promote apoptosis in dopaminergic neuronal cells: relevance to epigenetic mechanisms of neurodegeneration . Mol Pharmacol . 2010 ; 77 ( 4 ): 621 - 32 .