Epigallocatechin-3 gallate prevents pressure overload-induced heart failure by up-regulating SERCA2a via histone acetylation modification in mice

Heart failure is a common, costly, and potentially fatal condition. The cardiac sarcoplasmic reticulum Ca-ATPase (SERCA2a) plays a critical role in the regulation of cardiac function. Previously, low SERCA2a expression was revealed in mice with heart failure. Epigallocatechin-3-gallate (EGCG) can function as an epigenetic regulator and has been reported to enhance cardiac function. However, the underlying epigenetic regulatory mechanism is still unclear. In this study, we investigated whether EGCG can up-regulate SERCA2a via histone acetylation and play role in preventing heart failure. For this, we generated a mouse model of heart failure by performing a minimally invasive transverse aortic constriction (TAC) operation and used this to test the effects of EGCG. The TAC+EGCG group showed nearly normal cardiac function compared to that in the SHAM group. The expression of SERCA2a was decreased at both the mRNA and protein levels in the TAC group but was enhanced in the TAC+EGCG group. Levels of AcH3 and AcH3K9 were determined to decrease near the promoter region of Atp2a2 (the gene encoding SERCA-2a) in the TAC group, but were elevated in the TAC+EGCG group. Meanwhile, HDAC1 activity and binding near the Atp2a2 promoter were increased in the TAC group but decreased with EGCG addition. Further, binding levels of GATA4 and Mef2c near the Atp2a2 promoter region were reduced in TAC hearts, which might have been caused by histone hypoacetylation; this was reversed by EGCG. Together, upregulation of SERCA2a via the modification of histone acetylation plays a role in EGCG-mediated prevention of pressure overload-induced heart failure, and this might represent a novel pharmacological target for the treatment of heart failure.


Introduction
Heart failure is defined as "a clinical syndrome characterized by systemic perfusion which is inadequate to meet the body's metabolic demands as a result of impaired cardiac function". a1111111111 a1111111111 a1111111111 a1111111111 a1111111111

Minimally invasive transverse aortic constriction (TAC)
A minimally invasive TAC method [25] was used in our study. The mice were anesthetized via inhalation of 1.5-2.5% isoflurane, and their body temperature was maintained between 36 and 37˚C throughout the procedure using a heating pad. A horizontal skin incision of 7-10 mm in length was made at the level of the suprasternal notch. Then, the thyroid was retracted and the sternum was exposed. A 5-mm longitudinal cut was made down the sternum and the thymus was retracted to allow visualization of the aortic arch under low-power magnification. A 6-0 silk suture was passed through the aortic arch between the origin of the innominate and left common carotid arteries, guided by a curved 27-gauge needle. Another 27-gauge needle was placed next to the aortic arch, and the suture was tied neatly around the needle and aorta. After ligation, the needle was removed. The skin was closed with a 4-0 nylon suture, and the mice were allowed to recover fully in a clean cage on a heating pad (S1 Text). Buprenorphine was administered subcutaneously once per day for 3 days for post-operative analgesia. The animal's post-operative health and surgical site were observed and recorded twice per day for 7 days until the sutures was removed, then once per day for 3 months.

Echocardiography
Echocardiography studies were performed with a Vevo 2100 high-resolution imaging system (VisualSonics, Toronto, Canada). All measurements were performed by the same examiner. The mice were anesthetized with 1-1.5% isoflurane and placed on a heating pad to maintain body temperature at 36.5-37.5˚C. Hair on the precordial region was removed with depilatory cream. Bmode images were taken to measure the ventricular and aortic structure and M-mode images were taken to measure the ventricular function. P-mode images were taken to measure the velocity of blood flow (S2 Text). All data were analyzed to evaluate the effects of TAC treatment.
The efficacy of TAC was tested by transthoracic Doppler echocardiography 3 days after the procedure. The mice were sacrificed with isoflurane and the hearts were harvested after transthoracic echocardiography was performed 12 weeks after the operation.

Hematoxylin and eosin (HE) staining
Heart tissues were fixed with 4% paraformaldehyde, dehydrated with alcohol, embedded in paraffin, sectioned into 5-mm slices, and then stained with hematoxylin and eosin. Photographs of the ventricular sections from the four groups of mice were taken at 400× magnification (Nikon, Tokyo, Japan)

Quantitative real-time PCR (qPCR)
Total RNA was extracted using the RNA extract kit (Bioteck, Beijing, China). Single-strand cDNA was reverse transcribed from 500-1,000 ng of RNA using oligo dT-adaptor primers and the AMV reverse transcriptase kit (TaKaRa, Otsu, Japan). cDNA was detected by performing a quantitative real-time polymerase chain reaction (RT-PCR) assay with the SYBR Green RealMasterMix kit (Tiangen, Beijing, China). The mRNA expression levels of SERCA2a were quantified and GAPDH was used as an endogenous 'housekeeping' gene to normalize RNA levels across samples. The procedures were performed in accordance with the manufacturer's instructions. The primer sequences were 5 0 -TCGACCAGTCAATTCTTACAGG-3 0 and 5 0 -CAGGGACAGGGTCAGTATGC-3 0 for SERCA2a, and 5 0 -AAGAAGGTGGTGAAGCAGGCATC-3 0 and 5 0 -CGGCATCGAAGGTGGAAGAGTG-3 0 for GAPDH.

Western blot analysis
In brief, total protein was extracted from the cardiac tissue using a protein extraction kit (Key-GEN Bio-TECH, China) and then quantified using a BCA assay (BioTeke Biotechnology, China). Total protein (50 μg per lane) was separated on a 12% SDS-PAGE gel and transferred to a PDVF membrane. Proteins bound to the PDVF membrane were analyzed by western blotting using primary antibodies specific for SERCA2a (Abcam, USA) and GAPDH (Arigo, Taiwan). The band intensity was analyzed and quantified using a G-BOX imaging system (Syngene, UK).

Chromatin immunoprecipitation (ChIP) assay
ChIP experiments were conducted using a ChIP assay kit (ChIP Kit-one step, Abcam, USA). After the homogenization of cardiac tissues, 1% formaldehyde (SigmaAldrich, St. Louis, MO, USA) was added to the samples to cross-link proteins to DNA. The cross-linked DNA was then fragmented into small fragments (500-1000 bp) using an ultrasound (Bioruptor UCD-200). Next, the protein-DNA complexes were precipitated using monoclonal antibodies specific for acetylated histone 3 (AcH3), lysine 4 in H3 (AcH3K4), lysine 9 in H3 (AcH3K9), HDAC1, GATA4, and Mef2c (Abcam, Cambridge, USA). Anti-RNA polymerase antibodies were used as a positive control and anti-mouse IgG was used as a negative control. The total column input also served as a positive control. Next, cross-linked protein-DNA complexes were removed and the DNA was extracted. Specific quantitative real-time PCR (qPCR) primers were designed to determine levels of AcH3, AcH3K4, and AcH3K9 near the proximal promoter regions of Atp2a2. The qPCR primer sequences used to amplify the SERCA2a promoter were as follows: 5 0 -AGCCAAGGACACCAGTGC-3 0 and 5 0 -GGGATAGAGCGCGGAGTT-3 0 .

HDAC1 activity assay
HDAC1 activity was determined using a HDAC1 Activity Assay kit (BioVision, 10Mountain View, CA, USA) according to the manufacturer's instruction. Briefly, after protein extraction and protein concentration measurement, 6 microliters of HDAC1 antibody or control antibody were added to each 500 microliter reaction, and the reaction was incubated overnight at 4˚C. Protein-A/G (25 microliters) was added to each reaction prior to incubation for 1 h at 4˚C. Each reaction was then centrifuged at 14,000 × g for 10 s at 4˚C, and the supernatant was discarded. The reaction compound containing HDAC Assay Buffer and HDAC1 substrate was subsequently added to each reaction and incubated for 2 h at 37˚C. Then, 20 microliters of the Developer was added to each tube, which was incubated for 30 min 37˚C. The AFC (7-amino-4-trifluoromethy coumarin) Standard was diluted as described by the manufacturer's instructions, and for 100 microliters of each reaction, the fluorescence at Ex/Em = 380/500 nm (SYN-ERGY/H1 micropalte reader, BioTek) was read. The AFC standard curve was plotted and the sample reading was applied to the AFC Standard Curve to obtain B pmol of AFC in the sample wells. The calculation of sample HDAC1 activity is shown as follows: HDAC1 activity = 2 × B / TS (pmol/min/mg = mU), where B = AFC amount from the Standard Curve, T = reaction time = 120 min, and S = protein amount.

Data analysis
Quantitative data are shown as the means ± SE. Statistical significance associated with differences between mean values was analyzed by performing two-way or three-way ANOVA with a post hoc Bonferroni-Dunn test for multiple comparisons. P < 0.05 was considered significant.

Evaluation of the TAC operation in mice
Echocardiograph images clearly showed reduced aortic diameter at the transverse constriction site between the innominate and left carotid arteries after TAC ( Fig 1A). The constriction was further confirmed by anatomical dissection images of the aorta in TAC mice ( Fig 1B). The reduced transverse aorta diameter achieved the same level after TAC between the TAC and EGCG+TAC groups (Fig 1D), resulting in the same increase in transverse aorta blood flow between these groups ( Fig 1E).

EGCG prevents heart failure in TAC mice
A non-invasive echocardiograph measurement was used to assess changes in heart wall thickness before and 12 weeks after the operation. Compared to that before the operation, the thickness of the left ventricular anterior wall (LVAW) and left ventricular posterior wall (LVPW) increased in TAC group hearts, but did not differ significantly in the other groups (Table 1, Table). Heart weight normalized to body weight (HW/BW) is an index to evaluate cardiac hypertrophy. Consistent with the changes in wall thickness, HW/BW increased at 12 The transverse aorta diameter decreased significantly after the TAC operation with or without epigallocatechin-3-gallate (EGCG) treatment (S1 Table). E) The velocity of the transverse aorta blood flow (P-mode ultrasound image) increased significantly after TAC with or without EGCG (S1 Table). Values are presented as the mean ± SE. Ã , P < 0.05 by two-way ANOVA; n = 8 per group.
https://doi.org/10.1371/journal.pone.0205123.g001 weeks after TAC in the TAC only group, indicating a response to increased afterload; however, in the TAC+EGCG group, only a marginal insignificant increase in HW/BW was observed, indicating a compensatory effect of EGCG in response to long-term high afterload stress ( Fig  2B). This was confirmed by HE staining of heart sections (Fig 3A and 3B). Before TAC, the non-invasive echocardiograph did not detect any functional differences among the groups. The left ventricular chamber was dilated, and the ejection fraction (EF) and fraction shortening (FS) were decreased in the TAC group but not in TAC+EGCG group, suggesting that EGCG preserved heart function ( Table 1, Fig 2A, S2 Table).

EGCG prevents the TAC-induced decrease in SERCA2a expression
As shown in Fig 4A, SERCA2a mRNA expression was decreased in the TAC group compared to that in the SHAM group. However, this effect was almost completely reversed with EGCG treatment, as SERCA2a mRNA levels returned to those observed in the SHAM group. The expression of SERCA2a in the SHAM+EGCG group was not significantly changed as compared to that in the SHAM group. Western blot assays showed similar results ( Fig 4B). These results indicate that EGCG can prevent the downregulation of SER-CA2a induced by TAC.

Histone acetylation near the promoter region of Atp2a2 (encoding SERCA-2a)
Modification of histone acetylation or deacetylation is a reversible dynamic process, and it is a crucial component of epigenetic regulation. To explore the potential mechanism underlying the reduced SERCA2a expression in TAC mice, we assessed the acetylation levels of H3, H3K4, and H3K9 near the promoter region of Atp2a2 using ChIP-qPCR assays. As shown in Fig 5A, AcH3 levels near the promoter elements of SERCA2a were decreased in the TAC group compared to those in the SHAM group. In the TAC+EGCG group, levels were enhanced and reached those observed in the SHAM group. The pattern of AcH3K9 was similar to that of AcH3 among the groups, indicating that reduced SERCA2a expression was mediated by modified histone acetylation ( Fig 5B). However, the levels of AcH3K4 were not significantly different among the groups (Fig 5C).

HDAC1 enzyme activity and binding level of HDAC1 near the Atp2a2 promoter
We next determined HDAC1 activity in each group. Fig 6A shows that HDAC1 activity in the TAC groups was enhanced compared to that in the SHAM group, and decreased after EGCG treatment. Thus our data indicates that EGCG can suppress TAC-induced HDAC1 activation.    However, this effect was incomplete in TAC mice. EGCG was previously reported to inhibit the binding of HDAC1 to promoters that activate gene expression [26]. Therefore, we detected HDAC1 binding near the promoter region of Atp2a2 in the four groups. As shown in Fig 6B, these levels were increased in the TAC group, which might correlate with the observed downregulation of AcH3 and AcH3K9. However, binding levels were found to decrease significantly in the TAC+EGCG group.

Binding of transcription factors near the Atp2a2 promoter
Cardiac core transcription factors, GATA4 and Mef2c, were shown to play roles in the regulation of Atp2a2 transcription [27]. Thus, the binding levels of these transcription factors near the Atp2a2 promoter were determined by ChIP assays. As shown in Fig 7A and 7B, binding levels of GATA4 and Mef2c near the Atp2a2 promoter region were reduced in heart tissue of the TAC group, which might have been caused by hypoacetylation of histones. This was  reversed by the effect of EGCG on the acetylation of histones associated with Atp2a2, as the binding of GATA4 and Mef2c was increased significantly after EGCG treatment.

Discussion
Heart failure continues to be a leading cause of mortality and morbidity worldwide. Conditions that damage or overwork the heart muscle such as hypertension, coronary heart disease, and diabetes can result in heart failure. Hypertension is one of the most important causes of heart failure, as pressure overload exposes cardiac myocytes to elevated mechanical stress and neurohormones, which increases myocardial mass and results in left ventricular hypertrophy. These cardiac changes can further progress to heart failure [28]. Although several types of drugs have been developed over many years, gaps in the treatment of this disease remain.
Recently, epigenetic disorders have been reported to play a role in heart failure [18]. Therefore, a better understanding of associated epigenetic mechanisms is required to uncover novel pharmacological agents. Our data indicate that increased HDAC1 activity and binding to the promoter of the gene encoding SERCA2a (Atp2a2), which might result in the hypoacetylation of histone 3 lysine 9, in addition to chromatin regulation, result in decreased transcription factor binding to this promoter region. Further investigation showed that EGCG could inhibit the activity and binding of HDAC1 to the Atp2a2 proximal promoter region to rescue the low expression of SERCA2a and improve cardiac function. SERCA2a plays a critical role in regulating cardiac function; accordingly, several studies have demonstrated low expression of this protein in heart failure [4][5][6][7][8][9][10]. Similar to previous reports, we found that the protein and mRNA levels of SERCA2a were decreased after heart failure induced by TAC. However, the underlying epigenetic mechanism of this downregulation of SERCA2a remained unclear. Recently, a study demonstrated that histone modification underlies the reprogramming of SERCA2a promoters in adult murine left ventricles under conditions of chronic pressure overload [18], indicating a role for the histone-associated epigenetic regulation of SERCA2a and epigenetics in the etiology of heart failure. Histone epigenetic modifications include acetylation, methylation, phosphorylation, ubiquitination, and sumoylation [29]. Acetylation is one of the most important histone modifications that can result in chromatin remodeling and the activation of gene expression; moreover, hypoacetylation near the proximal promoter of the gene results in the compaction of chromatin and might affect the binding affinities of transcription factors with the key cis-elements of the proximal promoter [30]. In the present study, our results showed that the binding of total AcH3, as well as the subtype AcH3K9, was reduced, and that the binding of active transcription factors GATA4 and Mef2c was decreased near the Atp2a2 promoter region after heart failure. However, this pattern was not observed with the AcH3K4 subtype, indicating the selective control of H3 acetylated lysine sites during the regulation of SERCA2a after heart failure. The assessment of other acetylated lysine sites should be performed in future studies.
HDACs are a class of enzymes that remove acetyl groups from ε-N-acetyl lysine amino acids on histones, allowing histones to wrap the DNA more tightly. HDACs can be classified into four classes depending on sequence homology to the original yeast enzymes and domain organization. HDAC1 is one of the most important class I HDACs that regulate histone acetylation; it can enhance electrostatic attraction between DNA and histones and increase chromatin compaction, leading to reduced gene expression. The inhibition of class I HDACs has been reported to suppress pressure overload-induced ventricular hypertrophy and improve systolic function significantly [31,32], indicating that class I HDACs perform a crucial function during heart failure. Our results showed increased activity and binding of HDAC1 to the Atp2a2 promoter region, indicating that it might be a causal factor of SERCA2a downregulation mediated by hypoacetylation during TAC-induced heart failure in mice. With respect to the regulation of SERCA2a expression, other subtypes of HDACs should also be assessed in future studies.
Tea polyphenols are the main chemical components of green tea, which contains many types of catechins such as epicatechin, epicatechin-3-gallate, epigallocatechin, and EGCG [33]. EGCG, which has been approved as a health-promoting product for sale, is an ester that forms from the reaction between epigallocatechin and gallic acid, and this compound is associated with important health benefits [34]. EGCG has been reported to exert lipid-lowering effects, in addition to effects on angiogenesis and osteogenesis; it also affects many abnormal pathophysiological changes, as it has been demonstrated to have anti-cancer, anti-inflammatory, anti-collagenase, autoxidative, and anti-fibrotic effects [35][36][37][38][39][40][41][42][43][44], and possess therapeutic activity for a number of diseases such as cancer, oral disease, diabetes, and especially cardiovascular diseases [19,20]. Previously, several mechanisms were suggested for EGCG with respect to heart failure, such as alleviating cardiac fibrosis [45], reducing desensitization of the β1 adrenoceptor [46], modulating the Nrf2/ARE antioxidant system [47], and altering myofilament Ca2+-sensitivity [26,48]. Recently, studies have demonstrated that EGCG exerts a tremendous effect on events associated with epigenetic regulation, including histone acetylation, methylation, and DNA methylation [22][23][24]. Pan [26] found that EGCG could inhibit HDAC1 to up-regulate cTnI in aging mice and improve cardiac function, highlighting the detailed mechanisms through which EGCG contributes to epigenetic modifications. Since EGCG is a major epigenetic controller, whether epigenetic regulation by this compound contributes to the prevention of heart failure is not clear. In the present study, we found EGCE could reverse the low expression of SERCA2a observed after TAC; meanwhile, acetylated H3 and H3K9 were upregulated after EGCG treatment. We further found that HDAC1 activity and binding were decreased with EGCG treatment. All of these findings indicate that EGCG could inhibit HDAC1 activity and binding to the promoter of the gene encoding SERCA2a, resulting in increased expression through the enhanced binding of AcH3 and AcH3K9 to the proximal promoter of this gene.
In conclusion, upregulation of SERCA2a via the modification of histone acetylation plays a role in the EGCG-mediated prevention of pressure overload-induced heart failure, and thus might represent a novel pharmacological agent for the treatment of heart failure.  Table. Echocardiographic parameters before and 12 weeks after operation. Compared to that before the operation, the thickness of the left ventricular anterior wall (LVAW) and left ventricular posterior wall (LVPW) increased in TAC group hearts. The left ventricular chamber was dilated, and the ejection fraction (EF) and fraction shortening (FS) were decreased in the TAC group but not in TAC+EGCG group hearts. (XLSX)