A total of 120 pathogen-free Kunming male mice (weight, 25-30 g; age, 8-10 weeks) were purchased from the Animal Center of Zunyi Medical University (Guizhou, China). All animal experiments were approved by the Animal Protection and Use Committee of Zunyi Medical University and complied with Directive 2010/63/EU of the European Parliament. Animal studies were performed in the Physiology Research Laboratories of Zunyi Medical University, Guizhou, China. The mice were maintained in individually ventilated cages (25°C and 55-65% humidity) with a 12/12-h light/dark cycle and free access to standard laboratory mouse chow and water. Mice were exposed to pressure-overload by thoracic aortic banding (TAB); this procedure decreased the thoracic aortic diameter by 70%, as previously described (24,25). Prior to TAB surgery, 0.8% sodium pentobarbital (50 mg/kg) was intraperitoneally injected into each mouse for general anesthesia. Anesthesia was confirmed by the disappearance of the righting and pedal withdrawal reflex. Mice received pre- and post-operative analgesia (6 h post-surgery) via subcutaneous injection of 0.02 ml (0.3 mg/ml) buprenorphine hydrochloride solution (Merck KGaA), as previously described (26) Additional analgesia was administered as required. Mice were randomly divided into five groups (n=24/group): Normal, Sham, Vehicle (Veh), TAC and TAC + EGCG. For each animal, the left side of the chest was opened along the median of the sternum to expose the aortic arch. The transverse aortic arch was then ligated between the brachial and left common carotid arteries. Prior to ligation, a curved needle (diameter, ~0.4 mm) was used to narrow the vessel. Finally, the chest was sutured. The postoperative survival rate of TAC mice was 60-70%. After 4, 8 and 12 weeks of TAC, the hearts of experimental mice were collected to evaluate the effect of HDAC5-mediated modification on histone H3K27ac acetylation during myocardial remodeling induced by pressure overload. In addition, the effect of EGCG on myocardial remodeling induced by pressure overload was investigated, and different concentrations (0, 25, 50, 75 and 100 mg/kg) EGCG were used to screen the optimal dose of EGCG. Following the completion of TAB, each mouse received an intraperitoneal injection of EGCG (Selleck Chemicals; PubChem CID 65064) at a dose of 50 mg/kg/day for 12 weeks. Mice in the Sham group received surgery but were not ligated. In the Veh group, normal saline was injected intraperitoneally at the same dose and timepoints beginning 1 week after sham operation for 12 weeks. After 12 weeks, mice were sacrificed using CO₂ overdose; death was verified by applying pressure on the mouse nail bed (toe-pinch reflex) and the heart from each animal was collected for analysis.

The heart and lungs of mice were collected, and cardiac (heart weight/body weight) and lung mass index (lung weight/body weight) were tested. Subsequently, the morphology of hearts and lungs of mice were observed by stereoscope.

Following harvesting, the heart tissue was fixed in 4% paraformaldehyde for 48 h, embedded in paraffin wax and cut into serial sections (4 µm thick). Following dewaxing and dehydration in a series of alcohol concentrations, the sections were washed, soaked with tap water for 5 min and then stained with 0.5% hematoxylin dye for 5 min (to highlight the nucleus), faded in 1% hydrochloric acid with ethanol for 5 min and then placed in 0.5% eosin solution for 2 min (Beijing Solarbio Scince & Technology Co., Ltd.). Additional sections were washed with distilled water and then stained with 0.7% Masson Lichun red acid compound red solution for 5 min (Beijing Solarbio Science & Technology Co., Ltd.). The sections were then stained in 2% glacial acetic acid solution for 3 min. Next, sections were differentiated with 1% phosphomolybdate aqueous solution for 2 min, stained with 0.5% aniline blue for 5 min and then soaked with 0.2% glacial acetic acid aqueous solution for 1 min. H&E- and Masson’s-stained sections were successively treated with 95 and 100% ethanol and xylene I and II, then sealed with neutral resin. Sections were observed under a fluorescence microscope (H&E, magnification, ×4; Masson’s, magnification, ×40). All processes were completed at room temperature.

Transthoracic echocardiograms were performed on experimental mice using a Vevo 770 High-Resolution echocardiograph (Visual Sonics), as described previously (27,28). Blood flow velocity was measured using p-mode images. The efficacy of TAC was detected by chest Doppler echocardiography 4 weeks after the operation.

ChIP trials were performed using a ChIP Assay kit (Merck KGaA). First, a glass homogenizer was used to prepare homogenate from heart tissue. This homogenate was then mixed with formaldehyde (1%) to cross-link DNA/protein complexes at room temperature for 15 min. After cross-linking, 1% SDS Lysis Buffer (cat. no. 20-163, Merck KGaA) was added and then the DNA was fragmented by sonication. Following centrifugation at 100 × g for 2 min for 30 min at 4°C with rotation, pre-cleared solution was used as a DNA input control. A total of 1,800 µl ChIP Dilution Buffer (cat. no. 20-153; Merck KGaA) was added to 200 µl sonicated cell supernatant for a final volume of 2 ml. Next, the protein-DNA complex was precipitated using monoclonal antibodies (anti-MEF2A; 4 µl, 1:500, cat. no. sc-17785; Santa Cruz Biotechnology, Inc.). The antibody-chromatin complexes were precipitated by overnight incubation with Protein A Agarose/Salmon Sperm DNA (60 µl) at 4°C. The agarose pellet was obtained by centrifugation (1,500 × g) at 4°C for 1 min. The immunoprecipitated complexes of Ab-protein-DNA were collected and washed with low-salt, high-salt and LiCl buffer and Tris-EDTA, and then buffered with an elution buffer. The cross-linking of protein-DNA complexes was reversed by incubation with 5 M NaCl at 65°C for 4 h, and the DNA was digested with proteinase K for 1 h at 45°C. A DNA Fragment Purification kit (Merck KGaA) was used to extract the DNA in accordance with the manufacturer’s instructions. All experiments included a positive (precipitated by anti-RNA polymerase II antibody) and a negative control (precipitated by normal mouse IgG) groups. Quantitative (q)PCR was performed after ChIP detection using ChIP Assay kit (Merck KGaA). SYBR-Green Master Mix II (Takara Biotechnology Co., Ltd.) was used to perform qPCR. The thermocycling conditions were as follows: Initial denaturation 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing/elongation at 58°C for 30 sec. β-actin was used as an internal reference and the 2^−ΔΔCq method was used to determine relative gene expression (29). Lertpa-v1.0 software (Applied Biosystems, Thermo Fisher Scientific, Inc.) was used to analyze the results. The following primers were used for RT-qPCR: ANP forward, 5′-TCC TTG GTG TCT CTC GCT CT-3′ and reverse, 5′-CGC TGG CTT GCT TGT TGT A-3′; BNP forward, 5′-TGC TGT CCC TCT ATG CTT CC-3′ and reverse, 5′-CGC TGG CTT GCT TGT TGT A-3′; β-actin forward, 5′-CCT TTA TCG GTA TGG AGT CTG CG-3′ and reverse, 5′-CCT GAC ATG ACG TTG TTG GCA-3′.

Co-IP trials were performed using a Co-IP Assay kit (Thermo Fisher Scientific, Inc.). A total of 300 µl IP binding Buffer was added to 40 mg heart tissue with 1 mM PMSF protease inhibitor on ice for 15 min. Lysates were centrifuged at 14,000 × g for 10 min at 4°C. Then, ~25% of the supernatant was subjected to input assays and the remaining supernatant was used for the Co-IP assay. IP was performed by combining primary anti-HDAC5 (1 µl, 1:250; cat. no. 16166-1-AP; ProteinTech Group, Inc.), anti-H3K27ac (2.5 µl, 1:100; cat. no. ab4729; Abcam) and anti-MEF2A rabbit polyclonal anti-bodies (1 µl, 1:250; cat. no. sc-17785; Santa Cruz Biotechnology, Inc.), with Dynabeads protein G (1:250; cat. no. 2729S; Invitrogen; Thermo Fisher Scientific, Inc.) and western blotting. All procedures were performed in accordance with the manufacturer’s instructions. First, the primary antibody was combined with protein G magnetic beads. Then, a magnet was used to immunoprecipitate the target antigen (HDAC5) into immunoprecipitation buffer containing 1% Triton X-100, 0.5% NP-40, 20 mmol/l HEPES, 50 mmol/l NaCl and protease inhibitor, at pH 7.4. Next, the samples were washed three times with IP Washing Buffer for 2 min. The immobilized protein complex was eluted at 95°C in 1X SDS-PAGE Loading Buffer (25 µl) for 10 min. The supernatant was collected after magnetic separation. Western blotting was then performed with anti-HDAC5, anti-MEF2A and anti-H3K27ac. IgG was used as a negative control. The HDAC5 IP experiments were performed in triplicate.

Myocardial tissues were dissected. Nucleoproteins were extracted using a Nuclear Extraction kit (Merck KGaA). Protein concentration were tested by BCA method. An equal amount of nucleoprotein extracts (40 µg/lane) were separated by 8/12% SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes (Merck KGaA). Next, the PVDF membranes were blocked with 5% non-fat milk in tris-buffered saline containing 0.1% Tween-20 for 2 h at room temperature and incubated at 4°C overnight with monoclonal antibodies [anti-HDAC5, 1:1,000, cat. no. 16166-1-AP; ProteinTech Group Inc.; anti-H3K27ac, 1:200, cat. no. ab4729, Abcam; anti-myocyte enhancer factor (MEF) 2A, 1:1,000, cat. no. sc-17785; Santa Cruz Biotechnology, Inc.; anti-brain natriuretic peptide (BNP), 1:1,000, cat. no. ab239510; Abcam; anti-atrial (A)NP, 1:1,000, cat. no. ab236101; Abcam; anti-H3, 1:2,000, cat. no. ab1791; Abcam and anti-β-actin, 1:1,000; cat. no. TA-09; OriGene Technologies, Inc.]. The next day, membranes were washed in Tris-buffered saline containing 0.1% Tween-20. The membranes were then incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (1:2,000, cat. no. Sc-47778; Santa Cruz Biotechnology). Protein bands were visualized by Enhanced Chemiluminescence System (GE Healthcare). Positive bands were quantified using Quantity One (version 4.4) software package (Bio-Rad Laboratories, Inc.).

Total RNA was extracted from myocardial tissue using an RNA Extraction kit (BioTeke Corporation) in accordance with the manufacturer’s instructions. Single-stranded complementary DNA (cDNA) was synthesized from total RNA using primers and PrimeScript RT reagent (Takara Biotechnology Co., Ltd.). cDNA was subjected to RT-qPCR using gene-specific primers and SYBR-Green Master Mix II (Takara Biotechnology Co., Ltd.). The thermocycling conditions were conducted as follows: Initial denaturation 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing/elongation at 57°C for 30 sec. β-actin was used as an internal reference and the 2^−ΔΔCq method was used to determine relative gene expression (29). Lertpa-v1.0 software was used to analyze the results. The following primers were used for RT-qPCR: MEF2A forward, 5′-CAC TTC CTT GGA CTA CTT GTT TCG T-3′ and reverse, 5′-GTT CCT GCT TTT CTA CTG CTC TGT T-3′ and β-actin forward, 5′-AGA AAA TCT GGC ACC ACA CC-3′ and reverse, 5′-CAG AGG CGT ACA GGG ATA GC-3′.

Paraffin-embedded sections were treated with EDTA antigen repair buffer (pH 8.0; Beijing Solarbio Science & Technology Co., Ltd.) for antigen repair at 37°C for 20 min. After cooling, slides were washed in PBS buffer. Next, WGA dye (1:1,000; GeneTex) was added dropwise, then washed with PBS buffer. After the slides had partially dried, DAPI (5 ng/ml) solution (Beijing Solarbio Science & Technology Co., Ltd.) was added and incubated at room temperature for 3 min in the dark. The slides were then PBS washed and incubated with Autofluorescence Quenching Agent kit (Thermo Fisher Scientific, Inc.). The sections were then shaken dry and sealed with anti-fluorescence quenching sealing tablets. Finally, sections were observed under a fluorescence microscope (magnification, ×20; Olympus Corporation) and images were captured.

All data are expressed as the mean ± SD of 3 independent experiments. All statistical analysis was performed using SPSS software version 18.0 (SPSS, Inc.). Differences between multiple groups were analyzed by one-way ANOVA followed by Tukey’s post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Echocardiography indicated that the TAC model was successfully generated via TAB (Fig. 1A). Color Doppler imaging showed that blood flow was blocked in the aortic arch (Fig. 1B). The diameter of the transverse aorta decreased following TAC; the diameter of this vessel was similar between the TAC and EGCG + TAC groups (~0.4 mm; Fig. 1C), thus resulting in a similar increase of blood flow in the transverse aorta in both groups (Fig. 1D). In the present study, stereoscopy and H&E staining showed that the hearts of TAC mice at 4, 8 and 12 weeks after TAC were enlarged compared with the Sham group (Fig. 1E and F). Masson’s staining further revealed interstitial fibrosis and collagen accumulation in the myocardial tissue of TAC mice. Myocardial fibrosis and collagen deposition became more notable from 4 to 12 weeks after TAC (Fig. 1G). Echocardiography further showed that the left ventricular (LV) wall in TAC mice thickened between 4 and 8 weeks after TAC compared with the Sham group. At 8-12 weeks after TAC, the LV volume of TAC mice began to gradually increase; this was accompanied by a decrease in myocardial tissue (Fig. 1H). To determine morphological changes of lung in TAC mice, stereoscopy was performed. The results of stereoscopy showed that the lungs at 4, 8 and 12 weeks after TAC were enlarged compared with the Sham group (Fig. 1I). There was a decrease in the LV anterior wall thickness (AWT) in mice at 12 weeks after TAC. The LV ejection fraction (EF) also increased significantly in TAC mice over time. However, 8 weeks after TAC, LVEF showed a significant decrease, indicating that heart failure may have occurred (Table I).

Pathological myocardial remodeling is a key pathophysiological process of heart failure that is regulated by HDACs. Hence, protein expression levels of HDAC5 and H3K27ac were assessed by western blotting. Results showed that expression levels of HDAC5 in the heart of TAC mice increased gradually from 4 to 12 weeks after TAC (Fig. 2A). Western blotting showed that the acetylation levels of histone H3K27ac in TAC mice at 4 and 8 weeks after TAC were significantly higher than at 12 weeks (Fig. 2B). In addition, the expression levels of ANP and BNP at 4 and 8 weeks were significantly higher than those of the Sham group. However, the expression levels of ANP and BNP at 12 weeks after TAC were significantly lower than that at 4 and 8 weeks (Fig. 2C and D).

First, the optimal exposure dose for EGCG was determined. According to previous data (30,31), mice were injected intraperitoneally with various concentrations of EGCG (0, 25, 50, 75 and 100 mg/kg). The optimal concentration (50 mg/kg) was selected based on the lowest levels of HDAC5 in the heart of TAC mice (Fig. 3A). Next, the effect of EGCG on myocardial remodeling in TAC mice was investigated. EGCG (50 mg/kg/day) was intraperitoneally injected into mice after TAC for 12 weeks, and the hearts were collected for analysis. Western blotting showed that EGCG reversed the hypoacetylation of H3K27ac in the heart of TAC mice (Fig. 3B). EGCG attenuated the overexpression of HDAC5 in TAC mice (Fig. 3C). Analysis showed that the mRNA and protein expression levels of MEF2A were lower in the TAC group than those in the Sham group, while EGCG improved the mRNA and protein expression levels of MEF2A in TAC mice treated with EGCG (Fig. 3D and E). In addition, Co-IP was performed to verify the formation of a complex between H3K27ac and MEF2A; this demonstrated that HDAC5 may interact with H3K27ac and regulate gene expression levels of MEF2A (Fig. 3F).

Next, ChIP-PCR was performed to investigate the regulatory association between MEF2A and downstream genes associated with heart development (ANP and BNP). Data showed that MEF2A bound to the promoter of ANP and BNP in the mouse heart (Fig. 3G). Western blotting was performed to investigate the expression levels of ANP and BNP during myocardial remodeling. Data showed that the expression levels of ANP and BNP in the heart of TAC mice were significantly lower compared with the Sham group. Data also showed that EGCG treatment rescued the decreased levels of ANP and BNP in the heart of TAC mice (Fig. 3H and I).

Stereoscopy and H&E staining were used to detect the preventive and restorative effects of EGCG in the heart of TAC mice. Stereoscopy showed that the heart of mice in TAC group was larger compared with the Sham group and EGCG reversed cardiac dilatation (Fig. 4A). H&E staining showed that LV volume was higher in the TAC group than in the Sham group. However, EGCG treatment attenuated the thickening of LV in TAC mice (Fig. 4B). Masson’s staining revealed a greater extent of interstitial fibrosis and collagen deposition in the TAC group than in the Sham group. Following treatment with EGCG, the extent of interstitial fibrosis and collagen deposition was notably decreased (Fig. 4C). Furthermore, echocardiography showed that the LV end diastolic dimension (EDD) and end systolic dimension (ESD) in the TAC group were significantly higher than those in the Sham group. However, EGCG treatment attenuated the increased LVEDD and LVESD in the mouse heart. A significant decrease in LVEF was observed in TAC mice; however, EGCG treatment improved LVEF in the heart of TAC mice (Fig. 4D; Table II). In addition, EGCG significantly decreased the cardiac mass index of TAC mice, although the lung mass index remained unchanged (Table III). These data suggested that EGCG attenuated pathological myocardial remodeling in TAC mice.

Next, wheat germ agglutinin experiments were performed to evaluate the surface area of cardiomyocytes in TAC mice. Data showed that the surface area of cardiomyocytes in the TAC group increased significantly compared with the Sham group. However, EGCG treatment attenuated the increased surface area of cardiomyocytes in the heart of TAC mice (Fig. 5A and B). For the HDAC inhibitor EGCG to be used clinically, it is important to evaluate survival rate following administration. To investigate this issue, mice were subjected to TAC or sham operation and treated with EGCG (50 mg/kg/day) for 8 weeks, a period that corresponds to 6-8 years in humans (8). The data showed that EGCG treatment improved the survival rate of TAC mice [Sham + Veh, 90; TAC, 51; TAC + Veh, 51% and TAC + EGCG, 67% (n=35/group); Fig. 5C].