The study was approved by the Ethics Committee of Jilin University, and all experimental procedures conformed to the guidelines for the Animal Care and Use Committee of Jilin University, Changchun, China.

A total of 80 healthy male Wistar rats (weighing, 180–220 g) were purchased from the Animal Centre of Jilin University (certificate number SCXK (JI) 2007-0003). In total 40 Wistar rats first were subjected to MI by ligating the left anterior descending branch of the coronary artery and to 30 min of ischemia, followed by 0, 6, 12 or 24 h reperfusion in order to determine the effects at different time points on FoxO3a expression; 10 rats were used at each time point. The other 40 rats were randomly divided into 5 groups as follows: i) the sham-operated (control), treated with saline (n=8); ii) the I/R group, in which rats were subjected to I/R and treated with saline (n=8); iii) the TSA 1 group, in which rats were subjected to I/R and treated with 0.2 mg/kg (I/R+T1) (n=8); iv) the TSA 2 group, in which rats were subjected to I/R and treated with 0.1 mg/kg (I/R+T2) (n=8); and v) the TSA 3 group, in which rats were subjected to I/R and treated with 0.05 mg/kg (I/R+T3) (n=8). The rats in the 5 groups were injected with TSA or saline by intraperitoneal administration once daily for 5 days. Standard rat chow and water were available ad libitum. On day 5, the animals in the 5 groups underwent 30 min left anterior descending (LAD) coronary artery ligation and 24 h of reperfusion according to the method previously described by Li et al (22). Briefly, the rats were anesthetized by an intraperitoneal injection of 10% chloral hydrate (0.35 g/kg) before undergoing a thoracotomy. After ensuring the appropriate depth of anesthesia, the rats were intubated without incision using a rodent ventilator. The thoracic cavity was opened and the heart was rapidly exposed. The left anterior descending coronary artery (LAD) was ligated by a 6-0 silk suture (2–3 mm below the left auricle) and the chest was immediately closed. Successful I/R injury was monitored by standard lead II electrocardiography (ECG). The sham-operated rats only underwent the suture around the LAD, but were not ligated. Following reperfusion, the myocardial infarct size, serum myocardium enzyme activities and protein expression were evaluated.

The H9c2 rat myocardial cell line was obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). The cells were cultured in a 5% CO₂ humidified atmosphere at 37°C in 10% fetal bovine serum (FBS)-containing Dulbecco's modified Eagle's medium (DMEM). The cells were seeded in 6-well-plates for 24 h and were then incubated with/without TSA (50 nmol/l) for 1 h and then incubated with/without H₂O₂ (400 µM) for 2 h.

TSA was purchased from TCI Development Company Ltd. (Shanghai, China). Polyclonal rabbit antibody against catalase was purchased from Abcam (ab76110; Cambridge, CA, USA). Polyclonal rabbit antibodies to FoxO3a (12829) and SOD2 (13141) were purchased from Cell Signaling Technology (Danvers, MA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 60004-1-lg) antibody was from the ProteinTech Group, Inc. (Chicago, IL, USA). Goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody (A0208), goat anti-mouse IgG HRP-labeled secondary antibody (A0216), the cell counting kit-8 (CCK-8), ROS assay kit and mitochondrial membrane potential detection kit were from the Beyotime Institute of Biotechnology (Jiangsu, China). The EZ ChIP™ Chromatin Immunoprecipitation kit was purchased from Millipore Corp. (Billerica, MA, USA). Enhanced chemiluminescence (ECL) detection reagents were from Thermo Fisher Scientific (Waltham, MA, USA). Other chemical reagents were commercially available and analytically pure.

The viability of the H9c2 cells (in 96-well plates) was measured by CCK-8 assay according to the manufacturer's instructions. Briefly, the cells were exposed to H₂O₂ at various concentrations for different durations. CCK-8 solution (10 µl) was added followed by further incubation for 2 h and the absorbance were measured at 450 nm using a microplate reader (Infinite M200 PRO; Tecan Corp., Mannedorf, Switzerland).

Following 0, 6, 12 and 24 h of reperfusion, the animals were euthanized and the hearts were rapidly removed. The MIS size was assessed by the 2,3,5-triphenyltetrazolium chloride (TTC) method as previously described (23). Briefly, the excised hearts were washed using 0.9% saline. The right ventricular tissues were removed and left ventricles were frozen and stored at −20°C for 5 min and then were sliced into 1-mm-thick sections, giving 5 slices of equal thickness along the long axis from the apex to the base. The slices were incubated for 15–30 min in TTC at pH 7.4 at 37°C to identify the viable myocardium stained red, while the infarct tissue remained white. The slices were fixed in 10% formaldehyde solution and photographed with a digital camera. Areas stained in red and white were measured by a BI-2000 Medical Image Analysis System (Chengdu TME Technology, Chengdu, China). The infarct size was calculated using the following equation: %Infarct volume = infarct volume/total left ventricle volume ×100%.

At the end of the experiment, blood samples were obtained from the abdominal aorta, incubated at 37°C for 30 min and then centrifuged at 3,000 rpm for 10 min. The supernatant from each group was collected and frozen at −80°C for further analysis. The activities of CK, LDH and AST were measured using CK, LDH and AST kits (Jiancheng Bioengineering Institute, Nanjing, China) with an automatic biochemistry analyzer (EOS880; Hospitex Diagnostics, Italy) according to the manufacturer's instructions.

At the end of the experiment, the myocardial tissue was homogenated for 5 min in the ice and then centrifuged at 2,000 rpm for 5 min. The supernatant from each group was collected for measuring the levels of MDA and the activities of SOD using respective kits (Jiancheng Bioengineering Institute).

Proteins were separated by 5–18% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% skimmed dry milk for 2 h at room temperature and washed 3 times in Tris-buffered saline with Tween (TBST); the membranes were then incubated with the primary antibodies to FoxO3a, SOD2, catalase and GAPDH overnight at 4°C. After washing with TBST again, HRP-labeled secondary antibodies goat anti-rabbit (1:1,000) or goat anti-mouse (1:2,000) were added followed by incubation for 1.5 h at room temperature. Immunoblots were developed by ECL detection kits and the bands were quantified by Quantity One software.

The cultured H9c2 cells were fixed with 4% paraformaldehyde for 30 min and then washed 3 times in phosphate-buffred saline (PBS). Blocking solutions (0.2% Triton X-100 and sheep serum in PBS) were added to the fixed cells followed by incubation for 30 min at 37°C. The cells were then incubated with primary antibody against FoxO3a overnight at 4°C. After washing with PBS, the appropriate secondary antibody was added followed by incubation for 1 h in the dark at room temperature. The nuclei were then stained with Hoechst 33342 for 20 min. After washing with PBS, immunofluorescence was examined under a fluorescence microscope.

Intracellular ROS of H9c2 was measured by 2′7′-dichlorodihydrofluorescein diacetate (DCFH-DA) as a fluorescent probe according to the manufacturer's instructions. Briefly, H9c2 cells were loaded with DCFH-DA (10 µM) for 30 min at 37°C and washed 3 times with serum-free medium. The fluorescence image was observed under a fluorescence microscope.

The dye, JC-1, was used as a fluorescent probe to detect Δψm as previously described (24). CCCP (10 mM) provided in the kit was added to the cell culture medium at 1:1,000 for 20 min at room temperature, then the H9c2 cells were incubated with JC-1 dye (10 µM) for 30 min at 37°C and washed twice with JC-1 dyeing buffer (1X). The red and green fluorescence were observed under a fluorescence microscope.

Chromatin immunoprecipitation assay (25) was performed in accordance with the manufacturer's instructions with slight modifications. In brief, protein/DNA complexes of H9c2 cells (5×10⁶) were cross-linked with formaldehyde and were sonicated to 200–1000 bp of DNA fragments. The crosslinked protein/DNA was then immunoselected with specific antibodies and the protein/DNA complexes were then eluted. Protein-DNA crosslinks were reversed during incubation at 65°C and DNA was purified to subject to quantitative PCR using the following primers: 5′-AAAGGCATCCCAAGGTAT-3′ (forward) and 5′-CCA GCTCTACAGCTCGTAC-3′ (reverse).

Data are shown as the means ± SD. Analysis of variance (ANOVA) was used to compare quantitative variables between more than 2 groups. Intergroup differences were assessed by the Chi-square test and Student's t-test for quantitative variables. A p-value <0.05 (two-sided) was considered statistically significant. Data were analyzed with GraphPad Prism 5.0 software.

In order to observe the effects of oxidative stress on H9c2 rat myocardial cells, H₂O₂ was used as an inducer. The results from CCK-8 assay revealed that cell viability decreased following exposure to H₂O₂ in a dose- and time-dependent manner. The cell survival rate at H₂O₂ 400 µM for 2 h was 53.96±5.6% (Fig. 1A), compared with the control group, and the cell viability was significantly decreased (p<0.01). Subsequently, intracellular ROS levels were detected using the DCFH-DA probe; the strength of the green fluorescence directly reflects intracellular ROS. The levels of ROS increased gradually following exposure to H₂O₂ compared with the untreated group, and the fluorescence intensity was significantly increased at 2 h (Fig. 1B).

To investigate the role of FoxO3a under myocardial oxidative stress conditions, we performed the experiments in vitro and in vivo. We examined the expression of FoxO3a in the H9c2 cells exposed to H₂O₂ at 400 µM for different periods of time. A significant downregulation in FoxO3a expression was observed following exposure of the cells to H₂O₂ at 400 µM for 2 h (Fig. 2A). We also detected the expression of FoxO3a in rats subjected to MI and 30 min of ischemia, followed by 0, 6, 12 or 24 h of reperfusion. The expression of FoxO3a was also decreased in the myocardial tissue in the rats with I/R injury (Fig. 2B).

To examine the effects of TSA on myocardial injury mediated by oxidative stress, we performed the experiments in vitro and in vivo. We measured the MIS by the TTC method. Compared with the I/R group, the MIS was significantly reduced in the TSA 0.2 mg/kg (T1)- and 0.1 mg/kg (T2)-treated groups, from 53.36 to 15.89% (p<0.01) and 20.06% (p<0.001) respectively; however, the MIS between the I/R and TSA 0.05 mg/kg (T3)-treated groups was not significantly altered (Fig. 3A). Subsequently, in order to evaluate the extent of myocardial injury in the rats, we measured the activities of CK, LDH and AST in serum after 24 h of reperfusion. In accordance with MIS, TSA significantly decreased the activities of CK, LDH and AST in serum (Fig. 3B). In addition, the MDA level was decreased and the SOD activity was increased in the TSA-treated groups compared with those in the sham-operated group (Fig. 3C).

In order to investigate whether TSA reduces the ROS levels following exposure of the H9c2 cells to H₂O₂, intracellular ROS production was detected by the DCFH-DA probe. Compared with the H₂O₂-exposed group, treatment with TSA reduced the intracellular ROS level (Fig. 3D). It has been reported that oxidative stress induces mitochondrial dysfunction (26). Thus, in this study, to investigate the protective effect of TSA against oxidative stress-induced mitochondrial damage, Δψm in the H9c2 cells was assessed using the JC-1 probe. The Δψm of the H9c2 cells declined markedly following exposure to H₂O₂, whereas treatment with TSA increased the Δψm in the H9c2 cells (Fig. 3E).

FoxO3a is an important regulator of the resistance to oxidative stress and the downregulation of FoxO3a increases cell death in response to oxidative stress in human chondrocytes (27). Resveratrol, a potent antioxidant, has previously been demonstrated to protect photoreceptor cells from apoptosis by upregulating the FoxO family in experimental retinal detachment (28). Our experimental results revealed that the expression of FoxO3a in the H9c2 cells was markedly reduced in the presence of H₂O₂ compared with that of the control group (p<0.01). However, TSA elicited a significant increase in FoxO3a expression compared with the H₂O₂ alone-treated group (p<0.01) (Fig. 4A and B). In line with the in vitro experimental results, TSA increased the expression of FoxO3a following 24 h of reperfusion in rats (Fig. 4C; p<0.01 vs. sham-operated group; p<0.01 vs. I/R group).

Mitochondrial SOD and catalase as endogenous enzymes are known to be the target proteins of FoxO3a that contributes to their protective effects against oxidative stress (29,30). The levels of SOD2 and catalase in the H9c2 cells markedly decreased following exposure to H₂O₂, whereas treatment with TSA elicited a significant increase in SOD2 and catalase levels in the H9c2 cells (p<0.01) compared with cells exposed to H₂O₂ alone (Fig. 5).

To investigate whether TSA affects the levels of histone acetylation of the FoxO3a gene promoter and then FoxO3a gene expression, we performed in vitro experiments to observe the changes in the H3 and H4 acetylation levels of FoxO3a. The H4 acetylation of the FoxO3a promoter region was markedly decreased in the H₂O₂-exposed cells compared with that of the control group (p<0.01), whereas TSA elicited a significant increase in H4 acetylation of the FoxO3a promoter region compared with that of the H₂O₂ group. The level of H3 acetylation of FoxO3a was not significantly altered (Fig. 6). The above-mentioned results thus indicate that TSA alters the H4 acetylation of the FoxO3a promoter region, thus affecting the expression of FoxO3a.