All experimental animals were provided by the Experimental Animal Center of Chongqing Medical University. Clean and healthy neonatal Kunming male and female mice were used (1–3 days-of-age, weighing 2.3–2.7 g). All animal experiments were approved by the Animal Protection and Use Committee of Zunyi Medical University (Zunyi, China), and complied with Directive 2010/63/EU of the European Parliament (19).

Under aseptic conditions, Kunming mice (1–3 days-of-age) were sacrificed by decapitation. The ventricular tissue was immediately removed using ophthalmic scissors and cut into 1–2 mm³ pieces. The tissue was then ground in 1 ml 0.05% collagenase type II (Worthington Biochemical Corporation) for 5 min at room temperature (repeated 8–10 times). Next, the supernatant was centrifuged at 1,500 × g for 10 min at room temperature, the supernatant was discarded and the cells resuspended in DMEM/F12 supplemented with 20% FBS (HyClone; Cytiva). After 1.5 h of culture in an incubator at 37°C with 5% CO₂, the adherent fibroblasts were discarded and the cell suspension was transferred to a new culture flask. 5-BrdU (Beijing Solarbio Science & Technology Co., Ltd.) was added to a final concentration of 0.1 mmol/l to prevent the growth of fibroblasts, and the cells were further cultured in an incubator. According to previous studies (20–24), the cells were treated with 100 µmol/l PE (MedChemExpress; cat. no. HY-B0769), 50 µmol/l AA (Sigma-Aldrich; Merck KGaA; cat. no. 16611-84-0) or 10 µmol/l U0126 (Selleck Chemicals; cat. no. S1102). Cardiomyocytes were treated with the aforementioned drugs individually or in combination, dependent on the treatment groups.

Cardiomyocytes were plated in a 96-well plate at a density of 2×10⁴ cells/well. Next, 10 µl Cell Counting Kit-8 (CCK-8) solution (Beijing Solarbio Science & Technology Co., Ltd.) was added and cells were cultured for 4 h at 37°C in the dark. Finally, the absorbance was measured at 450 nm using a Universal Microplate Spectrophotometer (Bio-Rad Laboratories, Inc.).

Cardiomyocytes were used in western blotting at a density of 3–4×10⁶ cells/well. Nuclear protein was harvested from mouse cardiomyocytes using a nuclear protein extraction kit (Merck KGaA). Nucleic proteins were loaded on a 6 or 12% SDS-gel, resolved using SDS-PAGE and then transferred to a PVDF membrane (Merck KGaA). Membranes were blocked at 4°C with 5% BSA for 1 h and then incubated with a series of rabbit polyclonal antibodies, which included anti-ANP (1:5,000; Abcam; cat. no. ab189921), anti-H3K9ac (1:5,000; Abcam; cat. no. ab4441), anti-H3 (1:5,000; Abcam; cat. no. ab1791), anti-β-MHC (1:5,000; Abcam; cat. no. Ab207926) anti-BNP (1:1,000; Abcam; cat. no. ab239510), anti-β-actin (1:1,000; Abcam; cat. no. ab8226) and anti-PCAF (1:2,000; Abcam; cat. no. ab176316), anti-ERK (1:2,000; Cell Signaling Technology, Inc.; cat. no. 4695) or anti phospho-(p-)ERK (1:2,000; Cell Signaling Technology, Inc.; cat. no. 4370); β-actin and H3 were served as an internal controls. All antibodies were diluted in Tris-buffered saline containing 5% skimmed milk, and incubated with the membrane overnight at 4°C. HRP-labeled goat anti-rabbit antibody (1:2,000; Santa Cruz Biotechnology, Inc.; cat. no. sc2004) was used as the secondary antibody and incubated with the membrane at 4°C for 2 h. The results were detected with enhanced chemiluminescence reagents (Wanleibio Co., Ltd.). Finally, membranes were scanned using a Bio-Rad image analyzer; and densitometry analysis was performed using Quantity One version 4.4 (Bio-Rad Laboratories, Inc.).

Cardiomyocytes were used for qPCR at a density of 2–3×10⁶ cells/well. Total RNA was extracted from myocardial cells using an RNA Extraction kit (BioTeke Corporation), according to the manufacturer's protocol. Total RNA was then reverse transcribed into single-stranded cDNA using an AMV Reverse Transcription system, according to the manufacturer's protocol (Takara Bio, Inc.). cDNA was amplified using a SYBR Green dye kit and gene-specific primers (Takara Bio, Inc.). The primer sequences of MEF2C and β-actin were: MEF2C forward, 5′-CCTTTTCCTTTTCTGGGGACTTGTT-3′ and reverse 5′-TGCCGCTGTGAGCCTCTATTTG-3′; and β-actin forward, 5′-CCTTTATCGGTATGGAGTCTGCG-3′ and reverse, 5′-CTGACATGACGTTGTTGGCA-3′. β-actin was used as a standardized reference, and the 2^−ΔΔCq method was used to determine relative gene expression (25).

Cardiomyocytes were seeded into 6-well plates (1×10⁵ cells/well) for 24 h and incubated with 50 µmol/l AA for 1 h, then 10 µmol/l U0126 was added. After 48 h, 100 µmol/l PE was added. Subsequently, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature (RT), treated with 0.3% Triton X-100 in PBS at RT for 20 min, and then incubated with 10% goat serum (Solarbio Science & Technology Co., Ltd.; cat. no. SL038) at 37°C for 30 min. Subsequently, the cardiomyocytes were incubated at 4°C overnight with primary antibodies against α-actin (1:100; ProteinTech Group, Inc.; cat. no. 23660-1-AP), H3K9ac (1:1,000 Abcam; cat. no. ab4441) and anti-PCAF (1:250; Abcam; cat. no. ab176316). The following morning, the cells were incubated at 37°C in the dark for 1 h with Alexa Fluor 594 goat anti-mouse IgG secondary antibody (1:1,000; Thermo Fisher Scientific, Inc.; cat. no. A-11005) and Alexa Fluor 488 goat anti-rabbit IgG (1:200; Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. A-11008) secondary antibody. Finally, the cardiomyocytes were counterstained with DAPI for 5 min at RT. All images were obtained using a confocal microscope (magnification, ×40) with standardized imaging parameters. Finally, the images were quantified based on fluorescence using ImageJ 1.49 software (National Institutes of Health).

Co-IP was performed as described previously (26), using anti-phospho-(p-)ERK, anti-PCAF and anti-H3K9ac rabbit polyclonal antibodies with Dynabeads protein G magnetic beads (Invitrogen; Thermo Fisher Scientific, Inc.) for the immunoprecipitation and western blotting of primary cardiomyocytes. First, the primary antibody was bound to Protein G magnetic beads (according to the manufacturer's protocol). Next, at pH 7.4, a magnet along with 1% Triton X-100, 0.5% NP-40, 20 mM HEPES, 50 mM NaCl and protease inhibitor (1:50; Beijing Solarbio Science & Technology Co., Ltd.), were used to immunoprecipitate the target antigen (p-ERK) in the buffer. The sample was then washed three times with a lysis buffer. The immobilized protein complex was eluted, denatured in 5X SDS sample buffer at 95°C for 10 min, and then subjected to western blotting analysis with anti-p-ERK (1:2,000; Cell Signaling Technology, Inc.; cat. no. 4370), anti-PCAF and anti-H3K9ac antibodies. IgG served as a negative control.

ChIP was performed as described previously (26). Formaldehyde (1%) was added to the homogenized cardiomyocytes to cross-link the DNA-protein complex, and incubate at 37°C for 15 min. ChIP determination was then performed using a specific kit (Merck KGaA). Next, the cross-linked complex DNA was sheared with ultrasound and precipitated with monoclonal antibodies (anti-MEF2C, 1:1,000; anti-H3K9ac, 1:2,000; and anti-PCAF, 1:2,000) overnight at 4°C. DNA purification kits (Merck KGaA) were then used to extract the final DNA, according to the manufacturer's protocol. Normal mouse IgG (1:500; Sigma-Aldrich, Merck KGaA) was used as a negative control.

All experiments were repeated six time with six independent samples. All data are expressed as the mean ± standard deviation. All statistical analyses were performed using SPSS version 18.0 (SPSS Inc.). Comparisons among multiple groups were analyzed using one-way ANOVA followed by Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

In order to establish a model of PE-induced cardiomyocyte hypertrophy, first, the optimal PE exposure concentration (100 µmol/l) was determined based on a previous study (20). The treatment time of PE was 48 h in cultured myocardial cells, and the effects were determined using western blotting. Data arising from the western blotting experiments showed that the levels of biomarkers for myocardial hypertrophy (ANP and β-MHC) in the PE group were significantly higher than those in the vehicle group (Fig. 1A and B). Additionally, the cell-surface area was also assayed using immunofluorescence analysis. The results showed that myocardial cells in the PE group were significantly larger than those in the vehicle group (Fig. 1C and D). These data showed that a mouse model of PE-induced cardiomyocyte hypertrophy was successfully established.

Studies have confirmed that the p-ERK1/2 signaling pathway plays an important role in pathological myocardial cell hypertrophy (27,28). Thus, the effects of the ERK1/2 signaling pathway on the manner by which AA attenuates PCAF mediated-H3K9ac hyperacetylation in PE-induced hypertrophic cardiomyocytes was determined. First, according to the previous literature (21–24), the optimum concentration of the histone acetylase inhibitor AA was determined (50 µmol/l), as well as for the ERK inhibitor U0126 (10 µmol/l), in accordance with H3K9ac levels, and the viability of myocardial cells using western blotting and CCK-8 examination, respectively (Fig. 2A and B). Additionally, the inhibitory activity of AA decreased as the concentration of AA was increased. As the concentration of the inhibitor increases, its inhibitory effect will gradually increase, but when the concentration of the inhibitor is higher than a certain level, its inhibitory effect will be weakened (29,30). After hypertrophic cardiomyocytes had been treated with AA and/or U0126, the expression of total-(T-)ERK1/2 and p-ERK1/2 was determined by western blotting, and the results showed that the expression levels of p-ERK1/2 in the PE group were significantly higher than those in the control group. Additionally, it was also found that the HATs inhibitor AA, and the ERK inhibitor U0126 could ameliorate the increase in p-ERK1/2 levels induced by PE in primary cultured myocardial cells; however, the expression of T-ERK1/2 remained unchanged (Fig. 2C). In addition, our previous study found that an imbalance in the modification of histone H3K9ac, as mediated by PCAF, was involved in the pathological hypertrophy of myocardial cells (31). Next, whether the ERK1/2 signaling pathway interacted with PCAF in order to regulate the modification of histone H3K9ac acetylation and further enhance pathological cardiac hypertrophy was determined. Co-IP experiments were not used as an analytical method, but instead performed to verify the formation of a complex between the ERK1/2 signaling pathway and PCAF mediated-H3K9ac acetylation, and it was successfully demonstrated that there was an interaction in the primary cultured myocardial cells. Collectively, these data indicated that the ERK1/2 signaling pathway may interact with PCAF mediated-H3K9ac acetylation (Fig. 2D).

Our previous study showed that PCAF plays a critical role in pathological cardiac hypertrophy in a manner that is dependent on the modification of histone acetylation, and it was confirmed that p-ERK1/2 could interact with PCAF mediated-H3K9ac acetylation (31). Thus, in the present study, the expression of PCAF was assessed by immunofluorescence and western blotting, and a notable increase in PCAF expression in hypertrophic cardiomyocytes was observed when induced by PE. In contrast, exposure to AA reversed the upregulation of PCAF in primary myocardial cells, as did the ERK inhibitor, U0126 (Fig. 3).

Our previous study demonstrated that an imbalance in the modification of histone H3K9ac acetylation can result in pathological cardiac hypertrophy (32). In the present study, immunofluorescence and western blotting data showed that the levels of H3K9ac acetylation in the PE group were significantly higher than that in the control group. Additionally, it was shown that the HATs inhibitor AA, or the ERK inhibitor U0126, could downregulate the hyperacetylation of H3K9ac induced by PE in primary myocardial cells (Fig. 4).

MEF2C is a nuclear transcription factor in cardiac cells that is involved in pathological cardiac hypertrophy and several other cardiovascular diseases (33,34). Therefore, the binding of PCAF and histone H3K9ac acetylation in the promoter region of MEF2C was assessed using ChIP-qPCR. The data showed that the levels of PCAF promoter binding of MEF2C in the PE group was higher than that in the control group. Additionally, it was also found that the HATs inhibitor AA, and the ERK inhibitor U0126 downregulated the binding of PCAF to the promoter region of MEF2C. Meanwhile, the levels of histone H3K9ac acetylation in the promoter region of MEF2C was increased in the PE group. It was also evident that the HATs inhibitor AA, and the ERK inhibitor U0126, could attenuate the levels of histone H3K9ac acetylation in the promoter region of MEF2C (Fig. 5A and B). The mRNA expression levels of the MEF2C gene was determined using RT-qPCR, and the results showed there was a notable increase in gene expression in myocardial cells treated with PE. Exposure to AA or U0126 reduced the overexpression of MEF2C mRNA in primary cultured myocardial cells treated with PE (Fig. 5C).

To investigate the effects of MEF2C on downstream genes associated with cardiac hypertrophy in cardiomyocytes, the regulatory relationship between MEF2C and downstream genes associated with cardiac hypertrophy, including ANP, BNP and β-MHC were investigated. The binding affinity between MEF2C and the promoters of ANP, BNP and β-MHC, were detected by PCR following ChIP. The results showed that MEF2C could bind to the promoters of ANP, BNP and β-MHC (Fig. 5D). These results indicate that MEF2C is involved in regulating cardiac hypertrophy-related ANP, BNP and β-MHC gene expression. The levels of biomarkers of cardiac hypertrophy (ANP, BNP and β-MHC) at the protein level in the PE group were significantly higher than those in the control group. Additionally, it was found that AA and U0126 could reduce the increase in ANP, BNP and β-MHC levels in myocardial cells treated with PE (Fig. 6A, B and C). AA and U0126 could also significantly reduce the surface area of cardiomyocytes treated with PE (Fig. 6D and E).