The H9c2 rat cardiomyocyte cell line, obtained from the Shanghai Institutes for Biological Sciences (Shanghai, China), was routinely cultured in Dulbecco's modified Eagle's medium (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, China), 100 U/ml penicillin and 100 µg/ml streptomycin in a humidified atmosphere at 37°C with 5% CO₂. When the confluence of H9c2 cells was ~80%, PA (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at doses of 100, 200 and 400 µM was added to the medium and incubated for 12 h. An ER stress inhibitor, 4-phenyl butyric acid (4-PBA; 5 mM), was administrated 90 min before 400 µM PA treatment.

Oil Red O staining was used to measure the accumulation of intracellular lipids. Briefly, 150 mg Oil Red O powder (Sigma-Aldrich; Merck KGaA) was dissolved in 50 ml 60% isopropanol to prepare the Oil Red O reserve solution (3 mg/ml) and further diluted by distilled water (3:2) to prepare a working solution. H9c2 cells were fixed in 4% paraformaldehyde for 30 min at room temperature, washed with PBS three times, then followed by incubation with the Oil Red O working solution for 30 min at room temperature. After washing with PBS three times, nuclei were counterstained with 50% hematoxylin for 2 min at room temperature. Finally, the result of Oil Red O staining was observed under an optical microscope (Olympus Corporation, Tokyo, Japan).

MTT assay was used to determine the cell viability following PA treatment. Briefly, H9c2 cells were seeded in a 96-well plate and treated with various concentrations of PA as described above. After 12 h, the medium was removed and replaced with 0.5 mg/ml MTT solution (Sigma-Aldrich; Merck KGaA), 200 µl per well. After incubation at 37°C for 4 h, the MTT solution was removed and each well was washed with PBS three times. The precipitated formazan in each well was solubilized by dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA) and the optical density value was detected by an automated microplate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 490 nm.

Lactate dehydrogenase (LDH) activity in the culture medium was detected to determine the injury of H9c2 cells using a commercial LDH assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocols.

Apoptosis was detected by flow cytometry analysis using the PE-labeled Annexin V/7-aminoactinomycin D apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocols.

Cells were harvested after PA treatment for 12 h and mixed with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Nanjing, China) and protease inhibitor phenylmethanesulfonyl fluoride on ice for 20 min. Proteins were extracted from cells following 5 min of centrifugation of 13,000 × g at 4°C. The concentration of protein was measured using Enhanced BCA Protein Assay kit (Beyotime Institute of Biotechnology). For western blot analysis, 50 µg protein denatured by heating was subjected to 10% SDS-polyacrylamide gel electrophoresis for separation and then transferred to polyvinylidene difluoride membranes. The membrane was blocked using 2% bovine serum albumin (BSA) for 1 h at room temperature and then incubated overnight at 4°C with the specific primary antibodies including monoclonal anti-78 kDa glucose-regulated protein (GRP78; cat no. 3183; 1:1,000), anti-protein kinase R-like endoplasmic reticulum kinase (PERK; cat no. 3192; 1:1,000), anti-phospho (p)-PERK (cat no. 3179; 1:1,000), anti-eukaryotic initiation factor 2 α (eIF2α; cat no. 5324; 1:1,000), anti-p-eIF2α (cat no. 3398; 1:1,000) (all from Cell Signaling Technology, Inc., Danvers, MA, USA), anti-cleaved caspase-12 (cat no. ab18766; 1:1,000), anti-C/EBP homologous protein (CHOP; 1:1,000, cat no. ab11419) (both from Abcam, Cambridge, UK), and anti-β-actin (TA-09; 1:2,000; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit (cat no. sc-2004; 1:2,000) or rabbit anti-mouse immunoglobulin G (cat no. sc-358914; 1:2,000) (both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 37°C for 2 h. Detection of protein band was performed using an enhanced chemiluminescence kit (Santa Cruz Biotechnology, Inc.) according to the manufacturer's protocols. The levels of phosphorylated proteins were normalized to their corresponding total protein levels. Relative densitometry was calculated using ImageJ software version 2× (National Institutes of Health, Bethesda, MD, USA).

The data were expressed as the mean ± standard deviation. Statistical analysis was performed by software SPSS 17.0 version (SPSS, Inc., Chicago, IL, USA). Differences between groups were initially evaluated using one-way analysis of variance, and if the differences were significant, multiple comparison analysis was further performed using Fisher's least significant difference test. P<0.05 was considered to indicate a statistically significant difference.

The result of Oil Red O staining demonstrated a gradual increase in the extent of lipid accumulation in H9c2 cells following treatment with increasing concentrations of PA (0, 100, 200 and 400 µM; Fig. 1), suggesting PA induced excessive lipid accumulation in H9c2 cells.

An MTT assay demonstrated a significant decrease in cell viability following treatment with 200 and 400 µM PA compared with the control. Furthermore, PA reduced cell viability in a dose-dependent manner (Fig. 2).

An LDH assay indicated that LDH activity was significantly increased by 200 and 400 µM PA compared with the control. Furthermore, LDH activity in the 400 µM PA treatment group was higher than in the 200 µM PA group, which suggested that PA induced H9c2 cell injury in a dose-dependent manner (Fig. 3).

PA (200 and 400 µM) significantly increased H9c2 cell apoptosis compared with the control, demonstrated by increased apoptosis rate. In addition, apoptosis induced by PA was also displayed in a dose-dependent manner (Fig. 4).

As presented in Fig. 5, the expression of GRP78, a well-established ER stress marker, was increased by various dose of PA in a dose-dependent manner. Furthermore, the phosphorylation of eIF2α and PERK were also increased in a dose-dependent manner (Fig. 5), which indicated that PERK/eIF2α signaling may be activated by PA.

Various doses of PA significantly increased the expression of CHOP compared with the control; however, no significant differences in the expression of CHOP were detected among the different PA-treated groups. Furthermore, various dose of PA significantly increased the expression of cleaved caspase-12 in a dose-dependent manner (Fig. 6).

In order to reconfirm the role of ER stress in PA induced apoptosis, 4-PBA, a specific ER stress inhibitor, was administered to examine the changes in cell apoptosis rate and the expression of CHOP and cleaved caspase-12 following ER stress inhibition. The results revealed that 400 µM PA significantly increased H9c2 cells apoptosis rate, but this effect was reversed by treatment with 4-PBA (Fig. 7). Similarly, the expression of CHOP and cleaved caspase-12 were increased by 400 µM treatment, but were also reversed by 4-PBA treatment (Fig. 8).