The following reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA): 4-PBA, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolyl carbocyanine iodide (JC-1) and dimethyl sulfoxide (DMSO). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and phosphate-buffered saline (PBS) were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). A terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeling (TUNEL) kit was purchased from Invitrogen; Thermo Fisher Scientific, Inc. Primary antibodies against glucose-regulated protein 78 (Grp78; sc-13968), ATF6 (sc-22799), PERK (sc-13071), CHOP (sc-7351), JNK (sc-7345), phosphorylated (P)-JNK (sc-6254), BAX (sc-493), Bcl-2 (sc-492), and β-actin (sc-47778) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Secondary antibodies were purchased from Beijing ComWin Biotech Co., Ltd. (Beijing, China).

Cells of the rat H9c2 cardiomyocyte cell line were cultured in DMEM with 10% FBS and 1% antibiotic-antimycotic (penicillin and streptomycin; Gibco; Thermo Fisher Scientific, Inc.). When cell confluency reached to 70–80%, the H9c2 cells, maintained at 37°C with 5% CO₂, were used for experiments. For all the treatment groups, the H9c2 cells (100,000 cells/ml) were pre-incubated with or without different concentrations (0.5, 1, 2.5 and 5 mM) of 4-PBA, dissolved in PBS, for 24 h prior to initiating I/R. A simulated I/R cell model was used, as described previously (19) with modifications. The H9c2 cells were exposed to ischemia by replacing the DMEM with ischemic buffer (DMEM without D-glucose; pH 6.3; Gibco; Thermo Fisher Scientific, Inc.). Subsequently, the cells were incubated in a Modular Incubator Chamber (Billups-Rothenberg, Del Mar, CA, USA) in an atmosphere of 95% nitrogen and 5% CO₂ at 37°C for 6 h. Following ischemia, reperfusion was initiated by incubating the cells in DMEM at 37°C with 5% CO₂ for different durations. In the normoxic control groups, the cells were incubated with DMEM in an atmosphere of 5% CO₂ at 37°C.

Cell viability was determined using an MTT assay and cell death was measured using a lactate dehydrogenase (LDH) assay, as previously reported (20). Briefly, the H9c2 cardiomyocytes were seeded at a density of 5×10³ cells per well into 96-well plates and, at the end of the respective treatments, the cells were incubated with MTT solution (1 mg/ml final concentration stock solution in PBS per well) at 37°C for 4 h. The medium was then removed, and the formazan crystals were dissolved with 150 µl DMSO. The absorbance at 570 nm was determined using a microplate reader (Spectra Fluo, Tecan, Sunrise, Austria). The experiments were repeated in triplicate and data were expressed as the percentages of the control. The LDH assay was used to evaluate myocardial cell death. Following I/R treatment with or without 4-PBA incubation, the medium of the cardiomyocytes was collected to measure LDH release using LDH assay kits, according to the manufacturer's protocol.

Myocardial apoptosis was assessed using TUNEL staining, according to the manufacturer's protocol. The apoptotic index was expressed as the number of apoptotic cells of all cardiomyocytes per field, following the assessment of 20 randomly-selected fields per specimen. Positive (DNase-treated) and negative (no addition of terminal transferase) control tissue sections were incorporated into each assay. Individual nuclei were visualized at 200× magnification, and all measurements were performed in a blinded-manner.

The changes in Ψm were detected using JC-1 staining. The H9c2 cardiomyocytes were cultured (1×10⁵ cells/well) in poly-L-lysine-coated six-well plates. Following the pretreatment, the cells were incubated with JC-1 (2 µM final concentration) at 37°C in the dark for 15 min. The cells were then washed three times with PBS and examined under a Nikon TE300 confocal fluorescence microscope equipped with an argon laser (Nikon Coproration, Tokyo, Japan).

For immunoblotting, the cells were lysed with RIPA buffer, containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl and 1% Triton X-100, with protease inhibitors (aprotinin, leupeptin, phenylmethylsulfonyl fluoride and pepstatin) and phosphatase inhibitor (Sigma cocktail; Sigma-Aldrich). Following the lysate centrifugation for 15 min at 12,000 × g at 4°C, the supernatants were collected by removing insoluble materials. Protein was quantified using a BCA Protein Assay Kit (Beijing ComWin Biotech Co, Ltd.). For Western blot analysis, an equal quantity of protein (50–100 µg) was loaded in each well and separated by 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were then transferred from the gel onto polyvinylidinene fluoride membranes (EMD Millipore, Bedford, MA, USA) and blocked in 5% non-fat dry milk prepared in 1X Tris-buffered saline with 20% Tween-20 (TBST; Beijing ComWin Biotech Co., Ltd.) for 2 h. The membranes were incubated overnight at 4°C with the following primary antibodies: Polyclonal rabbit anti-Grp78 (1:500), monoclonal mouse anti-CHOP (1:200), polyclonal rabbit anti-BAX (1:200), polyclonal rabbit anti-Bcl-2 (1:200), polyclonal rabbit anti-PERK (1:500), polyclonal rabbit anti-ATF6 (1:500), monoclonal mouse anti-P-JNK (1:200), monoclonal mouse anti-JNK (1:200) and monoclonal mouse anti-β-actin (1:1,000). Following washing of the membranes three times with 1X TBST, the membranes were probed with the following secondary antibodies (1:1,000) for 2 h at room temperature: Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (CW0103M) and HRP-conjugated goat anti-mouse IgG (CW0102M). Following 30 min of washing with TBST, the membranes were visualized using an ECL Plus detection system (Beyotime Institute of Biotechnology, Haimen, China) and the relative band densities were measured using a Fluor Chem FC2 system (NatureGene Corp., Beijing, China).

The mean values were calculated from the data obtained from three investigations in each group and at least three separate in vitro experiments. GraphPad Prism version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) was used to perform all statistical analyses. The results were compared using one-way analysis of variance and Tukey's post-hoc test to identify specific differences between groups. P<0.05 was considered to indicate a statistically significant difference.

In order to investigate whether 4-PBA was able to protect against cardiac injury induced by I/R in vitro, the present study first determined the effect of reperfusion duration on cell death. The H9c2 cardiomyocytes were exposed to hypoxia for 6 h, as previously reported (19), followed by reoxygenation for different periods of time (0.5, 1, 2, 4, 8 and 12 h) to mimic in vitro I/R conditions. Cell viability was then detected using an MTT assay and cell death was evaluated using an LDH assay. As shown in Fig. 1A, the cells in control group were considered 100% viable. Hypoxia and reoxygenation caused a decrease in cell viability in a time-dependent manner. The viability of the cardiomyocytes at 12 h post-reoxygenation was ~57%. In addition, LDH leakage was measured as a biomarker of cell death. As shown in Fig. 1B, reoxygenation induced the release of LDH in a time-dependent manner, compared with the untreated control group. LDH leakage increased rapidly between 0.5 and 8 h post-reoxygenation and reached a peak at 12 h, which was ~8-fold of that in the untreated control group. Based on these results, ischemia for 6 h and reperfusion for 12 h were selected for the in vitro I/R conditions in the following experiments.

Treatment with 4-PBA prevented I/R injury-mediated myocardial cell death. To evaluate the effect of 4-PBA (Fig. 2A) on I/R injury on the rat H9c2 cells, the cardiomyocytes were incubated with 4-PBA at a range of concentrations between 0 and 5 mM for 24 h, prior to mimicking I/R injury in vitro. MTT and LDH assays were performed to determine the role of 4-PBA in the prevention of I/R-induced damage. The data demonstrated that cell viability was significantly improved following treatment with 4-PBA at concentrations between 0.5 and 5 mM, in a dose-dependent manner, in triplicate experiments (Fig. 2B). No statistically significant differences were found in cell viability or cell death between the cells incubated with 5 mM 4-PBA for 24 h and the control group (Fig. 2B). The level of LDH leakage in the I/R group was significantly higher, compared with that in the control group (P<0.001), whereas 4-PBA pretreatment decreased the levels of LDH (Fig. 2C). The results confirmed that 4-PBA pretreatment had the ability to inhibit the H9c2 cell death induced by I/R, and pretreatment of the cells with 4-PBA at 5 mM for 24 h was used in the subsequent experiments.

Treatment with 4-PBA prevented the cell apoptosis induced by myocardial I/R injury. To examine whether 4-PBA has any effect on I/R-induced apoptosis in H9c2 cells, the present study performed a TUNEL assay and JC-1 staining. The nuclei of normal cells were stained by propidium iodide and those of apoptotic cells were stained green. The number of TUNEL-positive cells was manually counted in five randomly-selected fields, with the apoptotic index expressed as a percentage of the total counted cells (Fig. 3A). TUNEL staining showed that I/R increased the apoptotic index between 5.6 and 16.7% (P<0.01), compared with the control group. However, 5 mM 4-PBA pretreatment reduced this value to 7.5% (P<0.01, compared with I/R group; Fig. 3B). No significant differences were identified in apoptotic index between the 4-PBA-only treated group and the control group. As the change in Ψm is one of the first signs of apoptosis, the present study determined the levels of mitochondrial depolarization using JC-1 staining (Fig. 3C). I/R significantly decreased the mitochondrial red:green fluorescence intensity ratio, indicating altered Ψm and depolarization. Treatment with 4-PBA partly recovered the mitochondrial red:green fluorescence intensity ratio, suggesting that it provided protection against the mitochondrial depolarization induced by I/R. In conclusion, these data confirmed that exposure of cardiomyocytes to I/R induced apoptotic cell death, however, pretreatment with 4-PBA reversed this effect and protected the cardiomyocytes.

ER stress-induced apoptosis was involved in the protective effect of 4-PBA on the myocardial I/R injury. To investigate whether the cardioprotective effect of 4-PBA is associated with the apoptosis induced by ER stress signaling during I/R injury, the protein levels of pro-apoptotic CHOP, BAX, JNK, and P-JNK, anti-apoptotic Bcl-2, and the ER stress response proteins, Grp78, PERK and ATF6, were measured using Western blot analysis. CHOP is a critical pro-apoptotic factor in ER stress-associated apoptosis. As shown in Fig. 4A and B, the present study found that the myocardial protein expression of CHOP in the I/R group was significantly upregulated, compared with that in the control group (P<0.01). However, 4-PBA attenuated the I/R-induced upregulation in the expression of CHOP, compared with the I/R group (P<0.01). In addition, the I/R-induced increases in the expression levels of BAX (Fig. 4C) and P-JNK (Fig. 4D) were significantly inhibited by 4-PBA treatment. However, the inhibition of I/R on the protein expression of anti-apoptotic Bcl-2 (Fig. 4E) was attenuated by 4-PBA. To further understand the mechanism underlying the effect of 4-PBA on ER stress, the present study also examined the effect of 4-PBA on Grp78, PERK and ATF6, the major transcription factors involved in ER stress. The immunoblotting results indicated that the protein levels of Grp78, ATF6 and PERK were significantly increased following I/R treatment, which were attenuated by 4-PBA (Fig. 5). These data suggested that the ER stress response was activated acutely in I/R injury, whereas treatment with 4-PBA inhibited the activation of ER stress-associated proteins, compared with the respective control group. Taken together, these results demonstrated that the protective role of 4-PBA in I/R injury was associated with the inhibition of apoptosis signaling pathways induced by ER stress.