SQS was kindly provided by Professor Yongming Luo from Jiangxi Chinese Medical University (Nanchang, China), and its identity and purity (>99%) were determined by nuclear magnetic resonance spectroscopy and high-performance liquid chromatography tandem mass spectroscopy analyses. εV1–2, a specific inhibitor of PKCε, was purchased from Anaspec (cat. no. AS-62186; Fremont, CA, USA). Anti-PKCε and anti-phosphorylated (p)-PKCε (Ser 729) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-β-actin antibody was purchased from the Jiancheng Bioengineering Institute of Nanjing (Nanjing, China). The horseradish peroxidase-labeled immunoglobulin G secondary antibody was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Cardiomyocytes from 30 Sprague-Dawley rats (weight, 10–15 g; age, 1–3 days; Nanchang University School of Medicine, Nanchang, China) were prepared according to the protocol of a previous study (17). The rats were treated in accordance with the National Institute of Health's Guide for the Care and Use of Laboratory Animals, and all procedures were approved by the ethics committee of Nanchang University (Nanchang, China). The rats were anesthetized by intraperitoneal injection with 10% chloral hydrate (0.03 ml/kg; Sigma-Aldrich, St. Louis, MO, USA), after which they were sacrificed by cervical dislocation prior to the removal of the rat hearts. The hearts were removed and placed in phosphate-buffered saline (PBS). The ventricles were digested with 0.1% trypsin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and then collected by centrifugation at 60 × g for 10 min, re-suspended in plating medium, including 85% Eagle's minimum essential medium, 15% fetal calf serum and 100 U/ml penicillin and streptomycin (Beijing Solarbio Science & Technology Co., Ltd.), seeded in a culture dish and incubated for 2 h in an atmosphere containing 95% air and 5% CO₂ to remove non-myocytes. The supernatant was collected and plated in 60-mm gelatin-coated culture dishes at 1×10⁶ cells per dish. After 24 h, cardiomyocytes were washed and the medium was replaced, followed by incubation for three days prior to the experiments.

The neonatal primary rat cardiomyocytes were divided into various experimental groups as follows: i) In the control group, cardiomyocytes were incubated under normal conditions for an additional 24 h; ii) in the sI/R group, cardiomyocytes were subjected to anoxia by incubating them in fresh anoxic medium [0.9 mM NaH₂PO₄, 6.0 mM NaHCO₃, 1.8 mM CaCl₂, 1.2 mM MgSO₄, 40 mM sodium lactate, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 98.5 mM NaCl, 10.0 mM KCl, pH 6.8; Sigma-Aldrich] at 37°C in a chamber containing 95% N₂ and 5% CO₂ for 3 h. Subsequently, the medium was replaced with re-oxygenation medium (129.5 mM NaCl, 5.0 mM KCl, 0.9 mM NaH₂PO₄, 20 mM NaHCO₃, 1.8 mM CaCl₂, 1.2 mM MgSO₄, 5.5 mM glucose, 20 mM HEPES, pH 7.4; Sigma-Aldrich) and the cells were incubated at 37°C in an atmosphere of 95% air and 5% CO₂ for 2 h; iii) In the SQS+sI/R group, cells were pre-treated with SQS at 10 µM for 24 h prior to sI/R; iv) In the SQS+εV1–2+sI/R group, cells were pre-treated with εV1–2 (1 µM) and SQS (10 µM) for 24 h prior to sI/R. The doses of SQS and εV1–2 used were selected on the basis of a preliminary study by our group and previous reports (8,18,19).

A colorimetric MTS assay was used to assess the viability of cardiomyocytes subjected to sI/R. MTS is a pale yellow substrate that is converted into a dark blue formazan product by living cells. Primary cardiomyocytes were seeded into 96-well plates at 1×10⁴ cells/well. After sI/R treatment, cells were incubated with 20 µl MTS (5 mg/ml; Promega Corp., Madison, WI, USA) in 100 µl medium at 37°C for 2 h. The absorbance of each well at a wavelength of 490 nm was then measured using a microplate reader (no. 680; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The results were expressed as a percentage of the control.

Following sI/R treatment, the culture media of the control groups and the supernatants of cell lysates from each group were collected to the activities of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) were assessed using kits (A020-2 and A032, respectively; Jiancheng Bioengineering Institute of Nanjing), according to the manufacturer's instructions.

ROS levels were determined using the fluorescent probe 2′7′-dichlorodihy-drofluorescein diacetate (DCFH-DA; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), which is cleaved by cellular esterases to non-fluorescent DCFH and oxidized by intracellular ROS to the fluorescent product dichlorofluorescein. ROS production is proportional to the fluorescence ratio of the treatment vs control group. After the indicated treatments, the cells were incubated with 10 µM DCFH-DA for 20 min at 37°C prior to being harvested and analyzed for green and red fluorescence intensity using a flow cytometer (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA) at wavelengths of 485 and 528 nm, respectively.

The measurement of [Cl⁻]_i was performed according to the method described in a previous study by our group with minor modifications (8). Briefly, after the indicated treatments, cardiomyocytes were washed twice with Cl⁻-free solution (NaCl was replaced by an equimolar amount of D-glucuronic acid, MgCl₂ by MgSO₄ and KCl by potassium gluconate), and incubated with 10 mM N-(ethoxycarbonylmethyl)-6-methoxyquinolinium (MQAE; Thermo Fisher Scientific, Inc.) in the dark for 20 min at 37°C. Cl⁻-free solution was then used to remove any excess dye. Cells were suspended in Cl⁻-free solution immediately subjected to flow cytometric analysis (FACSCalibur).

The fluorescent dye JC-1 was used to assess the mitochondrial membrane potential as described in the study by Tang et al (18). Briefly, after the indicated treatments, cardiomyocytes were incubated with JC-1 (200 µM; Beijing Solarbio Science & Technology Co., Ltd.) at 37°C for 20 min. Cells were then washed with ice-cold PBS to remove remaining dye. Fluorescence was measured by flow cytometry (FACSCalibur) with excitation/emission wavelengths of 530/580 nm (red) and at 485/530 nm (green). The ratio of red to green fluorescence intensity of cells reflected the Δψm.

Western blotting was performed as described previously (18). The protein concentrations were measured using the DC Protein Assay kit II (cat. no. 500-0112; Bio-Rad Laboratories, Inc.). Equal quantities of protein (30 µl/lane) were then separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. After blocking with 5% non-fat milk in Tris-buffered saline containing 0.2% Tween 20 (TBST), the blots were probed with rabbit anti-PKCε (C-15; 1:500; sc-214), goat anti-p-PKCε (Ser 729; 1:500; sc-12355) and rabbit anti-β-actin (1:1,000; #21338) polyclonal antibodies overnight at 4°C. After washing with TBST, the blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit (1:5,000; #7074) and rabbit anti-goat (1:5,000; #7075) secondary antibodies for 1 h at room temperature. After washing, the blots were saturated with enhanced chemiluminescence mixture (Pierce Biotechnology, Inc., Rockford, IL, USA) for 1 min, and images of the blot were captured by exposure to pre-flashed X-ray film (Kodak, Rochester, NY, USA). for 300 sec. Densitometric scanning was performed using Quantity One software, version 4.62 (Bio-Rad Laboratories, Inc.). Results were expressed as a percentage of the control. The bands were normalized to β-actin.

Data are expressed as the mean ± standard error of the mean. Statistical analyses were performed using SPSS software, version 11.0 (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance was applied to test the significance of differences between groups, followed by post-hoc testing for individual differences. P<0.05 was considered to indicate a statistically significant difference between values. For each assessment, at least three independent experiments were performed.

Primary cardiomyocytes were incubated with or without 10 µM SQS for 24 h, followed by sI/R, and the levels of total and phosphorylated PKCε (Ser 729) were assessed by western blotting. As shown in Fig. 1, the levels of p-PKCε Ser 729 were significantly increased in the SQS pre-treated group (P<0.01 vs. control group; P<0.01 vs. sI/R group), whereas the levels of total PKCε remained unchanged. These results suggested that SQS treatment led to the activation of PKCε in cardiomyocytes following sI/R.

The release of CPK and LDH into the culture medium was analyzed to evaluate the effects of SQS and PKCε inhibitor εV1–2 on cardiomyocyte death. As shown in Fig. 2, a significant increase in CPK and LDH levels was observed in the sI/R group compared to that in the control group (P<0.01), which was significantly inhibited by pre-treatment with SQS (P<0.01 vs. sI/R group). Of note, the release of CPK and LDH in the group additionally pre-treated with εV1–2 prior to sI/R was enhanced when compared with that in the SQS+sI/R group (P<0.01) (Fig. 2), indicating that SQS may exert its protective effects via PKCε. Furthermore, the results of the MTS assay showed that SQS significantly increased cardiomyocyte viability following sI/R (P<0.01), which was significantly inhibited by co-treatment with εV1–2 (P<0.01 vs. SQS+sI/R group) (Fig. 3). These data suggested that PKCε is required for SQS to elicit its cardioprotective effects.

To investigate whether PKCε is implicated in the inhibitory effects of SQS on the elevation of [Cl⁻]_i induced by sI/R, cardiomyocytes were treated with PKCε inhibitor εV1–2 in combination with SQS and [Cl⁻]_i was assessed via MQAE staining and flow cytometric analysis. As shown in Fig. 4, SQS pre-treatment abrogated the increases in [Cl⁻]_i in cardiomyocytes following sI/R (P<0.01 vs. sI/R group), which was inhibited by the selective PKCε inhibitor εV1–2 (P<0.01 vs. SQS+sI/R group). These results indicated that activation of PKCε is required for SQS to inhibit sI/R-induced elevation of [Cl⁻]_i.

As the decrease of Δψm induced by Cl⁻ influx reflects the opening of the mPTP, which results in a ROS burst, the present study assessed the effects of SQS on sI/R-induced changes in Δψm in cardiomyocytes as well as the possible involvement of PKCε. As shown in Fig. 5, sI/R decreased the Δψm as indicated by a decrease in the ratio of red to green fluorescence intensity (P<0.01 vs. control group). However, pre-treatment with SQS significantly attenuated the loss of Δψm (P<0.01 vs. sI/R group). As expected, the restorative effect of SQS on the Δψm was inhibited by εV1–2 (P<0.01 vs. SQS+sI/R group), indicating that PKCε is required for SQS-mediated attenuation of Δψm loss following sI/R in cardiomyocytes.

Intracellular ROS levels were assessed by measuring cDCF fluorescence intensity. As shown in Fig. 6, sI/R induced marked intracellular ROS production, while pre-treatment with SQS inhibited ROS production induced by sI/R. Co-treatment with εV1–2 attenuated the inhibitory effects of SQS on ROS production induced by sI/R, indicating that PKCε is required for SQS to inhibit sI/R-induced ROS production.