Primary rat neonatal cardiocytes were derived from 1-2-day-old Wistar rats (Experimental Animal Holding Facility of Jilin University, China). Briefly, the hearts were removed under aseptic conditions and the ventricles were homogenized in D-Hank’s buffer. The tissue fragments were digested by stepwise exposure to 0.125% pancreatin (Gibco Life Technologies, Carlsbad, CA, USA). The dissociated cells were pre-plated for 90 min to remove fibroblasts, and the non-adherent cardiocytes were then plated at a density of 1×10⁵ cells/mm². Cells were cultured in Iscove’s modified Dulbecco’s medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (BHKT Clinical Reagent Co., Ltd, Beijing, China), and maintained at 37°C in an incubator with 95% O₂ and 5% CO₂. All animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Jilin University (Changchun, China).

2,7-Dichlorofluorescein diacetate (DCFH), Fluo-3/AM, Rhodamine 123 and Annexin V were obtained from Sigma-Aldrich (St. Louis, MO, USA). The mitoK_ATP inhibitor 5-hydroxydecanoate (5-HD) was also obtained from Sigma-Aldrich.

To induce oxidative stress, cells were cultured in serum-free medium for 12 h when the cells were semiconfluent and beating synchronously. For the peroxidation challenge, the cells were cultured in medium with 0.2 mmol/l H₂O₂ (Beijing Chemical Factory, Beijing, China) and maintained at 37°C for 24 h.

The present study utilized five experimental groups of cardiocytes as follows: i) The dimethyl sulfoxide (DMSO, Beijing Chemical Factory, Beijing, China) group, maintained in 0.1% DMSO; ii) the H₂O₂ group, treated with 0.2 mmol/l H₂O₂ for 24 h; iii) the AsIV+H₂O₂ group, pretreated with 30 mg/l AsIV (provided by Academy of Chinese Medical Sciences of Jilin Province, Changchun, China) for 30 min prior to H₂O₂ injury; iv) the AsIV+H₂O₂+5-HD group, pretreated with 50 μmol/l 5-HD for 5 min prior to the AsIV pretreatment; and v) the nicorandil (NIC; Changchun Dazheng International Trade Group Pharmaceutical Co., Ltd, Changchun, China)+H₂O₂ group, pretreated with 120 mg/l NIC for 30 min prior to H₂O₂ injury. The NIC+H₂O₂ group was used as a positive control. For all AsIV treatments, a 0.1% AsIV stock solution was dissolved in DMSO.

Cardiocyte survival was assessed using an MTT assay. Briefly, cells were incubated with 200 μl medium in 96-well plates, 5 μl MTT stock solution (5 mg/ml) was then added to each well and the cells were incubated for a further 4 h. Blue formazan precipitate was produced from the cells by adding 100 μl DMSO and gently shaking, and the absorbance was measured at 570 nm using an A-5082 ELISA reader (Chengdu Taimeng Technology Co., Ltd., Chengdu, China). LDH release was measured using a GF-200 Semi Automatic Biochemical Analyzer (Shandong Gaomi Caihong Instruments Co., Ltd., Gaomi, China).

Intracellular ROS levels were analyzed using a flow cytometer with DCFH staining. DCFH was dissolved in ethanol (Beijing Chemical Factory, Beijing, China) to produce a 1 mmol/l stock solution. Cells were subsequently washed three times with phosphate-buffered saline (PBS) prior to the addition of DCFH (10 μM). Following a 10-min incubation in the dark, the cells were washed three times then resuspended in PBS. Fluorescence was measured using a fluorescence microplate reader at 488/525 nm (Gemini XPS; Shanghai Spectrum Instruments Co., Ltd., Shanghai, China).

The non-adherent myocytes were digested by treatment with 0.25% pancreatin for 10 min. The cells were then washed twice with PBS, and the percentage of apoptotic cells was determined with an Coulter Epics XL flow cytometer (Beckman Coulter, Brea, CA, USA) after double staining with the Annexin V-FITC apoptosis detection kit (Tianjin Sungene Biotech Co., Ltd. China). Annexin V was used as an apoptosis indicator and propidium iodide (PI) as a necrosis indicator. An⁺PI⁻ represents viable apoptotic cells in the lower right quadrant.

Fluo-3 was used as a Ca²⁺ indicator during fluorescence imaging to assess [Ca²⁺]i. Changes in [Ca²⁺]i were analyzed using a Fluoview FV500 confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan). Cells in 24-well plates were washed with HEPES buffer and treated with Fluo-3/AM (10 μmol/l) for 40 min in the dark. Following removal of Fluo-3/AM, the cells were washed three times with HEPES, and fresh HEPES was added with the various drug treatments as described. Baseline fluorescence in normal cells was recorded for 1 min, then H₂O₂ was added and the change in fluorescence was measured for 5 min. The results were analyzed using software which accompanied the confocal microscope. Alterations in the levels of green fluorescence were used to evaluate [Ca²⁺]i.

Fluorescence imaging of ΔΨm was conducted using Rhodamine 123 as an indicator. Rhodamine 123 is a mitochondria-selective dye, which in a reaction driven by ΔΨm in normal polarized mitochondria, assembles into red fluorescence-emitting dimers forming J-aggregates. Changes in MMP were analyzed using the confocal microscope. Cells in 24-well plates were treated with Rhodamine 123 (5 μg/ml) for 10 min away from light. Following removal of Rhodamine 123, the cells were washed with PBS. The level of Rhodamine 123 was measured and analyzed with the Fluoview Viewer software, version 1.7a (Olympus Corporation).

Data are expressed as the mean ± standard error of the mean. Statistical significance was calculated using one-way analysis of variance with Newman-Keuls post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Pretreatment with AsIV significantly increased cardiocyte viability compared with that of the H₂O₂ group (P<0.05), while pretreatment with 5-HD resulted in reduced viability (P<0.05 vs. the AsIV group) (Fig. 2). As indicated in Fig. 3, cardiocyte damage resulting from H₂O₂ exposure was reflected by increased LDH release (P<0.01 vs. the DMSO control group); which was reduced by AsIV pretreatment (P<0.01 vs. the H₂O₂ group) and significantly increased in the 5-HD pretreatment group (P<0.05 vs. the AsIV group). These results indicated that AsIV may protect cardiocytes against H₂O₂ injury, while the mitoK_ATP inhibitor 5-HD may partially reverse this effect.

Elevated levels of ROS may contribute to ischemia-reperfusion injury. Therefore, the present study evaluated whether AsIV-mediated cardioprotection may be partially attributed to a reduction in oxidative stress. ROS generation was assessed by DCFH fluorescence. The results indicated that H₂O₂ induced significant ROS generation in cardiocytes compared with the DMSO control group (P<0.01) (Fig. 3), which was reduced following AsIV treatment (P<0.05 vs. the H₂O₂ group). This protective effect was significantly attenuated by 5-HD pretreatment (P<0.05). These results indicate that AsIV may protect cardiocytes against H₂O₂ injury by reducing oxidative stress; and this effect may be associated with the mitoK_ATP.

Ca²⁺ overload is a key contributor to the mitochondrial permeability transition leading to ischemia-reperfusion injury. As displayed in Fig. 4, AsIV-pretreated cells presented significantly lower [Ca²⁺]i compared with that of the H₂O₂ group (P<0.01). 5-HD pretreatment prior to AsIV exposure resulted in significantly higher [Ca²⁺]i compared with that of the AsIV group (P<0.01). These results indicated that AsIV may relieve Ca²⁺ overload, an effect which is also associated with the mitoK_ATP.

MPTP opening may be critical for the transition from reversible to irreversible myocardial ischemia-reperfusion injury. Therefore, the present study investigated MPTP dynamics in cardiocytes using Rhodamine 123. The MMP was (93.80±19.53) 30 min subsequent to AsIV treatment, which was significantly higher than that in the H₂O₂ group (P<0.01) (Fig. 5). 5-HD pretreatment prior to AsIV led to an MMP that was significantly lower than that of the AsIV group (P<0.01). These results demonstrated that AsIV may protect cardiocytes by stabilizing the MMP, and this effect may be associated with the mitoK_ATP.

The effect of AsIV on cell apoptosis was analyzed by measuring Annexin V positivity using flow cytometry. Pretreatment with AsIV led to significantly reduced Annexin V-positivity compared with the H₂O₂ group (P<0.01). By contrast, pretreatment with 5-HD resulted in a significant increase in the number of Annexin V-positive cells compared with the AsIV group (P<0.01) (Fig. 6). These results demonstrated that AsIV treatment may reduce apoptosis, and this may be associated with the mitoK_ATP.