Animal experiments were conducted following the guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The present study was approved by the Animal Use and Care Committee of Shandong University. Male Wistar rats weighing 380–430 g, aged ~14 weeks were purchased from the Department of Experimental Animals of Shandong University. The rats were housed in independent ventilation cages at an ambient temperature of 23±1°C and a humidity of 55±5% under a 12-h light/dark cycle. They were allowed access to food and tap water ad libitum.

A previously published rat model of CA was used in the present study. Briefly, rats were anesthetized with 5% isoflurane in room air (21% oxygen) in a plastic induction box. The trachea was orally intubated once the rats were fully anesthetized (no response to pain absence of corneal reflex). The rats were mechanically ventilated and maintained under anesthesia with 2% isoflurane. A PE-50 catheter was advanced through the left femoral artery into the aorta for measurement of the mean aortic pressure (MAP). A microcatheter was inserted into the left femoral vein for drug administration. A conventional lead II electrocardiogram was continuously recorded. Pancuronium bromide (2 mg/kg) was intravenously administered for complete muscle relaxation. Following pancuronium bromide administration, the rats were administered 0.5% isoflurane in room air (21% oxygen) for 5 min.

The rats were randomly allocated to three groups as follows (Fig. 1): i) In the CA+PGE1 group, the rats underwent 8 min asphyxia followed by CPR, and 1 µg/kg Lipo-PGE1 (Qilu Pharmaceutical Co., Ltd.) was intravenously administered at the onset of ROSC (19,20); ii) in the CA group, the rats underwent 8 min of asphyxia and CPR; and iii) in the sham group, the rats underwent the same surgical procedure without PGE1 or asphyxia/CPR. The rat model of asphyxia-induced CA was established as described previously (21,22). Following pancuronium-bromide administration, the rats were administered 0.5% isoflurane in room air (21% oxygen) for 5 min. CA was induced by asphyxia, which occurred after turning off the ventilator and clamping the endotracheal tube. The hypotension and heart rate dropped quickly. CA was defined as a MAP of <20 mmHg. Following 8 min of asphyxia, chest compression (23,24) was initiated at a rate of 200 beats/min with a pneumatically driven mechanical chest compressor (Fig. S1). The depth of compressions (1–1.5 cm) was adjusted to maintain the MAP >25 mmHg during resuscitation. Mechanical ventilation with 100% oxygen and a bolus injection of 10 µg/kg epinephrine via a venous line were initiated 30 sec prior to chest compression. Chest compressions were continued and epinephrine was injected every 5 min until ROSC was achieved. ROSC was defined as the return of supraventricular rhythm with an increase in MAP >50 mmHg for 5 min. Only rats that achieved ROSC within 10 min were used for further studies (25–27). This criterion was to avoid non-homogeneous CA periods leading to pathophysiological differences and subsequently to the need for larger numbers of animals. In this CA model, most of rats could get back to supraventricular rhythm within 5 min after performing CPR and maintain MAP more than 50 mmHg. At 4 h post-ROSC, 21 rats (n=7/group) were euthanized with Euthasol (390 mg/kg pentobarbital sodium and 50 mg/kg phenytoin sodium; intraperitoneal injection) (28). Death was confirmed by the cessation of the heartbeat and respiration. The remaining 30 rats (n=10/group) were monitored for 72-h survival analysis.

Cardiac function was measured using an ultra-high frequency ultrasound system for small animals (Vevo 2100; VisualSonics, Inc.) at baseline and 1, 2, 3 and 4 h post-ROSC. Left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV) and heart rate (HR) were recorded from M-mode images. Ejection fraction (EF) was calculated as EF=[(LVEDV-LVESV)/LVEDV] ×100. Cardiac output was calculated as CO=(LVEDV-LVESV) × HR. All measurements were performed and reviewed by two investigators blinded to group assignment.

For the 72-h survival study, all catheters were removed and wounds were surgically closed at 4 h following ROSC. Rats received a subcutaneous injection of 1 mg/kg buprenorphine to relieve pain when they were returned to their cages. Rats were observed every 2 h within first 24 h after ROSC. Then rats were observed at 48 and 72 h. During the 72-h observation period, gasping or respiratory rate under 5 breaths/min required immediate euthanasia (29). At 72 h, all surviving rats were re-anesthetized with 5% isoflurane and had a vein catheter placed for blood sampling. At the end of the experiment rats were euthanized.

The H9c2 rat cardiomyoblast cell line was obtained from the Cell Bank of the Chinese Academy of Sciences. H9c2 cells were grown in high-glucose DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO₂.

In the present study, H9C2 cells were digested and divided into three groups. The control group was incubated in normal culture medium. The H/R group was treated as described previously (30,31). Briefly, H9c2 cells in glucose-free DMEM (Gibco; Thermo Fisher Scientific, Inc.) were exposed to 1% O₂, 94% N₂ and 5% CO₂ in an anaerobic chamber (Don Whitley Scientific, Ltd.) for 12 h to mimic ischemia. Subsequently, the medium was replaced with high-glucose DMEM and the cells were transferred to a regular incubator at 37°C with 5% CO₂ for 12 h to mimic reperfusion. To study the effects of PGE1 on H/R model, the H/R+PGE1 group was treated with 0.5 µM PGE1 at the start of reoxygenation.

Cardiomyocyte apoptosis was detected in rats using a TUNEL assay using with the in situ Apoptosis Detection Kit (EMD Millipore) according to the manufacturer's instructions. Briefly, the heart tissue was separated and fixed in 4% paraformaldehyde overnight at room temperature. Fixed heart tissue was embedded in paraffin and cut transversely into 5-µm sections. TUNEL reaction mixture (100 µl) was added to cover the tissue section and incubated in a humidified chamber for 1 h at 37°C. The nuclei were counterstained with 0.5% methyl green for 5 min at room temperature. The tissue sections were washed with xylene (three washes; each wash, 2 min) and covered with coverslips using neutral balsam mounting medium. The sections were examined under a BX41 light microscope (Olympus Corporation) and images of selected areas were captured.

H9c2 cell apoptosis was detected using a TUNEL assay with an Apoptosis Assay Kit (Roche Diagnostics), according to the manufacturer's instructions. Briefly, the H9c2 cells were fixed in 4% paraformaldehyde for 1 h at room temperature. The samples were incubated with the TUNEL reaction mixture (50 µl) at 37°C for 60 min. The nuclei were counterstained with DAPI for 5 min at room temperature. The samples were washed in PBS (three washes; each wash, 5 min) and coverslipped in fluorescence mounting medium. The cells were visualized under an IX73 fluorescence microscope (Olympus Corporation) using excitation and emission wavelengths of 540 and 580 nm, respectively. The cells exhibiting red fluorescence were defined as TUNEL-positive apoptotic cells.

The levels of apoptosis in tissue and H9c2 cells were calculated by counting the TUNEL-positive cardiomyocyte nuclei in three randomly selected fields in each slide (magnification, ×400). The levels were reported as a percentage of total cardiomyocyte nuclei.

The mitochondria derived from rat myocardium and/or from H9c2 cells were isolated by differential centrifugation using a Tissue/Cell Mitochondria Isolation Kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Fresh mitochondria were used for the measurement of mPTP opening, while the mitochondria-free cytoplasmic protein extract was used for the measurement of cytochrome c expression.

mPTP opening was measured using a Purified Mitochondrial Membrane Pore Channel Colorimetric Assay kit (Genmed). mPTP opening was induced by 200 µM CaCl₂ and presented as mitochondrial swelling, which results in a reduction of the absorbance at 520 nm (A₅₂₀). The changes in A₅₂₀ at various time points were measured for each sample. The value at −1 min was normalized to the value at 10 min and was used for statistical analysis.

An additional immunofluorescence method was used to measure mPTP opening in H9c2 cells. Briefly, cells were washed with PBS and subsequently stained with 1 µmol/l calcein-AM (Invitrogen; Thermo Fisher Scientific, Inc.) in the presence of 8 mmol/l CoCl₂ (Sigma-Aldrich; Merck KGaA) at room temperature for 20 min in the dark. CoCl₂ was added to quench the cytoplasmic signal so that only fluorescence in the mitochondria was captured. A change in fluorescence intensity corresponded to the level of mPTP opening.

Total protein was extracted from heart tissues and H9c2 cells using 1X RIPA buffer (Sigma-Aldrich; Merck KGaA) with a Protease Inhibitor Cocktail (Sigma-Aldrich; Merck KGaA). The protein concentration was determined using the Pierce BCA Protein Assay (Pierce; Thermo Fisher Scientific, Inc.). Total protein was used to measure the expression levels of GSK3β and caspase-3. Cytoplasmic protein without mitochondria was used to measure cytochrome c expression. The samples with equal amounts of protein (50 µg) were loaded onto 10% sodium dodecyl sulfate-polyacrylamide gels, subjected to electrophoresis and subsequently transferred onto 0.22-µM PVDF membranes (EMD Millipore). Following blocking with 5% nonfat milk for 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4°C: Specific for total GSK3β (1:1,000; cat. no. 12456; Cell Signaling Technology, Inc.), phosphorylated (p)-GSK3β (1:1,000; Ser9; cat. no. 55558; Cell Signaling Technology, Inc.), caspase-3 (1:1,000; cat. no. 9662; Cell Signaling Technology, Inc.), cleaved-caspase-3 (1:1,000; cat. no. 9664; Cell Signaling Technology, Inc.), cytochrome c (1:1,000; cat. no. 11940; Cell Signaling Technology, Inc.). β-actin (1:1,000; cat. no. 60008-1; ProteinTech Group, Inc.) was used to detect reference protein expression. After incubation with an HRP-conjugated anti-rabbit (1:20,000; cat. no. ab205718; Abcam) or anti-mouse secondary antibody (1:20,000; cat. no. ab205719; Abcam) for 1 h at room temperature, the labeled proteins were visualized using the Pierce ECL Western Blotting Substrate (Pierce; Thermo Fisher Scientific, Inc.). Signals from immunoblots were scanned using Amersham Imager 600 imagers (GE Healthcare). The relative band intensities were quantified using the ImageJ software (version 1.52a; National Institutes of Health).

All data are presented as the mean ± SD of three independent experiments. Multi-group comparisons were performed using one-way ANOVA followed by Tukey's post hoc test. Student's t-test was used to compare two groups. The comparisons between time-based measurements within each group were performed using repeated-measures ANOVA. Survival was analyzed using Kaplan-Meier curves and log-rank tests. Two-sided P<0.05 was considered to indicate a statistically significant difference. Statistical analysis was carried out using GraphPad Prism 7.0 (GraphPad Software, Inc.).

In total, 58 rats were used in the present study, of which 51 rats achieved ROSC within 10 min and were included in further studies. Two rats died for the anesthesia induction before CA, and 5 rats were euthanized because that they did not get back to supraventricular rhythm after 5 min CPR or could not maintain MAP more than 50 mmHg for 5 min within 10 min. The mortality rate during model establishment in this study was consistent with the previous report (27).

No significant differences were noted in the parameters body weight, baseline MAP and HR between the treatment groups (Table I). No significant differences were noted in the duration of CA, epinephrine dose, duration of CPR, MAP and HR at 1 h following ROSC between the CA and the CA+PGE1 groups (Table I).

CO and EF were measured in the three treatment groups at baseline and at 1, 2, 3 and 4 h following ROSC (Fig. 2). No significant differences were noted in baseline cardiac function between the groups. CO and EF were significantly decreased within 4 h following ROSC in the CA group compared with the sham group. However, CO was significantly increased following PGE1 treatment, compared with the CA group (P<0.05). A highly significant difference was also noted at 4 h following ROSC when comparing the mean EF values between the CA and CA+PGE1 groups (65.8±16.2 vs. 81.2±9.91%; P<0.01).

The survival rates were monitored for 72 h. Kaplan-Meier survival curves indicated a rapid decline in the survival rate of rats in the CA group within 24 h following ROSC (Fig. 3). The survival rate in the CA+PGE1 group was significantly higher than that in the CA group (P<0.05; Fig. 3).

Myocardial apoptosis was detected using TUNEL staining. In vivo, the levels of apoptosis in the CA group were significantly increased compared with those in the sham group (12.11±4.37 vs. 48.33±19.07%, P<0.05, Fig. 4A). In contrast to these findings, the levels of cardiac apoptosis decreased following treatment with PGE1, compared with the CA group (31.02±13.51 vs. 48.33±19.07, respectively; P<0.05; Fig. 4A). In vitro, the fraction of apoptotic H9c2 cells was significantly higher in the in H/R group than that in the control group (52.13±7.86 vs. 6.38±2.02%, respectively; P<0.01; Fig. 4B). The fraction of apoptotic H9c2 cells in the H/R+PGE1 group significantly decreased, compared with the H/R group (31.20±12.62 vs. 52.13±7.86%, respectively; P<0.01, Fig. 4B).

The mitochondrial swelling assay results demonstrated that the change of OD value A₅₂₀ in the CA group was lower compared with the sham group (0.61±0.11 vs. 0.82±0.13, respectively; P<0.05; Fig. 5A and B), which indicated the level of mPTP opening was increased in the CA group. PGE1 treatment reduced mPTP opening, compared with the CA group (0.76±0.11 vs. 0.61±0.12, respectively; P<0.05; Fig. 5A and B). Moreover, mPTP opening was decreased by PGE1 treatment in vitro, compared with the H/R group (0.77±0.13 vs. 0.58±0.10, respectively; P<0.05; Fig. 5C and D). Consistent with the mitochondrial swelling assay results, the relative fluorescence intensity of calcein-AM was significantly increased in the H/R+PGE1 group, compared with the H/R group (0.72±0.24 vs. 0.41±0.12, respectively; P<0.01; Fig. 5E-H).

GSK3β protein phosphorylation (Ser9) was also analyzed. In vivo, GSK3β phosphorylation decreased in the CA group compared with the sham group (P<0.01; Fig. 6A). GSK3β phosphorylation was higher in the CA+PGE1 group than in the CA group (P<0.01; Fig. 6A). In vitro, GSK3β phosphorylation was decreased in the H/R group (P<0.01), compared with the control group. However, it was partly restored following PGE1 treatment (P<0.01; Fig. 6B).

The cytoplasmic protein levels of cleaved caspase-3 and cytochrome c were significantly increased in the CA group in vivo, compared with the sham group (P<0.01; Fig. 6A). PGE1 treatment partially suppressed these effects (P<0.01; Fig. 6B). The results obtained in vitro were consistent with the in vivo data. Indeed, PGE1 treatment inhibited the increase in the expression levels of cleaved caspase-3 and cytochrome c induced by H/R (P<0.01; Fig. 6B).