Animals
Male Wistar rats weighing 380–430 g 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.
Asphyxia-induced CA model establishment and treatments
Rats were anesthetized with 5% isoflurane in room air (21% oxygen) in a plastic induction box. The trachea was orally intubated. 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 (ECG) was continuously recorded. Pancuronium-bromide (2 mg/kg) was intravenously administered for complete muscle relaxation. After pancuronium-bromide administration, the rats were administered 0.5% isoflurane in room air (21% oxygen) for 5 min.
Rats were randomly allocated to three groups (Fig. 1): (i) CA+PGE1 group: rats underwent 8 min of asphyxia followed by CPR, and lipo-PGE1 (1 μg/kg, purchased from Qilu Pharmaceutical, Jinan, China) was intravenously administered at the onset of ROSC (Fang et al, 2010; Zhu et al., 2017); (ii) CA group: rats underwent 8 min of asphyxia and CPR; and (iii) sham group: rats underwent the same surgical procedure without asphyxia or CPR. The rat model of asphyxia-induced CA was established as described previously (Kim et al, 2016; Wei et al, 2019). After 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. Cardiac arrest was defined as a MAP of <20 mmHg. After 8 min of asphyxia, chest compression was started at a rate of 200 beats/min. The MAP was maintained at >25 mmHg during resuscitation. Mechanical ventilation with 100% oxygen and a bolus injection of epinephrine (10 μg/kg) via a venous line were initiated 30 s before 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 mean artery pressure of more than 50 mmHg for 5 min. Only rats that achieved ROSC within 10 min (n = 42) were used for further study. At 4 h after ROSC, 21 rats (n = 7/group) were sacrificed and the hearts were collected. The remaining 21 rats were used for 72-h survival analysis.
Cardiac function monitoring
Cardiac function was measured using an ultra-high frequency ultrasound system for small animals (Vevo 2100; Visual Sonics, Toronto, Canada). 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) and cardiac output (CO) were calculated as follows: EF = (LVEDV–LVESV)/LVEDV ×l00%; CO = (LVEDV–LVESV) × HR. All measurements were reviewed and confirmed separately by two investigators.
Survival analysis
For the 72-h survival study, all catheters were removed and wounds were surgically closed at 4 h after ROSC. The rats were then returned to their cages and closely monitored during 72 h.
Hypoxia-reoxygenation (H/R) cell model establishment
Rat cardiomyoblasts (H9c2) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were grown in high-glucose DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in an atmosphere of 5% CO2 in air.
H/R procedures were performed as described previously (Huang et al, 2016; Pu et al, 2019). Briefly, H9c2 cells in no-glucose DMEM were exposed to 1% O2, 94% N2, and 5% CO2 in an anaerobic glove box (Don Whitley Scientific, Bingley, UK) for 12 h to mimic ischemia. Then, the medium was replaced with high-glucose DMEM and the cells were transferred to a regular incubator (95% air, 5% CO2, 37°C) for 12 h to mimic reperfusion. H9c2 cells were treated with PGE1 (0.5 μM) at the start of reoxygenation.
Terminal deoxynucleotide nick-end labeling (TUNEL) assay
Cardiomyocyte apoptosis in rats was detected by a TUNEL assay using an In Situ Apoptosis Detection Kit (Millipore, MA, USA) according to the manufacturer’s instructions. Heart tissue sections were stained with 3-3¢-diaminobenzidine to detect TUNEL-positive cells, then counter-stained with methyl-green and examined with light microscopy (Olympus BX41, Tokyo, Japan).
H9c2 cell apoptosis was detected by a TUNEL assay using an Apoptosis Assay Kit (Roche, CA, USA) per the manufacturer’s instructions. After TUNEL staining and DAPI staining, the cells were imaged under a fluorescence microscope (Olympus IX73, Tokyo, Japan) using 540-nm excitation and 580-nm emission. Cells exhibiting red fluorescence were defined as TUNEL-positive, apoptotic cells.
The levels of apoptosis were calculated by counting TUNEL-positive myocyte nuclei in three randomly selected fields (400×) in each slide. The levels were reported as a percentage of total myocyte nuclei.
Measurement of mPTP opening
Mitochondria of rat myocardium or H9c2 cells were isolated by differential centrifugation using a Tissue/Cell Mitochondria Isolation Kit (Beyotime, Beijing, China) according to the manufacturer’s instructions. Fresh mitochondria were used for the measurement of mPTP opening, and the rest components (cytoplasmic protein without mitochondria) were used for the measurement of cytochrome c.
mPTP opening was measured using a Purified Mitochondrial Membrane Pore Channel Colorimetric Assay kit (Genmed, Shanghai, China). mPTP opening causes mitochondrial swelling, which results in a reduction of the absorbance at 520 nm (A520). Changes in A520 at various time points were measured for each sample. The value at –1 min normalized to the value at 10 min was used for statistical analysis.
For H9c2 cells, we used an additional immunofluorescence method to measure mPTP opening. In brief, cells were washed with phosphate-buffered saline and then stained with 1 μmol/l calcein-AM (Invitrogen, Eugene, OR, USA) in the presence of 8 mmol/l CoCl2 (Sigma, MO, USA) 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 is an index of mPTP opening.
Western blotting analysis
Total protein was extracted from heart tissues and H9c2 cells. Total protein was used to measure the expression of GSK3β and cleaved-caspase3. Cytoplasmic protein without mitochondria was used to measure cytochrome c expression. Samples with equal amounts of protein (50ug) were loaded onto 10% sodium dodecyl sulfate–polyacrylamide gels, subjected to electrophoresis and subsequently blotted onto 0.22 μM polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocking with 5% nonfat milk, the membranes were incubated with the following primary antibodies: anti-total GSK3β (#12456, Cell Signaling Technology, USA), anti-phospho-GSK3β (Ser9, #55558, Cell Signaling Technology), anti-cleaved caspase-3 (#9664, Cell Signaling Technology, USA), anti-cytochrome c (#11940, Cell Signaling Technology, USA). Anti-β-actin (60008-1, Proteintech, China) was used to detect reference protein expression. Relative band intensities were quantified using the ImageJ software.
Statistical analysis
Data are expressed as the mean ± SD of at least three independent experiments.
Group comparisons were performed by one-way analysis of variances (ANOVA) with Tukey’s post hoc test or Student’s t-test. Comparisons between time-based measurements within each group were performed by repeated-measures ANOVA.
Survival analyses were conducted based on Kaplan–Meier plots and log-rank tests. Two-sided P-values less than 0.05 were considered statistically significant. Statistical analyses were performed using GraphPad Prism 7.0.