Myocardial ischemia and reperfusion injury mice model
The animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985). All experimental procedures involving animals were approved by the Ethics Committee of the Animal Care and the Ethics Committee of Sichuan University. For the type 2 diabetic mouse model, 8- to 10-week-old male C57BL/6J mice were fed a high-fat diet for 4 weeks and then given a single intraperitoneal injection of STZ (90 mg/kg, Sigma). One week after STZ injection, mice with a 12-hour fasting blood glucose level ≥ 11.1 mmol/L were considered to have developed diabetes mellitus (DM)[15,16]. For the MIRI model, the left anterior descending (LAD) coronary artery was ligated with a 7–0 suture for 1 hour and then reperfused for 24 hours. For the sham group, a similar surgical procedure was performed, but the LAD coronary artery was not ligated. RvD1 (Cayman Chemical) was administered at a dose of 5 µg/kg/day via intraperitoneal injection.
Echocardiography analysis was performed 14 days after MIRI surgery. Mice were anaesthetized with isoflurane, and cardiac function was assessed using a Vevo 3100. Measurements were performed at the midpapillary level from well-aligned M-mode images from the parasternal short axis view.
TTC staining and Evans blue staining
Following MIRI (1 h ischemia/24 h reperfusion), 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma-Aldrich) and Evans blue (Sigma-Aldrich) dye were applied for measurement of myocardial infarct size. Briefly, at the end of reperfusion, 1 mL 1% Evans blue was injected through the abdominal aorta after re-ligation of the LAD. The heart was then extracted and sectioned once it hardened at −20 °C, cut into slices (2–3 mm), and incubated in TTC staining solution for 15 minutes at 37°C. Subsequently, the slices were fixed in 4% formaldehyde for 1 hour. Generally, the area stained with Evans blue represented the non-MIRI myocardium, whereas the unstained area was the MIRI myocardium, the red area was at risk, and the white area indicated infarct size. Images were captured using a camera, and the area of the infarcted myocardium as a percentage of the risk area was analysed using ImageJ software.
Vascular permeability analysis
Vascular permeability was evaluated with Evans blue staining as previously reported[17-19]. In brief, following MIRI, at 3 hours before termination of the experiment, Evans blue dye (20 mg/kg) was injected intravenously, and the mice were then euthanized and immediately subjected to aorta perfusion with phosphate buffered saline (PBS). The hearts were then excised, and Evans blue dye was eluted in formamide for 18 hours at 70°C. The absorbance of Evans blue dye at 620 nm was measured with a Spectra Max 250 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA)
Human cardiac microvascular endothelial cells (HCMECs) were purchased from Shanghai Huzhen Biotechnology. HCMECs were cultured in DMEM containing 10% foetal bovine serum and 1% penicillin/streptomycin in an incubator with 5% CO2 at a temperature of 37°C. The cells were cultured with a final concentration of 30 mM glucose as a HG concentration, and 5.5 mM glucose served as a control. Permeability across endothelial cell monolayers was measured in Transwell units (0.4 µm pore; Corning Costar) under the following conditions in the presence or absence of RvD1 (100 ng/ml): (1) Hypoxic conditions (5% CO2, 94% N2, and 1% O2) used fresh DMEM with 1% FBS to mimic ischemic conditions for 12 hours, reoxygenation in a 5% CO2 incubator and DMEM containing 10% foetal bovine serum and 1% penicillin/streptomycin for another 12 hours, (2) H2O2 (400 μM) for 12 hours, (3) LPS (500 ng/ml) for 12 hours. FITC-dextran (0.5 mg/ml, average molecular mass 40,000; Sigma) was added as a fluorescent indicator to the upper chamber for 2 hours before the end experiments. A 100 μl sample was taken from the lower chamber, and the fluorescence of FITC-dextran was measured (485/535 nm, absorption/emission wavelengths) with a Spectra Max 250 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).
At the end of the cellular experiments, the medium was removed, and the cells were washed with PBS 3 times, fixed in 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100 in PBS. Then, 5% bovine serum albumin in PBS was used to block cells for 15 min. A primary antibody against VE-cadherin overnight and a secondary antibody for 30 minutes were used for immunostaining. DAPI was used to stain nuclei for 15 minutes, and images were captured with a confocal microscope (N-STORM & A1, NiKON).
HCMECs were seeded in 24-well plates at a density of 5 × 10 4 /ml. The cells were cultured with HG, and the 5.5 mM glucose served as a control. The cells were pretreated with RvD1 (100 ng/ml) for 12 hours, followed by 400 μM H2O2 for 4 hours. Then, the cells were washed three times with warm Hank's Balanced Salt Solution (HBSS). MitoSOX-Red (4 μM, Thermo Fisher Scientific) dye was added to each well, incubated with the cells for 30 minutes in the dark at 37°C, and then washed away three times with warm HBSS. Finally, fluorescence intensity was determined using a Celigo imaging cytometer, and images were captured with a confocal microscope (N-STORM & A1, NiKON).
Mitochondrial transmembrane potential (MMP) assay
Mitochondrial membrane potential detection was performed using JC-1 (Med Chem Express HY-K0601). According to the manufacturer’s instructions, JC-1 (2 μM) was added to each well and incubated with the cells at 37°C for 20 minutes. The cells were then centrifuged for 3 minutes at 400 g, washed twice with PBS and finally resuspended in 500 μL JC-1 fluorescence, which was measured with a flow cytometer.
HCMECs or heart tissue were lysed using lysis buffer (Beyotime) with protease inhibitors (CST). Protein lysates were separated via SDS‐PAGE, transferred to a PVDF membrane, and blotted with antibodies against the target proteins [VE‐cadherin (BIOSS), 1:1000 dilution; GAPDH (Affinity Biosciences), 1:1000 dilution], followed by incubation with a goat horseradish peroxidase (HRP)‐conjugated antibody (1:10,000 dilution). Bands were detected using an ECL system.
For immunostaining of heart sections, hearts were excised from mice after euthanasia, fixed in 4% paraformaldehyde overnight, embedded in paraffin, and sectioned. The sections were then deparaffinized, rehydrated, retrieved for antigens, and subjected to antigen retrieval. Immunohistochemical analysis of CD45 (Abcam) and CD68 (Abcam) expression was performed, followed by staining with a secondary antibody (goat anti‐rabbit antibody). Images were captured with a ZEISS IX83 microscope. For Masson staining, heart sections were deparaffinized, stained with Masson's composite staining solution and a bright‐green staining solution and dehydrated in absolute ethanol. Acetic acid was used for washing. Finally, the sections were subjected to transparency in xylene and then mounted with neutral gum for analysis. The smooth muscle cell cytoplasm was stained red, and the collagenous fibrous tissue was stained blue by Masson staining. Images were captured using ZEISS IX83.
Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from tissues using TRIzol (Invitrogen). After extraction using chloroform and precipitation with isopropanol, the RNA was washed twice with 75% ethanol, and the RNA concentration was measured after the sample was dissolved in RNase-free water. Subsequently, the RNA was reverse transcribed using a PrimeScript RT Reagent Kit (Takara). RT-PCR assays were performed on a CFX96TM Real-time PCR Detection System (BioRad) using an EvaGreen Supermix Kit (Bio-Rad). Relative mRNA expression was normalized to GAPDH expression. The RT-PCR primer sequences are shown in Table 1.
Transmission electron microscopy (TEM)
Heart tissues were harvested and quickly fixed in 2.5% glutaraldehyde for 1 hours, exposed to 1% osmium tetroxide, sectioned to 60 nm, mounted on Cu grids contrasted with uranyl acetate and lead citrate, and analysed using a JEM-1400 electron microscope.
All experimental data are presented as means ± SD. Differences between 2 groups of variables were compared using a two‐tailed, unpaired t-test. One-way analysis of variance followed by Bonferroni’s post hoc test was performed to compare more than three groups. A value of P < 0.05 was considered statistically significant.