RAW 264.7 mouse macrophages (SCSP-5036) were provided by the Chinese Academy of Sciences. Dulbecco’s modified Eagle medium (DMEM, C11995500BT), penicillin-streptomycin (15140122), collagenase type II (17101015), trypsin (25200072), and Lipofectamine 3000 transfection kit were purchased from Thermo Fisher Scientific. GM-CSF (51048-MNAH) and IFN-γ (50709-MNAH) were purchased from Sino Biological. Exosome-depleted fetal bovine serum (C3801-0050) was provided by VivaCell BIOSCIENCES. Fetal bovine serum (1913444) was provided by Biological Industries. Antibodies, including anti-CD63 (ab193349), phalloidin-iFluor 488 (ab176753), and anti-CD9 (ab92726), were purchased from abcam, and calnexin polyclonal antibody (10427-2-AP) was purchased from proteintech. Antibodies, including phospho-histone H3 (9713), ki-67 (9129), α-actinin (69758), phospho-Jak2 (3771), phospho-Stat3 (9145), and GAPDH (5174), were all purchased from Cell Signaling Technology. Antibodies, including anti-CD81 (sc-166029) and anti-IL-6Rα (sc-373708), were purchased from Santa Cruz Biotechnology. Peroxidase AffiniPure goat anti-mouse IgG (115-035-003) and peroxidase AffiniPure goat anti-rabbit IgG (111-035-003) were purchased from Jackson ImmunoResearch. Small interfering RNA (siRNA) was purchased from Genepharma.
Small interfering RNA
Mice and MI model
Male C57BL/6J mice aged 8–10 week were purchased from Guangzhou Dean Gene Technology Co., Ltd. and bred in the Animal Center of the Second Affiliated Hospital of Guangzhou Medical University. C57BL/6J mice aged 1–3 days were purchased from Guangzhou University of Chinese Medicine. All animal projects complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and they were approved by the Animal Care and Use Committee of Guangzhou Medical University (Ethics Number: 2019-014).
Mice were anesthetized with 2% isoflurane and connected to a small animal ventilator. The heart was exposed at the left third rib of the chest, and the left anterior descending coronary artery was ligated with 9 − 0 polyester suture. The anterior wall of the left ventricle became white, and the periapical beat weakened.
Induction of M1 macrophages
After the macrophages were grown to a density of 60–70%, the M0 macrophage group was added with DMEM containing 5% exosome-depleted FBS, while the M1 macrophage group was added with DMEM containing 5% exosome-depleted FBS, mouse GM-CSF (50ng/mL) and mouse IFN-γ (20ng/mL) for 24 h.
Culture and treatment of primary cardiomyocytes
The hearts of C57BL/6 neonatal mice aged 1–3 days were taken out and placed in pre-cooled PBS, and the atrial appendages and atria were removed to preserve the ventricles. The ventricular tissue was torn and pressed into thin tissue pieces, which were digested with 0.125% trypsin at 4°C for 12–13 h. An appropriate amount of collagenase II digestion solution was added, and the mixture was shaken and digested in 37°C water bath for 8 min. It was quickly and vigorously pipetted until no obvious tissue could be observed, and then the mixture was shaken and digested again for 8 min. It was vigorously pipetted until no tissue was visible and then centrifuged at room temperature at 950 rpm for 3 min. An appropriate amount of DMEM with 10% FBS and 1% penicillin-streptomycin was added to resuspend the cells and placed in the incubator for 1.5 h twice (37°C, 5% CO2, 95% air). The unattached cells were collected, centrifuged at 950 rpm for 3 min at room temperature, added with DMEM containing 10% FBS, 1% penicillin-streptomycin, and 100 uM Brdu to resuspend the cells. Next, they were transferred to a six-well plate, mixed well, and incubated for 48 h (37°C, 5% CO2, 95% air). The cardiomyocytes were treated with IL-6 at 50 ng/mL.
Isolation of exosomes
The medium of M0 macrophages and M1 macrophages was collected separately, and the exosomes in the medium were isolated by differential ultracentrifugation. The collected medium was centrifuged in 50 mL centrifuge tubes at 4°C for 300×g for 15 min, 2,000×g for 30 min, and 10,000×g for 60 min. The supernatant was transferred and centrifuged at 100,000×g for 90 min at 4°C. The pellets were resuspended with 10 mL of pre-cooled PBS and centrifuged again at the same speed. The pellets were exosomes, and they were resuspended in 200 µL pre-cooled PBS.
Uptake and treatment of exosomes
The operation was in accordance with Sigma’s PKH26 kit instructions. Four µL of PKH26 ethanolic dye solution was added to 1 mL of Diluent C, and the exosomes was resuspended with the mixture. After 5 min of incubation at room temperature in the dark, the reaction was terminated by adding 2 mL of 0.5% BSA. An appropriate amount of medium was added, and the mixture was centrifuged twice at 100,000×g for 90 min at 4°C to remove unbound dye. The labeled exosomes were resuspended in DMEM containing 5% exosome-depleted FBS, added to cardiomyocytes at 2 × 109/mL, and placed in a cell incubator (37°C, 5% CO2, 95% air) in the dark for 10 h. After the cells were fixed with 4% paraformaldehyde, the cytoskeleton was stained with phalloidin-iFluor488 at room temperature for 30 min in the dark, and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) at room temperature for 10 min in the dark. After the excess antibody was washed, an anti-fluorescence quencher was added, and the uptake was observed and photographed under a laser confocal microscope.
The exosomes were isolated in accordance with step 2.5, and their particle size was calculated by nanoparticle tracking analysis (NTA). Then, the concentration of exosome resuspension was calculated. The exosomes were added to DMEM containing 5% exosome-depleted FBS at 2 × 109/mL and co-cultured with cardiomyocytes for 24 h.
After the cardiomyocytes adhered and grew, the cells were transfected in accordance with the instructions of Lipofectamine 3000 transfection kit. The concentration of siRNA in the negative control group and miR-155 mimic group was 100 nM, while the same amount of PBS was added in the mock group. The transfection mixture was added into the target well and incubated for 8 h. Then, the medium was changed to DMEM containing 10% FBS and 1% penicillin–streptomycin and cultured for 40 h.
The sections were placed in xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% alcohol for 5 min, 75% alcohol for 5 min, and distilled water for washing. Then, they were placed in a retrieval box filled with EDTA antigen retrieval buffer for antigen retrieval in a microwave oven. They were heated for 8 min to boiling, heating was stopped for 8 min, and then heating resumed for 7 min. The sections were placed in PBS and washed them three times for 5 min after cooling. They were sealed at 3% BSA for 30 min after drying. After the primary antibody was added, the sections were placed flat in a humidified box at 4°C overnight. After washing three times with PBS, secondary antibody was added to cover the tissue, and incubated at room temperature for 50 min in the dark. The DAPI was incubated for 10 min in the same manner. A spontaneous fluorescence quenching reagent was added for 5 min and rinsed with running water for 10 min. After mounting with anti-fade mounting medium, images were observed and collected under a fluorescence microscope.
After soaking in 4% paraformaldehyde for 20–30 min, 0.5% TritonX-100 was added to permeabilize for 20 min at room temperature. Then, 5% BSA was blocked at room temperature for 1 h, and primary antibody was added to incubate at 4°C overnight. Incubation was performed with fluorescent secondary antibody for 1–2 h at room temperature, and then DAPI was added and incubated at room temperature for 10–15 min. Finally, images were observed and taken under a fluorescent inverted microscope.
Cells and exosome proteins were lysed on ice, and the cell membranes were disrupted by ultrasound to release the proteins. Then, they were centrifuged at 4°C at 12,000 rpm for 15 min. The protein sample concentration was measured by BCA method, and the concentrations of proteins of each group were adjusted to the same concentration. The proteins were placed at 100°C for 5 min and cooled on ice for 1 min. Subsequently, 10% gels were prepared and loaded in sequence for vertical protein electrophoresis. The amount of protein in each well was 15–30 ug. The PVDF membrane was activated with methanol and then transferred with a sandwich transfer clip. It was blocked by adding 5% nonfat dry milk and shaken slowly at room temperature for 1 h. The target band was placed into the corresponding primary antibody and incubated overnight at 4°C at 40 rpm. The corresponding secondary antibody was selected in accordance with the source of the primary antibody species and added to the corresponding target band to incubate at room temperature at 40 rpm for 1 h. After exposure and development in the darkroom, the corresponding target protein name, molecular weight, grouping, and other information were marked on the film, and the data were saved and analyzed statistically.
RNA extraction, reverse transcription, and RT-qPCR
One mL of TRIzol reagent was added to cells or exosomes and lysed for 10–15 min on ice. The cells were gently pipetted down with a sterile enzyme-free pipette tip and collected into a 1.5 mL EP tube. Then, 200 µL of chloroform was added to the lysate and shaken up and down to mix well. After standing at room temperature for 10 min, the lysate was centrifuged at 4°C for 12,000×g for 15 min. The liquid could be observed to be divided into three layers. It was then tilted 45°, and the upper transparent liquid was slowly pipetted into a new EP tube. Next, 500 µL of isopropanol was added, let stand at room temperature for 10 min, and centrifuged at 4°C for 12,000×g for 10 min. A white precipitate (RNA) could be seen at the bottom. Afterwards, 1 mL of pre-cooled 75% ethanol was added, the precipitate was dissolved, and centrifugation was performed at 4°C for 7500×g for 5 min. After air drying was conducted for 5–10 min, an appropriate amount of DEPC was added to resuspend the RNA, and a microplate reader was used to measure the RNA absorbance (A260/280) and concentration. Reverse transcription and RT-qPCR were performed in accordance with the instructions of TaKaRa’s Prime Script RT reagent kit (Perfect Real Time) and TB Green Premix Ex Taq II (Tli RNaseH Plus) kit.
Dual luciferase reporter assays
When the growth density of HEK293T cells reaches approximately 50–60%, co-transfection of siRNA and plasmids was carried out following the instructions of Lipofectamine 3000 reagent. The siRNA of the negative control and miR-155 mimic were transfected at 20 nM, while plasmids were transfected at 150 ng. The transfection mixture was added to the corresponding groups and placed in an incubator for 6–8 h, and then the medium was replaced with DMEM containing 10% FBS and 1% penicillin–streptomycin for another 40 h. The cells were removed, and the operation was performed in accordance with the instructions of the dual luciferase reporter assay kit. First, 65 µL of prepared 1 × PLB was added to the cells, which were then shaken slowly at room temperature for 15 min. Next, the cell lysate was collected, and 50 µL of prepared LAR II and 10 µL of cell lysate were added to the detection tube. Afterwards, the firefly fluorescence value was measured using the detector and recorded as Read1. After the detection, 50 µL of 1 × Stop & Glo was added to the detection tube and mixed well to detect the fluorescence value of Renilla immediately. The fluorescence value of Renilla, recorded as Read2, was used as an internal reference for data analysis with Read1/Read2.
Statistical analysis of data was performed on GraphPad Prism 8.0 software, and all data are expressed as mean ± standard error. Comparisons between two groups were performed using T test, and comparisons between multiple groups were performed using one-way ANOVA or two-way ANOVA. If statistical significance was found, the Bonferroni method was used for further statistical analysis, and the difference was considered to be statistically significant when P < 0.05.