2.1. Animal studys and mice model of MIRI
All animal experiment procedures after appraisals, feasibility studies were approved by the Shanghai Ninth People’s Hospital institutional ethics committee (SH9H-2017-A39-1). All experimental contents acted in accordance with the guidelines of the Directive 2010/63/EU of European Parliament. Six weeks old male specific pathogen free(SPF) Wild-type (WT) C57BL/6 mice were purchased from Experimental Animal Center of Shanghai Ninth People’s Hospital. The mice were fed normally under SPF conditions in experimental animal center until eight weeks. Mice were firstly induced anesthesia in an anesthesia box with 3% concentration of isoflurane(Sinopharm Chemical reagent co.,Ltd, Shanghai, China) for 1 minites and then mice were maintained anesthesia with 1.5-2% concentration of isoflurane through an anaesthetic mask. After mice were under complete anesthesia, invasive experiments were allowed to carry out. In order to minimize potential errors, animal operations(model of MIRI, Evensblue/TTC stain) were performed by Professor Erhe gao(MD,PhD,Director of Small Animal Surgery and Physiology Core, Lewis Katz School of Medicine, Temple University, USA). Mice were euthanized with excessive carbon dioxide inhalation.
A total of 80 healthy (WT C57BL/6) male mice (eight weeks old, ~ 24 g) were kept under SPF experimental animal feeding surroundings (temperature, 20-24oC; humidity, 50–60), and fed with standard mouse chow and water ad with ad libitum feeding. Mice model of MIRI was operated under anesthesia with 1.5-2% concentration of isoflurane through an anaesthetic mask by ligating the left coronary artery (LCA) for 30 min with a silpknot, then released the slipknot to induce reperfusion. Successful model of MIRI was confirmed by electrocardiograph(ECG) dynamic changes (ST segment elevation). After releasing the slipknot ligating LCA with a certain period of time, that is blood reperfution, hearts were harvested for further studys. A sham operated group was used as a control and underwent the same procedure, with the exception that the knot was left untied. For a detailed description of the procedure, please review Gao et al., 2010 .
2.10. Analysis of exosomes uptake into cardiomyocytes
Exosomes and mice cardiomyocytes were labeled with PKH67 (MINI67, Sigma-Aldrich,USA) and phalloidin (A12379s, ThermoFisher,USA), respectively, according to the manufacturer’s protocol. A total of 5 µL of PKH26-stained exosomes was added to the sample, followed by a 2 hours incubation for internalization by the cell. After washing twice with PBS, slides were fixed in 4% paraformaldehyde and cell nuclears were stained with DAPI (C1005, Beyotime, China). Fluorescent images were collected by using a inverted microscope (BX63, Olympus, Japan) at x̍630 magnification (Fig. 1F).
2.11 Cell transfection
Cells were seeded in a 6- or 24-well plate, and transfected with miR-224-5p mimics, inhibitors, or plasmid DNA using a Lipofectamine 3000 transfection reagent (Cat. 11668019,Invitrogen,USA) following the manufacturer’s protocol. Experiments were performed 24 h after transfection.
2.12 Immunofluorescence assay
Cells cultured on confocal plates were fixed in 4% paraformaldehyde for 15 min, then washed with PBS for 3 times, and cells were permeabilized with 0.5% Triton X-100(C1016, Beyotime, China) for 15 min. Cells were blocked with a primary antibody dilution buffer for 1 hour at room temperature. Next, cells were incubated with mice anti-α-actinin primary antibody (1:1000, ab9465, Abcam,USA) overnight at 4 °C. Primary antibody was removed and the cells were washed twice with PBS. Then, the cells were incubated with a secondary antibody (1:1000, 4409, CST,USA) for 2 hours at room temperature. The cells nuclears were then stained with DAPI for 5 min at room temperature. Fluorescent images were collected by using a inverted microscope (BX63, Olympus, Japan) at x̍630 magnification.
2.13 Dual luciferase reporter assay
HEK 293T cells were cultured in a 24-well plate. When cell confluence reached approximately 70%, cells were transfected with PGL3 luciferase plasmids containing WT, NC and mutated TXNIP 3’UTR and miR 224-5p mimic (Ribobio, Shanghai, China). Then cells were re-cultured in a lucifugal 96well plates 12 hours later. After 36 hours, the ratio of fireflyluciferase and ranilla luciferase was detected using the Dual-GLO™ Luciferase Assay System (E2920,Promega,USA).
2.14 Real-time PCR
Total RNAs were extracted with RNAiso Plus extraction reagent following the manufacturer's instructions (No. 9108, Takara, Dalian, China). Stem-loop primers(purchased from Ribobio Biotech, guangzhou, China) were used to produce cDNA of miRNA by using PrimeScriptTM RT reagent Kit (RR047A, Takara, Dalian, China). The miRNA-cDNA was amplified by using TB Green® Premix Ex Taq™ II (RR820L, Takara, Dalian, China) through ABI-7500 Real-Time PCR Detection System (Applied Biosystems, USA). U6 was used as an internal control.
2.15 Western blotting
Protein was extracted from cardic tissues, cardiomyocytes. 10ug total protain was separated on 12% SDS-PAGE at 80 V for 1.5 hour, and then transferred to a PVDF membrane (IPVH00010,Millipore,USA) at 300 mA for 1 hour. After blocking. Membranes were incubated with primary antibodies at 4 °C overnight and secondary antibodies at room temperature for 1 hour. The primary antibodies used in the study included rabbit anti-TSG101 (1:2000, ab125011, Abcam,USA), rabbit anti-CD9 (1:2000, ab92726, Abcam,USA), rabbit anti-TXNIP (1:1000, ab188865, Abcam,USA), rabbit anti-ASC (1:1000, 67824, CST,USA), rabbit anti-caspase-1 (1:1000, ab179515, Abcam,USA), rabbit anti-GATA4 (1:1000,ab134057,Abcam,USA), rabbit anti-BCL-2 (1:1000, #3498, CST, USA), rabbit anti-BAX (1:1000, #2772, CST, USA), rabbit anti-caspase-3(1:1000, #14220, CST, USA), rabbit anti-α-actinin (1:1000, ab9465, Abcam, USA), and rabbit anti-α-tubulin (1:1000, #2125, CST, USA). Immunoreactive proteins were visualized using ECL substrate kit (ab65623, Abcam, USA) and two-color infrared fluorescence imaging system(Odyssey CLX, LICOR, USA). Tubulin-α was applied as internal control.
2.16 Statistical analysis
Data analysis was performed through SPSS 19.0 software. Shapiro-Wilk test was applied to assess whether data conformed to normal distribution. Statistical analysis was performed using Pearson Chi square test(n ≥ 5) or Fisher exact test(n༜5)with subsequent multiple comparisons using Chi square with Bonferroni correction for categorical variables. One-way ANOVA with subsequent post hoc multiple comparisons using the Student-Newman-Keuls test for continuous variables. Kruskal–Wallis test was applied to nonparametric testing of multiple independent samples and a Dunn-Bonferroni test for post hoc comparisons.