Ethics statement
Animal experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animal (8th Edition, National Institute of Health, 2011) and with the approval of the Animal Ethics Committee of North Sichuan Medical College. Extensive measures were taken to minimize animal suffering in the study.
Myocardial I/R mouse model establishment
Male healthy C57BL/6 wild type mice (aged: 7–11; weighing 18 g; Shanghai SJA Laboratory Animal Co., Ltd., Shanghai, China) were individually caged in the SPF laboratory of 60–65% humidity, at 22–25 °C, under a 12-h light/dark cycle, with ad libitum access to food and water, for 1 week before the experiment. The health status of the mice was monitored prior to the experimentation. The mouse model of I/R was developed by a 30-min occlusion procedure of the LAD and reperfusion based on existing protocols [55]. Briefly, the mice (n = 30) were anesthetized by intraperitoneal injection with 3% pentobarbital sodium (Takara Bio Inc., Otsu, Shiga, Japan) in the induction room, and then supported using oxygen via a small-animal ventilator. A horizontal incision was then made between the fourth and fifth ribs of the left chest to expose the heart and was subsequently sutured with a 6-0 silk knot at the proximal LAD (2–3 mm). The ligation area was white with evident arrhythmia, thus indicative of successful MI injury. The knot was untied 30 min post-MI. Successful reperfusion was validated by epicardial congestion. Another group of ten mice were sham-operated without ligation of LAD as control. In addition, the I/R-rendered mice were further injected with 10 μg of Exo-mimic-NC or 10 μg of exo-miR-182-5p mimic which was dissolved in 10 μL phosphate buffer saline (PBS) prior to use, at the front and outside of the visible injury area [56].
Measurement of cardiac function
Three hours after reperfusion, the mouse cardiac function was assessed via echocardiographic and hemodynamic measurements by left ventricular catheterization. Briefly, mice (n = 3) in each group were intraperitoneally given 50 mg/kg pentobarbital sodium (57-33-0; Shanghai Beizhuo Biotechnology Co., Ltd., Shanghai, China) for anesthesia, intubated, and then mechanically ventilated with oxygen. A catheter was inserted in the left ventricle through the mitral valve for connection with a pressure transducer (AD Instruments, New South Wales, Australia). Data were obtained by a MacLab/4 s data acquisition system (AD Instruments) and a Power Macintosh 7200/90 computer (Apple Inc., Cupertino, CA). End-diastolic pressure in the left ventricle was adjusted to 4–6 mmHg prior to the real-time data recording. Left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-systolic diameter (LVESD), and left ventricular end-diastolic diameter (LVEDD) were measured. The relative data are listed in Supplementary Table 1.
Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) double staining
After 24 h of reperfusion, the mice were anesthetized and the LAD was re-occluded. Evans blue dye (2%) was delivered to the heart via the carotid artery for identification of the ischemic regions. The non-ischemic myocardium was stained blue while the ischemic regions remained unstained. The mice were then euthanized using an intravenous injection of 10% KCl solution. The heart was cautiously harvested and the left ventricle was sliced into 3–4 mm-thick sections. Freshly prepared 5% TTC was utilized to incubate with the sections in dark for 15 min at 37 °C to distinguish the infarcted regions and non-infarcted regions (infarcted regions were not stained, and non-infarcted regions were stained red). Each sample was documented with a digital camera through which the MI size was quantified using the Sigma Scan Pro 4.0 software (Aspire Software International, Ashburn, VA) as described previously [57].
Establishment of the H/R-induced myocardial cell models
Myocardial cells were isolated utilizing the methods as stated in the previous literature [58]. Briefly, the left ventricular tissues, obtained from C57BL/6 suckling mouse pups (aged 1–3 days; Shanghai SJA Laboratory Animal Co., Ltd) were incised into 1 mm3 blocks. Post detachment using 0.1% trypsin, the cells were filtered through a 40 μm cell strainer. After centrifugation, the myocardial cells were isolated and suspended in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL, Life Technologies Inc., Grand Island, NY) replenished with 20% fetal bovine serum (FBS) and 0.1 μM 5-Bromo-2-deoxyuridine (Gibco). Subsequently, the myocardial cells were purified using the differential adherence method, stained with trypan blue, and counted. Myocardial cells (5 × 105 cells/mL) were then seeded into a 24-well plate and subjected to incubation at 37 °C with an incubator (Thermo Fisher Scientific, Waltham, MA) that contained 5% CO2 and saturated humidity.
The purity of myocardial cells was assessed by immunofluorescence detection of cTnI. The culture medium was removed after 3 days of culture, and then myocardial cells were fixed using 4% paraformaldehyde for 10–20 min. The cells were probed employing the polyclonal antibody to cTnI diluted at 1:10 (ab209809, Abcam, Cambridge, UK) and subsequently re-probed with the fluorescein isothiocyanate (FITC)-coupled secondary antibody. After sealing, the myocardial cells were observed under a fluorescence microscope. Six fields were screened at randomization and photographed to count the fluorescence-positive cells as myocardial cells (n1). The total number of cells was counted as N1. The ratio of myocardial cells was expressed as n1/N1 × 100%.
Myocardial cells were then exposed to hypoxic conditions for 24 h in a hypoxic incubator containing a combination of 1% O2, 5% CO2, and 94% N2 followed by reoxygenation for 12 h in an incubator with a combination of 21% O2, 5% CO2, and 74% N2.
Trypan blue staining
Mouse tissue samples were taken, washed, and cut into 1 mm3 tissue pieces, which were then digested with 0.1% trypsin, filtered with a 40 μm pore size filter, and centrifuged. The precipitated cells were resuspended in a medium without phenol red and serum. Next, 5% trypan blue solution prepared using 5 g of trypan blue (T6146-5G, Sigma-Aldrich Chemical Company, St Louis, MO) added with sterile PBS were diluted to ten times with cell culture medium, and mixed with the cells. Lastly, the cells were counted in the captured microscopic images under an inverted microscope within 3 min.
HUVEC culture
The culture of HUVECs supplied by American Type Culture Collection (ATCC; Manassas, VA) was proceeded in Roswell Park Memorial Institute 1640 medium (Thermo Fisher Scientific) containing 20% FBS, 60 μg/mL of endothelial cell growth supplement (BD Biosciences, San Jose, CA), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Thermo Fisher Scientific) at 37 °C under saturated humidity with 5% CO2.
Immunohistochemistry staining
Paraffin-embedded mouse myocardium slices were dewaxed and antigen retrieved by the standardized procedures. Slices were treated with 3% H2O2 in methanol for 20 min to deactivate the endogenous peroxidase. After a 3-min rinse with 0.1 M PBS, the slices were blocked employing normal goat serum (C-0005) sourced from Haoran Bio Technologies Co., Ltd. (Shanghai, China). The rabbit anti-human GSDMD (1: 1000, ab219800, Abcam) served as the primary antibody for overnight incubation at 4 °C. On the following day, re-probing was completed with goat anti-rabbit immunoglobulin G (IgG) (ab6785, 1: 1000, Abcam) for 20 min at 37 °C. Next, incubation at 37 °C with horseradish peroxidase (HRP)-coupled streptavidin solution (0343-10000U, Imunbio Biotechnology Co., Ltd., Beijing, China) lasted for 20 min. The slices were then stained with diaminobenzidine (ST033, Guangzhou Whiga Technology Co., Ltd., Guangdong, China) and counter-dyed utilizing hematoxylin (PT001, Shanghai Bogoo Biological Technology Co., Ltd., Shanghai, China). After immersion in 1% ammonia, the slices were processed with conventional procedures. Finally, the dyed slices were documented utilizing microscopic images.
RNA isolation and quantification
Total RNA content was harvested from the tissues and cells as per the specifications of the TRIzol reagent (Invitrogen, Carlsbad, CA). For mRNA detection, after determination of the RNA concentration, the complementary DNA (cDNA) was generated after RT of RNA content (1 µg) by the PrimescriptTM RT Reagent Kit with gDNA Eraser (RR037A, Takara). For miRNA detection, PolyA Tailing Reverse Transcription Kit (B532451, Sangon Biotech Co., Ltd., Shanghai, China; containing universal PCR reverse primer for miRNA and U6) was adopted for RT, with PolyA-containing cDNA obtained. The polymerase chain reaction (PCR) was performed by an SYBR® Premix Ex TaqTM (Tli RNase H Plus) reagent kit (RR820A, Takara) and an ABI7500 real-time qPCR system (Thermo Fisher Scientific). miR-182-5p was quantitated by real-time qPCR using a TaqMan miRNA Assays Kit (Thermo Fisher Scientific). Primer sequences from GenePharma (Shanghai, China) are presented in Supplementary Table 2. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 were selected as endogenous controls for the GSDMD protein and miR-182-5p respectively. Relative quantification of the target genes was performed based on the 2−ΔΔCt method [59].
Western blot analysis
Total protein was extracted from the tissues and cells by RIPA lysis buffer with a 1% protease inhibitor. After centrifugation, the supernatant was collected. A bicinchoninic acid (BCA) kit (23227, Thermo Fisher Scientific) was adopted for the protein concentration determination. Protein separation was conducted by polyacrylamide gel electrophoresis and then loaded onto the polyvinylidene fluoride membrane. After blockade by 5% skimmed milk, an incubation was conducted with the use of corresponding primary antibodies against apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC) (1: 100, ab18193, Abcam), pro-caspase-1 (1: 1000, ab16883, Abcam), caspase-1 (1: 100, AF4005, Affinity Biosciences, CN), GSDMD (1: 1000, ab209845, Abcam), NLRP3 (1: 500, ab214185, Abcam), interleukin-1β (IL-1β; 1: 100, ab71495, Abcam), CD63 (1: 1000, ab216130, Abcam), heat-shock protein-70 (HSP70) (1: 000, ab2787, Abcam), tumor susceptibility gene 101 (TSG101) (1: 1000, ab125011, Abcam), Alix (1: 1000, ab186429, Abcam), Calnexin (1: 100, ab22595, Abcam), and GAPDH (1: 5000, ab8245, Abcam) overnight at 4 °C. HRP-tagged goat anti-rabbit IgG (1: 20000, ab205718, Abcam) was employed for 90 min of incubation. Subsequently, enhanced chemiluminescence (NCI4106, Pierce, Rockford, IL) was adopted to visualize protein bands. The gray intensity of each band was measured by the ImageJ 1.48U software (Bio-Rad, Hercules, CA). Data were presented as the relative ratio of the gray intensity between the target protein and GAPDH (loading control).
Measurements of LDH release and ROS production
In compliance with the instructions of the LDH assay kit available from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), the tissue block (0.1–0.2 g) was rinsed in ice-cold normal saline to remove the blood, dried with filter paper, loaded into a homogenization tube. Thereafter, the tube was added with homogenization medium (0.86% normal saline) at the ratio of weight (g): volume (mL) =1:9 for homogenization, and the tissues were chopped with small ophthalmic scissors in an ice water bath. The homogenate was produced in a homogenizer. For cells, they were centrifuged and the cell pellet was collected. The cell suspension was centrifuged at 1000 r/min for 10 min, with the supernatant discarded and the cell pellet harvested. The cell pellet was washed and prepared into a homogenate. The absorbance value at 450 nm was measured using a microplate reader.
The measurement of ROS was then conducted. Cells after transfection and myocardial tissues were collected. Myocardial tissues were fixed in 10% formalin, frozen in liquid nitrogen, and preserved at −80 °C. Next, the tissues were loaded on the tissue support with optimum cutting temperature compound-embedded and prepared into 10-μm-thick sections, which were then loaded onto the slides. Afterward, the sections were added with 10 umol/L of reactive oxygen fluorescent probe dihydroethidium diluted by PBS (pH 7.4) for 30 min of incubation at 37 °C, followed by washing with PBS. The cell red emission images through the N21 filter were captured under the fluorescence microscope. For cells, they were digested, washed, and loaded with 15 μM DCFH-DA to intracellularly convert DCFH for 15 min in the dark. The cells were subsequently scraped off in 1 mL of ice-cold PBS whereupon 2 × 106 cells were incubated. LPS-220B spectrofluorometer (Photon Technology International, Bermingham, NJ) was utilized for recording the fluorescence excited at 485 nm and emitted at 535 nm for 20 min. The difference between the end points and the start points was compared for the calculation of the DCF fluorescence units.
Cell viability and apoptosis measurement
Mouse tissue samples were taken, washed, and cut into 1 mm3 tissue pieces, which were then digested with 0.1% trypsin, filtered with a 40 μm pore size filter, and centrifuged to collect the precipitated cells. The cells were resuspended in a medium without phenol red and serum. Calcein-AM/PI Cell Viability/Cytotoxicity Assay Kit (C2015M, Beyotime) was applied for measuring the cell viability rate. Cells were mixed with 1× Assay Buffer, and dyed by 2 μM Calcein-AM and 4.5 μM PI at 37 °C for 30 min. A fluorescence microscope (Olympus IX51) was utilized for observing and Image Pro advanced software for evaluating the average fluorescence intensity [29].
The tissues were prepared into 4-μm-thick sections before experimentation. The apoptosis in cells and tissues was detected using TUNEL Fluorometric Kit (Promega, Madison, WI), with DAPI (D8200, Solarbio, Beijing, China) utilized for nuclear staining. Images were acquired with a fluorescence microscope at a magnification of ×400, and positive cells were counted at a magnification of ×200, with at least ten fields of view checked for each sample [29].
ELISA
IL-1β and IL-18 levels in the supernatant of cells and tissue homogenate were measured by IL-1β (ab100704, Abcam) and IL-18 (ab216165, Abcam) ELISA kits following the provided instructions. The optical density (OD) value at 450 nm was recorded using a Synergy 2 microplate.
Isolation, culture, and identification of MSCs
Well-grown C57BL/6 mice were euthanized and soaked in alcohol for 10 min. The femurs and tibias of the mice were removed in a sterile environment, with the meat discarded using the instrument, and then washed in a plate containing DMEM (Gibco). Both ends of the femur and tibia were removed with clean and sterile scissors, after which the bone marrow cells were delivered using a DMEM-contained syringe into a 15 mL centrifuge tube for 3-min centrifugation at 1500 r/min, followed by removal of the supernatant. Cells were resuspended in a 5% CO2 incubator at 37 °C employing DMEM replenished with 10% FBS (Gibco) and 100 U/mL penicillin-streptomycin (Gibco). After three days, the medium was renewed to get rid of non-adherent cells, and meanwhile, the changes of cell morphology were visualized, photographed, and recorded. The cells of 80–90% confluence were subcultured and those at passage 3 were collected for later use.
MSCs at the 80% confluence were collected, fixed overnight in 4% paraformaldehyde at 4 °C. The cells were directly labeled for 1 h with FITC-coupled antibodies to CD34, CD11b, CD45, CD90, CD105, and CD73 (all sourced from BD Biosciences Pharmingen, San Jose, CA), and then with FITC-coupled goat anti-mouse IgG (1:200; BD Biosciences) for 1 h. A sum of 1 × 104 FITC-marked cells were counted with BD FACS Calibur [60].
In addition, MSCs at passage 3–5 were cultured in OriCell osteogenic, adipogenic, or chondrogenic differentiation medium (Cyagen, Guangzhou, China). Alizarin Red S, Oil Red O, and Alcian Blue staining tests were proceeded for the identification of MSCs by assessing the osteogenic, adipogenic, and chondrogenic differentiation properties [61].
Extraction and characterization of MSC-derived exosomes
MSCs at passage 3 (1 × 107 cells, 1 × 106 cells/mL, 10 mL) were incubated overnight with serum-free DMEM in a petri dish (90 mm). Upon attaining 80–90% confluence, the MSCs were incubated for 24 h, followed by the collection of the supernatant. The supernatant was harvested after sequential centrifugations (at 350×g, 2000×g, and at 12,000×g) for 10 min at 4 °C. The attained supernatant was filtered on a 0.22-μm filter to eliminate any large particles. The pellet was obtained through centrifugation at 120,000×g for 70 min at 4 °C, resuspended in PBS, and purified by repeated centrifugation at 120,000×g for 70 min at 4 °C. The purified pellet (exosomes) was suspended in PBS, and preserved at −80 °C for subsequent experimentation with the exosome output shown in Supplementary Fig. 7 [62, 63].
The exosome surface marker CD63 (ab217345, Abcam) was assayed using flow cytometry. Exosomes were resuspended employing 1 mL PBS containing 1% bovine serum albumin (BSA) at ambient temperature for 30 min. Five minutes post centrifugation, the supernatant was discarded and exosomes were resuspended in EP tubes with 200 µL PBS and subsequently allowed to incubate with the phycoerythrin (PE)-conjugated anti-CD63 antibody, isotype control (PE-conjugated anti-human IgG), or blank control (no-antibody) for 30 min. After that, another 5-min of centrifugation was conducted, followed by resuspension in PBS containing 1% BSA.
TEM observation: the mixture (5 μL) of exosome suspension with 4% paraformaldehyde was added to the carbon-coated grid, and then stained with 1% phosphotungstic acid. Images were acquired using a TEM (HT7830, Hitachi, Tokyo, Japan).
NTA procedures: the collected exosomes were diluted using PBS to 106/mL−109/mL and loaded into the Nanosight NS300 analyzer (Malvern, UK) using a 1 mL syringe for analysis.
Western blot analysis of exosome markers: exosome particles were immersed in RIPA buffer, in which which the protein was quantified utilizing BCA kit (A53226, Thermo Fisher Scientific). The expression of CD63, HSP70, TSG 101, Alix, and Calnexin in exosomes was detected using Western blot analysis.
Dual-luciferase reporter assay
GSDMD 3′UTR sequences carrying putative miR-182-5p binding sites and its mutant sites were independently inserted into a pmirGLO luciferase reporter vector, regarded as the GSDMD-WT or GSDMD-MUT plasmid. The miR-182-5p mimic and mimic-NC were co-transduced with the constructed GSDMD-WT or GSDMD-MUT into the human embryonic kidney (HEK)-293T cells (ATCC; cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin in a 5% CO2, 37 °C incubator). Twenty-four hours post transduction, the cells were lysed. The lysate was centrifuged at 12,000 rpm for 1 min and the supernatant attained here was preserved for use. A Dual-Luciferase® Reporter Assay System (E1910, Promega) was adopted for luciferase activity measurement. Relative luciferase activity was presented as the ratio of Firefly to the internal reference Renilla luciferase.
Cell transfection
H/R-exposed myocardial cells in the logarithmic growth period were seeded into a six-well plate (4 × 105 cells/well). The myocardial cells of 80–90% confluence were manipulated with the overexpression plasmids, short hairpin RNAs (shRNAs), mimics, and inhibitors using Lipofectamine 2000 reagent (11668-019, Invitrogen) in strict compliance with the manufacturer’s instructions. The plasmids (sh-GSDMD, miR-182-5p mimic, miR-182-5p inhibitor, corresponding sh-NC, mimic-NC, and inhibitor-NC) were supplied by GenePharma (Shanghai, China).
MSCs were cultured at a density of 2 × 105 cells/well with the DMEM without penicillin-streptomycin for 24 h. Upon attaining 70–90% confluence, the MSCs were transfected with mimic-NC/inhibitor-NC or miR-182-5p mimic/inhibitor using the TransIT®-2020 transfection reagent (MIR5404, Mirus, Madison, WI). The medium was replaced 6 h following transfection. After 48 h, the MSCs were harvested and used for follow-up experimentation.
Co-culture of MSCs or exosomes with myocardial cells
MSCs were allowed to grow to a confluence of 80–90% after seeding into a six-well plate (1 × 106 cells/well). Then, the MSCs were then treated with 10% GW4869 (an inhibitor of exosome release) (D1692-5MG, Sigma-Aldrich) or 0.005% dimethyl sulfoxide (DMSO) (as the control). Twenty-four hours post-incubation, the MSCs and supernatants were preserved for further use.
MSCs (1 × 104 cells/well) treated with DMSO or GW4869 were cultured in the basolateral compartment of a 24-well transwell system (0.4 μm pore size). The apical compartment contained myocardial cells exposed to H/R and without treatment or transfected with the mimics or inhibitors. Twenty-four hours later, the myocardial cells were attained for miR-182-5p and GSDMD expression pattern characterization. Exosomes were stained with the red lipophilic fluorescent dye PKH26 (MINI67-1KT, Sigma-Aldrich). After staining, the PKH26-tagged exosomes were co-cultured with the myocardial cells at 50–60% confluence seeded into the 24-well plate for 48 h. H/R-exposed myocardial cells were manipulated with sh-NC, sh-GSDMD, or co-incubated with Exo-mimic-NC, exo-miR-182-5p mimic, Exo-inhibitor-NC, or exo-miR-182-5p inhibitor, or co-incubated with exo-miR-182-5p inhibitor and manipulated with sh-GSDMD in combination. Myocardial cells were then harvested and monitored under observation with an inverted fluorescence microscope.
RNA-FISH assay
The myocardial tissues or epidermal tissues of mice embedded in paraffin were sliced into sections, and heated at 65 °C for 4 h and at 73 °C for 2 min. After rehydration using xylene, ethanol, and distilled water, respectively, the sections were treated with SSC for 5 min, then with proteinase K for 30 min and with SSC three times (5 min/time) and finally fixed with formaldehyde for 10 min. Following treatment using ethanol, 1.5 μL of miR-182-5p probes designed by GenePharma (F22101/50; Shanghai, China) were applied for hybridization and a fluorescence microscope was employed for observation.
Statistical analysis
Data processing and analyses were implemented with the application of the SPSS 21.0 statistical software (IBM Corp., Armonk, NY). Measurement data were summarized as mean ± standard deviation. Comparison between two groups was performed using the unpaired t-test. Multigroup comparison was proceeded employing one-way analysis of variance (ANOVA), followed by fTukey’s post hoc test. Differences among multiple groups concerning time-based data were assessed with repeated-measures ANOVA. Pearson’s correlation analysis was utilized to verify the relevance between miR-182-5p and GSDMD expression in the I/R mouse models. Differences were deemed to be significant with the value of p < 0.05.
