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Cell culture
MSCs were purchased from Cambrex BioScience and cultured as previously described [10]. MSCs were cultured on Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FBS, Life Technologies, 16,000), 5 ng/mL fibroblast growth factor (bFGF, PeProTech, 100-18B), and 10 ng/mL epidermal growth factor (EGF, PeProTech, AF-100-15). MSCs at passage 3–4 were used in the current study. Neonatal mouse cardiomyocytes (NMCMs) were isolated and cultured as described previously [26], 37 °C in a 6-well culture plate containing 2 mL/per well Claycomb Medium (Sigma, 51800).
MSC-EXO extraction and characterization
MSC-EXO were isolated and characterized as previously reported [27]. Briefly, when MSCs reached 70–80% confluence, medium was replaced by DMEM supplemented with 10% exosome-depleted FBS (dFBS) (Systems Biosciences) and cultured for 48 h. For hemin pretreatment, MSCs were cultured for 24 h under normoxic conditions in complete medium containing 10 µM hemin. After 48 h, supernatant was collected and EXO isolated and purified by anion exchange chromatography. Subsequently, EXO were suspended in PBS and their concentration determined using a bicinchoninic acid (BCA) kit. The particle size of EXO was analyzed using nanoparticle tracking analysis (NTA). Transmission electron microscopy (TEM) and western blotting for CD63, CD81, TGS101 and Alix were used to characterize the collected MSC-EXO and hemin-MSC-EXO.
Internalization of EXO
MSC-EXO were labeled with Dil (Beyotime) and then co-cultured with NMCMs for 48 h. Next, NMCMs were washed with PBS three times and fixed in 4% paraformaldehyde. The internalized MSC-EXO in NMCMs were evaluated under a fluorescence microscope.
SA-β-gal assay
The senescence of NMCMs was evaluated by SA-β-gal staining (Beyotime, C0602). Briefly, NMCMs cultured in 6-well plates were treated with PBS, MSC-EXO (10 μg/mL) or Hemin-MSC-EXO (10 μg/mL) and then exposed to a serum deprivation/hypoxia (SD/H) challenge (94% N2, 5% CO2, and 1% O2) for 72 h. After washing with PBS three times, NMCMs were fixed for 15 min and incubated with SA-β-gal staining solution at 37 °C without CO2 overnight. Finally, SA-β-gal positive cells, stained blue, were randomly imaged. The percentage of senescent NMCMs was calculated as the ratio of SA-β-gal positive NMCMs to total number of NMCMs obtained from five different fields of view.
MitoTracker staining
The mitochondrial morphology of NMCMs was detected by MitoTracker Green FM (Invitrogen, M7514) according to the manufacturer’s protocol. Briefly, NMCMs were cultured in 24-well plates with cover slides and subjected to different treatments. After washing with PBS three times, they were incubated for 15 min at room temperature with DMEM supplemented with 20 nM MitoTracker Green FM. Finally, NMCMs were washed with PBS and imaged using a confocal microscope. Six fields were randomly observed and at least 300 cells per treatment group counted. The percentage of fragmented mitochondria among total number of cells was calculated.
Transfection of miR-183-5p inhibitor and mimic
miR-control, miR-183-5p mimic and inhibitor were commercially acquired from GenePharma (Shanghai, China). Briefly, 1 × 106 MSCs were plated on a 10-cm culture dish and transfected with 50 nM miR-183-5p mimic, inhibitor or miR-control using Lipofectamine 2000 transfection reagent (Invitrogen, 11668027) according to the manufacturer’s instructions. Subsequently, MSCs were cultured at 37 °C in a 5% CO2 incubator for 48 h and then harvested for further experiments.
Luciferase assay
The 3′-UTR of human HMGB1 was inserted into the pGL3 luciferase reporter vector (Promega, Madison, WI, USA). Mutations in the seed region of the miR-183-5p-binding site in the HMGB1 3′-UTR were generated by PCR. 293T cells were seeded in 24-well plates and then co-transfected with the reporter plasmid (pGL3-HMGB1-3′-UTR or mutant HMGB1-3′-UTR vector) and miRNA control or miR-183-5p mimics using Lipofectamine 2000 (Invitrogen, 11668027). According to the manufacturer’s protocol, luciferase activity was determined 48 h after transfection using a Dual-Luciferase Reporter Assay System Kit (E1910, Promega).
Real-time PCR
Total RNA from MSCs or MSC-EXO was isolated with TRIzol reagent (Takara, 2270A) and reverse transcription performed using a PrimeScript RT Reagent Kit (Takara, RR037A). RT-PCR of miR-183-5p and HMGB1 was performed using a One-Step TB Green® PrimeScript™ RT-PCR Kit (Takara, RR820A). The mouse HMGB1 primer was: F: 5′-GCTGACAAGGCTCGTTATGAA-3′, R: 5′-CCTTTGATTTTGGGGCGGTA-3′. GAPDH and U6 were used as the internal reference. The miR-183-5p and U6 primers were obtained from GenePharma. The expression of miR-183-5p was normalized to the expression of U6 using the 2−ΔΔCt cycle threshold method.
Western blotting
Total protein of differently treated NMCMs and heart tissue from different experimental groups were extracted using a total protein extraction kit (Bestbio, BB-3101). After measuring the concentration using a BCA assay kit (Thermo, 231227), 30 μg protein was resolved by 10% Tris–glycine gel electrophoresis and then transferred onto a PVDF membrane. Subsequently, the membrane was blocked by 5% fat-free milk in TBST and incubated overnight at 4 °C with the following antibodies: anti-CD63 (Abcam, ab134045), anti-CD81 (Abcam, ab109201), anti-TSG101 (Abcam, ab125011), anti-Alix (Abcam, ab186429), anit-Calnexin (Proteintech, 10427-2-AP), anti-p21 (Abcam, ab109199), anti-p53 (Abcam, ab26), anti-p-Drp1 (Ser616) (CST, 3455), anti-Drp1 (CST, 14647), anti-Mfn2 (Abcam, ab124773), anti-Mfn1 (Abcam, ab57602), anti-p-ERK (CST, 9101), anti-ERK (CST, 4695), anti-HMGB1 (Abcam, ab18256) and anti-GAPDH (CST, 2118). Next, after washing three times with TBST, the PVDF membrane was incubated with secondary antibodies (1:1000, CST) at room temperature for 1 h and exposed in a dark room. The quantification of western blotting was analyzed with GAPDH as the internal reference using Image J software (National Institutes of Health, Bethesda, MD, USA) in three independent experiments.
Exosomal miRNA sequencing
Total RNA was extracted from MSC-EXO and Hemin-MSC-EXO using a miRNeasy® Mini kit (Qiagen, 217004). Degradation and contamination of RNA were assessed and the concentration and purity of RNA measured. After cutting into 18-30nt, small RNAs were reverse-transcribed to cDNA and a cDNA library generated. Gene Denovo Biotechnology Co. (Guangzhou, China) sequenced cDNA using Illumina HiSeqTM 2500. Raw reads were further filtered, then microRNA aligned and identified. miRNA expression profiles, miRNA Principal Component, miRNA Expression Pattern Clustering, differentially expressed miRNA (DE miRNA), Target gene Prediction and Target gene functional enrichment were analyzed. DE miRNA was identified through fold change > 1.5 and Q value < 0.001 with the threshold set for up- and down-regulated genes.
MI model and transplantation of MSC-EXO
All animal experiments were approved by the Committee on the Use of Live Animals in Teaching and Research (CULTAR) of the Guangdong Provincial People’s Hospital for Laboratory Animal Medicine (No. KY-Z-2020-486-02). Male C57BL/6J mice (6–8 weeks, 20–25 g) were used for an acute MI model induced by ligation of the left anterior descending coronary artery (LAD) using an 8–0 nylon suture as previously described. After LAD ligation, mice were randomly assigned to one of the following treatments: (1) phosphate-buffered saline (PBS) (MI group, n = 12); (2) 20 μg MSC-EXO (MSC-EXO, n = 11); (3) 20 μg Hemin-MSC-EXO (Hemin-MSC-EXO group, n = 12); (4) 20 μg miR-183-5pKD-Hemin-MSC-EXO (miR-183-5pKD-Hemin-MSC-EXO, n = 12). All MSC-EXO were suspended in 30 μL PBS and intramuscularly injected 30 min after surgery into three sites at the border zone of the infarcted mouse heart. Another group of mice underwent thoracotomy without LAD ligation and served as the sham group (n = 6). Cardiac function in each mouse was assessed by transthoracic echocardiography (Ultramark 9; Soma Technology, Bloomfield, CT, USA) at baseline (before MI), and 1 and 28 days following MI. Left ventricle ejection fraction (LVEF) and left ventricle fraction shortening (LVFS) were calculated.
Masson’s Trichrome staining
After measuring heart function at 28 days post-MI, all mice were sacrificed and the hearts quickly harvested. After washing with PBS three times, hearts were fixed, embedded and sectioned into 5 µm slices. Masson’s Trichrome staining was performed on heart sections from different groups. Images from 6 mice for each group were captured by scanning electron microscope (SU8010, Japan) and analyzed using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA). The percentage infarct size was determined as the sum of infarcted area from all sections/the sum of LV area from all sections × 100%.
TEM assay
Mice heart tissue was harvested from the different experimental groups and fixed in 2% glutaraldehyde for 24 h. Samples were washed three times with cold 0.1 M phosphate buffer and fixed in phosphate acid buffer supplemented with 1% osmic acid for 2 h at room temperature. After dehydration with a gradient ethanol solution, samples were infiltrated with acetone-epoxy resin, embedded in epoxy resin and finally placed in an oven at 70 °C to polymerize. Embedded samples were sectioned into ultrathin sections (100 nm thickness) using a Leica EM UC7 microtome. Subsequently, sections were stained with 5% uranyl acetate for 10 min and Reynold’s lead citrate for 5 min. Finally, five randomly selected areas of mitochondria on each slide were captured using a 40–120 kV transmission electron microscope (Hitachi H600 Electron Microscope, Hitachi, Japan). Mitochondrial size (μm2) was analyzed with Image-Pro Plus software with measurements taken of at least 1000 mitochondria from six mice hearts in each group. Size < 0.6 μm2 was classified as mitochondria undergoing fragmentation.
Immunohistochemistry
To determine cardiomyocyte senescence in the heart tissue from different groups, heart sections were immunohistochemically stained with anti-Troponin (1:100; Abcam, ab209809) and anti-p21 (1:100; Abcam, ab109199). To determine the blood vessel density, the heart sections were immunohistochemically stained with anti-CD31 (1:100; Abcam; ab19898). Five randomly selected areas on each slide were photographed under a fluorescence microscope (n = 6 per group). The percentage of Troponin/p21 double-positive cells was calculated per DAPI positive cells. The capillary density was expressed as the average number of CD31-positive blood vessels per field.
Statistical analysis
Data are expressed as mean ± SD. Statistical analyses were performed using Prism 5.04 Software (GraphPad Software for Windows, San Diego, CA, USA). Comparison between two groups was analyzed by unpaired Student’s t-test and between multiple groups by one-way ANOVA followed by the Bonferroni test. A p value < 0.05 was considered statistically significant.
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