All experimental procedures were performed with the approval of the Ethics Committee as well as the Animal Care and Use Committee of Shanghai Chest Hospital affiliated to Shanghai Jiao Tong University.
Bone marrow-derived MSCs-Exo isolation and characterization
Mouse bone marrow-derived MSCs were purchased in Cyagen Biosciences Inc. (Santa Clara, CA, US). When MSCs reached 70%-80% confluency, culture medium was replaced by that containing 5% exosome-free fetal bovine serum and then MSCs were cultured for 48 h. MSCs-Exo were isolated using differential centrifugation using ExoEasy Maxi Kit (QIAGEN, Germantown, MD, US). The morphology of MSCs-Exo was observed using transmission electron microscope. After isolation, 10 µl MSCs-Exo were diluted in 1 ml of filtered PBS and size distribution was measured by Nanoparticle Tracking Analysis (Malvern Instruments Ltd., Malvern, UK). Then, MSCs-Exo were quantified by bicinchoninic acid (BCA) assay (Thermo Scientific) for measurement of total protein. MSCs-Exo were identified by the marker proteins CD63 using western blot. 1 µl MSCs-Exo were dissolved in 100 µl PBS. The diluted MSCs-Exo were pre-labeled by PKH26 (Sigma-Aldrich, San Louis, MO, US) and co-cultured for 12 h in the supernatant of fibroblasts that had reached 60%-70% confluence in the 24-well plate.
Isolation and culture of atrial fibroblasts
Primary atrial fibroblasts were isolated from 1- to 3-day-old BALB/c mice as previously described. Fibroblasts were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in 37°C humidified incubator with 5% CO2. Cells in passage 2–3 were used for in vitro study. Angiotensin II (Ang-II, 100 nM)、MSCs-Exo (50 µg/ml) were used to treat fibroblasts as indicated.
Culture of MSCs
DMEM plus 10% exosome-free FBS and 1% streptomycin/penicillin was used for the culture of MSCs. For transfection of miR-148a-3p inhibitors (100 nM), MSCs were seeded in 6-well plates and transfected with lipofectamine 3000 (Thermo Scientific).
Animals and treatments
8-week-old male BALB/c mice were purchased from Pengyue Laboratory Animal Co, Ltd (Shandong, China). For in vivo study, all animals were anesthetized with 2% isoflurane inhalation before euthanization. 30 mice were randomized into three groups, namely the sham procedure group, Ang-II treatment group, and Ang-II and MSCs-Exo co-treatment (Ang-II + MSCs-Exo) group. An osmotic pump (ALZET 2004, Cupertino, CA, US) filled with Ang-II (1.0 mg/kg/min) or PBS (200 µl) were implanted subcutaneously after anesthesia. In the Ang-II + MSCs-Exo group, 300ug/150ul of MSCs-Exo were injected into tail veins a week before Ang-II pump implantation and twice a week (every Monday and Thursday) for 28 days after establishing the experimental animal model.
Ultrasonic cardiogram analysis
On the 28th day post-Ang-II or saline infusion, echocardiography was performed with ultrasound instrument (Vivid 7, GE Healthcare). Mice were anesthetized with 2% isoflurane inhalation. The size of left atrium size was assessed by apical four-chamber view and was measured at end-ventricular systole from the tip of the mitral valve closure to the base of the LA [10, 11].
Electrophysiological investigation for AF inducibility
Programmed intracardiac stimulation was performed to evaluate AF inducibility as previously described [12, 13]. A 1.1F electrode catheter (Science, Canada) was advanced into the right atrium through the right external jugular vein. A burst of programmed electrical stimulation was used to evaluate the inducibility of AF. Each burst stimulation lasted for 5 s, starting from the cycle length of 50 ms, decreasing in each successive burst by a 2 ms decrement to the cycle length of 10 ms. Following these successive bursts, the incidence of AF and AF duration were analyzed. AF was defined as the occurrence of rapid and irregular atrial excitation lasting at least 1 s, and the time from the end of burst pacing to the first P wave was measured after rapid irregular atrial rhythm. The incidence of inducible AF was calculated as the percentage of the AF-inducible mice divided by the total number of mice.
The hearts were quickly removed under anesthesia.
The left atrium was fix with 4% formaldehyde after washing with PBS. Tissues were imbedded in paraffin and cut into slices of 5 mm thick. The slices were stained with Masson’s trichrome staining to assess the degree of atrial fibrosis. Images were acquired and analyzed by Image-Pro Plus 6.0 software. In each atrium, five visual fields with a magnification of 100× and 200× were tested and analyzed.
The atrial fibroblasts were seeded on confocal dish and were washed twice with PBS. The cells were fixed in 4% paraformaldehyde for 10 min at room temperature. Then, atrial fibroblasts were washed twice with PBS and permeabilized with 0.5% Triton X-100 for 20 min at room temperature. Cells were blocked with western blocking buffer (Beyotime, China) for 30 min at room temperature. The atrial fibroblasts were incubated with anti-α-SMA antibody (CST, 1:200) overnight at 4°C. Subsequently, atrial fibroblasts were washed twice with PBS and incubated with Alexa Fluor 647 (Beyotime, 1:500, China) and were protected from light. Finally, the DNA was stained with DAPI, and images of the cells were captured using a Leica SP5 confocal microscope (Leica Microsystems, Mannheim, Germany).
RNA isolation, PCR and quantitative real-time PCR
Total RNA and miRNA were extracted from cultured atrial fibroblasts and left atrium using RNAeasy Mini Kit (Qiagen, Netherlands) and miRcute miRNA isolation kit (TIANGEN Biotech, China) according to the manufacturer’s instructions. Further detection of miRNA and TGF-β1 levels as well as RNA reverse transcription to cDNA using miRcute Plus miRNA First Strand cDNA Synthesis Kit (TIANGEN Biotech, China) and RevertAid First Strand Kit (Thermo Scientific, USA) were respectively performed according to the protocols. The primers of miR-148a-3p, TGF-β1, ALK5, U6 and GAPDH were designed and synthesized by Sangon Biotech Co., Ltd (Shanghai). The real-time PCR (RT-PCR) assay was performed with the ABI 7500 RT-PCR System (Applied Biosystems, CA, USA). Fold change of miRNA and mRNA were normalized with U6 or GAPDH expression levels using the 2−ΔΔCT method. Primer sequences are listed in Supplemental Table 1.
Public data mining to identify highly expressed microRNAs in MSCs-Exo
We downloaded the two public MSCs-Exo miR abundance data from rat (PMID: 30715852) and from mouse (PMID: 30753344). For the first rat dataset, we first normalized the read count from multiple samples and log transformed the data. Then we selected the highly expressed miR by selected the miR with an expression level higher than the mean plus two standard deviation. For the second mouse study, the highly expressed miR list was obtained from their supplementary table directly with a TPM cutoff at 1000.
MiR-148a-3p targeted gene prediction and luciferase reporter gene assays
We used TargetScan 7.2 (http://www.targetscan.org/) and miRDB (http://mirdb/org/) to predict and analyze the binding site of miR-148a-3p and ALK5 as well as that of miR-148a-3p and Smad2 as shown in Fig. 7. To test if ALK5 and Smad2 were the targets gene of miR-148a-3p, we separately constructed the 3’UTR of wild type ALK5 and Smad2, and then cloned them to the downstream pGL3 plasmid vector of the dual-luciferase reporter gene. Furthermore, we constructed the 3’UTR of mutant type ALK5 and Smad2, which was replaced by the 6 ~ 7 bp synthesized at the seed region of miR-148a-3p and then inserted in the same plasmid vector. HEK 293T cells at their logarithmic growth phase were collected and transferred to the 96-well plate at 2×104 cells/well. The cells were incubated at 37℃ and 5% of CO2 in a cultivation chamber. MiR-148a-3p mimics or inhibitor was transfected for 48 h according to the user’s manual of Lipofectamine 3000 Transfection Reagent. The luciferase activity was tested with the dual-luciferase reporter system (Promega, USA).
Western blot analysis
The mice left atrium tissues were lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing 1% protease inhibitor and phospholipase inhibitor. The protein concentration of each sample was quantified using BCA kit (BioRad, CA, USA). Briefly, electrophoretic separation was performed using a 12.5% SDS-polyacylamide gel and subsequently transferred to a PVDF membrane. Blocking was performed with 5% nonfat milk in Tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature. The antibodies for TGF-β1, alpha-1 type I collagen (COL1a1), connective tissue growth factor (CTGF), Smad2, ALK5, p-ALK5 and GAPDH were purchased from Cell Signaling Technology (Danvers, USA). The membrane was exposed to an enhanced chemiluminescence kit (Millipore Corp, USA) and quantified using Image J software.
Numerical data were expressed as mean ± SEM. Two-tailed Student’s t-test was used for data comparison of two groups with normal distribution, and one-way ANOVA followed by Holm-Sidak’s test was used for multiple-group comparison with normal distribution. Mann-Whitney and Kruskal-Wallis test followed by Dunn’s test were used to compare groups when the data were not normally distributed. Fisher’s exact or Chi-squared tests were used to compare categorical data. A p-value < 0.05 is considered statistically significant.