All animal experiments were approved by the Ethic Committee of Soochow University (reference number: SZUM2008031233) and were conducted according to institutional animal ethics guidelines for the Care and Use of Research Animals established by Soochow University, Suzhou, China. Forty rats were purchased from the Laboratory Animal Center of Nanjing University (Nanjing, China) and maintained under specific pathogen free conditions.
Isolation, culture, and characterization of bone marrow MSCs
Bone marrow-derived mesenchymal stem cells (BM-MSCs) were isolated and cultured as described previously . Briefly, bone marrow was flushed from femora of SD rats. Cells were collected by centrifugation, seeded onto a culture dish, and cultured in DMEM/F12 supplemented with 10% fetal bovine serum (FBS). The culture medium was refreshed every 48 h and the colonies of MSCs should appear in 8–10 days. The phenotype of MSCs was identified by flow cytometry at passage 4, using antibodies against rat CD90-APC, CD11b/c-FITC, and CD45-PEcy7.
Hypoxia conditioning of MSCs in vitro
For hypoxic culture, MSCs were cultured in a tri-gas incubator (Thermo Fisher Scientific, Marietta, OH, USA) composed of 94% N2, 5% CO2, and 1% O2. To determine the optimal duration of hypoxic treatment, cells were cultured for 6, 12, 24, and 48 h under hypoxic and normoxic conditions. MSCs were then harvested for flow cytometric analysis of apoptosis and proliferation.
MSCs (3 × 105 cells) were seeded onto the 12 wells of a 12-well plate, incubated overnight, and then transfected with miR-133a agomir, miR-133a antagomir, or negative control using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions as described previously .
Quantitative RT-PCR assay
Total RNA was isolated from MSCs or left ventricular tissues using Trizol reagent (Invitrogen, USA) as described previously . Subsequently, RNAs were reverse-transcribed to cDNAs with the microRNA reverse transcription system (GenePharma, Shanghai, China) or the primescript RT reagent kit (TAKARA, Japan). The expression level of miR-133a was analyzed by quantitative RT-PCR (Q-PCR) using the miRNAs Quantitation Kit (GenePharma, China). For snail 1, Q-PCR was performed using SYBR PCR master mix in the ABI Step One-Plus Detection system (Applied Biosystems, USA) according to the manufacturer’s introductions. The primers used for snail 1 are as follows: sense, 5′-TTCTCCCGAATGTCCTTGCT-3′; and antisense, 5′-CTGCCTTCCATCAGCCATCT-3′. Relative gene expression quantifications were calculated according to the comparative Ct method using U6 as an internal standard. Each assay was performed in triplicate.
Viability and proliferation assay
Cell viability was evaluated by CCK-8 assay (Dojindo Laboratories, Japan) as described previously . Briefly, MSCs in 96-well plates were transfected with miR-133 agomir/antagomir and cultured for 6, 12, 24, and 48 h. The culture supernatants were then changed to fresh medium with 10% CCK-8 (Dojindo Laboratories, Japan). The absorbance at 450 nm indicating cell viability was measured in the Multi-Mode Microplate Reader (BIOTEK, USA), taking into account the background readings. The proliferation of MSCs was assessed with carboxyfluorescein diacetate succinimide ester (CFSE) (Invitrogen). MSCs were stained with CFSE at a concentration of 25 mM for 10 min, washed with PBS three times, and transfected with miRNAs in 12 h. MSCs were then collected at 6, 12, 24, and 48 h and analyzed by flow cytometry (Guava).
Cell cycle and apoptosis were analyzed by flow cytometry as reported previously . MSCs at passage 5 were placed in a 12-well plate at a confluence of 80% and then transfected with miR-133 agomir/antagomir or negative controls. At corresponding time points, cells were collected and stained with Annexin-V (eBioscience, USA) and propidium iodide (for apoptosis assay) or only propidium iodide (Sigma, for cell cycle assay) for 30 min. The cells were detected by flow cytometry, and data were analyzed by FlowJo software.
Reconstruction of plasmids and preparation of lentivirus
The vector pCDH-CMV-MCS-EF1-copGFP was used as a backbone plasmid to reconstruct lentiviral vector containing miR-133a. The vector and pre-miR-133a fragments amplified from rat genomic DNA were digested with XbaI and EcoRI (NEB, USA) and ligated with T4 ligase (TAKARA). The primers used for amplification of pre-miR-133a are as follows: sense, 5′-ATGTCTAGACCCTCTAATACTCGTCAT-3′; and antisense, 5′-GAATTCGACCGTTGTTAGTTGTTT-3′. The reconstruct plasmid was verified by Sanger sequencing. For lentiviral production, HEK293NT cells were cotransfected with control vector or lentiviral plasmid carrying pre-miR-133 fragments, along with lentiviral packaging mix. The culture medium was collected at 24 and 48 h after transfection, filtered through a 0.45-μm filter, and incubated overnight with polyethylene glycol 8000 (PEG 8000) before being concentrated by centrifugation (4000 × g for 20 min at 4 °C; Thermo). Lentivirus was titrated using a titer kit, and virus particles were stored at –80 °C until use. The ratio of lentivirus-infected MSCs was observed under an inverted fluorescence microscope (Olympus, Tokyo, Japan).
Rat model of acute MI and assessment of heart functions
Acute MI was induced as described previously [27, 28]. Briefly, young Sprague–Dawley rats (~250 g) were anesthetized with 10% chloral hydrate (w/v; Acros, Japan) by intraperitoneal injection. The left anterior descending artery was ligated between the pulmonary artery outflow tract and the left atrium. While sham-ligated rats were used as control, these rats under MI surgeries were divided into four groups receiving intramyocardial injection of PBS, normal MSCs, MSCs infected with empty lentivirus (vector-MSCs), or MSCs infected with miR-133-overexpressed lentivirus (miR-133-MSCs), respectively. A total of 1 × 106 MSCs in 20 μl of phosphate buffer (PBS) were transplanted by myocardial injection near the ligation site in the free wall of the left ventricle. Rats were anesthetized for echocardiography detection on days 0, 7, and 28 after MI, using the Vevo 2100 system (VisualSonics Inc., Toronto, ON, Canada) with an 80-MHz probe. Rats were finally sacrificed to harvest the heart tissue for Q-PCR analysis, hematoxylin and eosin (H&E) staining, and Masson staining.
To evaluate the effect of miR-133 on cell survival in vivo, normal MSCs, MSCs infected with empty lentivirus (vector-MSCs), or miR-133-overexpressed lentivirus (miR-133-MSCs) were dyed with chloromethylbenzamido (CellTrackerTM CM-Dil 11372053; Invitrogen) according to the manufacturer’s instructions, followed by cell transplantation. Rats were anesthetized for MSC survival detection on day 3 after MI, and CM-Dil+ MSCs were observed via fluorescence microscopy (Olympus, Japan).
Freshly isolated hearts were fixed in 4% paraformaldehyde (PFA, pH 7.4) and sectioned. H&E and Masson trichrome staining were performed to demonstrate myocardial fibrosis as described previously . For Masson trichrome staining, heart slices were produced perpendicular to the axis of the left anterior descending coronary artery. The severity of myocardial fibrosis was indicated by the average ratio of the fibrotic area to the entire LV cross-sectional area and the average ratio of fibrosis length to entire internal LV circumference, which was analyzed by ImageJ software (National Institutes of Health, USA).
MSCs were transfected with miR-133 agomir, miR-133 antagomir, or negative control, and subsequently cultured in DMEM/F12 with exosome-free FBS. Exosomes from culture supernatants were isolated by differential centrifugation. In brief, supernatants were centrifuged at 200 × g for 30 min to remove cell debris (991; Thermo). Subsequently, the supernatants were mixed with total exosome isolation reagent (Invitrogen, USA) overnight at 4 °C. After centrifuging at 10,000 × g for 1 h, the pellet was resuspended in PBS. miRNA in the exosome was isolated using a mirVana miRNA isolation kit (Invitrogen, USA) according to the manufacturer’s instructions. Subsequently, the expression level of miR-133a was analyzed by Q-PCR as already described. The protein level in the exosome was analyzed by BCA assay kit (Beyotime, China) and the secreted level of CD63 was detected by western blot analysis as described in the following.
Coculture of MSCs with cardiomyocytes
Cardiomyocytes were isolated from neonatal rat with 1 mg/ml collagenase II (Invitrogen, USA). Forty-eight hours later, the isolated cardiomyocytes were cocultured with MSCs transfected with miR-133 agomir/miR-133 antagomir or negative control at a 10:1 ratio. After 48 h of coculture, the cardiomyocytes were collected for RNA isolation and Q-PCR analysis.
Western blot analysis
Western blot analysis was performed according to previous description . Total protein (30 μg protein per lane) from MSCs transfected with different miRNAs was subjected to SDS-PAGE and immunoblotting with the antibody against poly ADP-ribose polymerase (PARP) (46D11; CST), CD63 (ab108950; Abcam), Collagen I (14695; Proteintech), α-SMA (ab5694; Abcam), and GAPDH (KM9002T; Sungene Biotech).
Data were expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA to compare data among three or more groups and Student’s unpaired t test to compare data between two groups. p < 0.05 was considered statistically significant. Statistical analysis was carried out using GraphPad Prism software (version 5.01; San Diego, CA, USA).