Ethics Statement
All animal experimental procedures were carried out in accordance with the guidelines of the Shanghai Experimental Animal Center and the Shanghai Tenth People's Hospital (SYXK: 2014-0026) “People and Animal Use in Research” and in accordance with the principles set out in the Helsinki Declaration. Experimental animals are treated humanely, and all efforts are made to alleviate their discomfort. All mice were housed in a specific pathogen-free animal vivarium and maintained on a 12h light, 12h dark cycle, with free access to standard rodent chow and water.
Isolation and Culture of ADMSCs
SD rats were purchased from Shanghai Research Center of the Southern model organisms (China). ADSCs were isolated from the subcutaneous adipose tissue of 8 to 12-week-old SD rats. Briefly, The adipose tissue was obtained from the subcutaneous fat pad around the ilium of the rats and was washed in phosphate-buffered saline (PBS) (CORNING, Manassas, USA) containing 1% Penicillin-Streptomycin(PS)solution (HyClone, Pasching, Austria). We minced the fat pad into pieces and then lysised them in Dulbecco's modified Eagle's medium (DMEM) (Thermo Fisher, Suzhou, China) containing 1 mg/ml collagenase I (YEASEN, Shanghai, China) and 1% PS at 37°C for 30 min. The digested tissue was filtered through a sterile 100 μm nylon mesh (Corning, NY, USA), and centrifuged at 1800 rpm for 5 min. After that, we discarded the supernatant and resuspended the pellet with culture medium twice. ADSCs were seeded onto culture dishes with growth medium DME/F-12 (HyClone, GE Healthcare Life Sciences, Logan, Utah, USA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich) and 1% PS at 37°C in a humidified atmosphere of 5% CO2 before being using for analysis.
Animal Study
8-weeks-old male C57BL/6 mice were purchased from Shanghai Research Center of the Southern model organisms (China). Type I diabetes was induced with the dose of streptozotocin (STZ, 70mg/kg in 0.1mol/L citrate buffer solution; Sigma-aldrich, St. Louis, Missouri, USA) intraperitoneally injected per two days. 8 weeks later, the blood glucose level (fasting blood glucose over 16.6mmol/L were considered as diabetic) were measured. The diabetic mice were tail vein injected with ADMSCs (2×106) or saline for 12 weeks. Body weight, random blood glucose levels and blood lipid level as well as echocardiography were tested at the time points. 30 weeks after the model establishment, all animals were sacrificed and heart tissues were isolated for further analysis.
Echocardiography
The echocardiography was performed by using a Vevo 2100 High Resolution Imaging System (Visual Sonics, Toronto, Canada). Briefly, mice were anesthetized by isoflurane (1%) and placed on the 37.C platform. Parasternal long axis (PSLAX) images were acquired and analyzed with a VevoStrain software. From the M-mode images, the diameter of the left ventricle (LV) at the end of the diastole (LVEDD) and systole (LVESD), LV fractional shortening (FS), and LV ejection fraction (EF) were calculated by the machine.
Cell culture and stimulation
H9c2 cardiomyocytes were purchased from the Institute of Biochemistry and Cell Biology (Shanghai, China). Cells were incubated in DMEM containing with 10% FBS and 1%PS. For high glucose administration, cells were incubated with culture media containing 33 mM glucose for 72 hours. H9c2 cardiomyocytes were treated with high glucose (HG, 33mM), IL-1β (20ng/ml, popertech, cat#400-01B), IL-1β and HG respectively after they were starved for 3 hours. And untreated cells were used as negative control.
Histopathology staining
Myocardial tissue was fixed with 4% paraformaldehyde and dehydrated and embedded in paraffin. Next, the samples were cut into 5‐μm‐thick sections that should be undertaken. Heart sections were stained with hematoxylin-eosin (H&E), Masson's trichrome and wheat germ agglutinin (WGA) staining. The muscle cells with clear nuclear nucleus and clear sarcolemma were selected for analysis of muscle cell cross-sectional area (CSA) in H&E staining and WGA staining. Fibrosis was detected on the heart tissue by Mason trichrome staining. 4 microscopic photographs of every section were shot randomly selected by NIS-elements v3.0 (Nikon, Japan). Cardiac CSA and myocardial fibrosis were analyzed Image-ProPlus6.0 software (Media Cybernetics, Silver Spring, Maryland, USA) for quantitative analysis. Date expressed as mean ± standard deviation.
Immunohistochemistry
The myocardial tissues were fixed in 4% paraformaldehyde and then embedded in paraffin after dehydration. Next, the samples were cut into 5‐μm‐thick sections. After that , the sections were deparaffinized and stained with F4/80(diluted concentration 1:200, Servicebio, Wuhan, China). For IHC analysis, each section was captured in at least 5 images by NIS-elements v3.0 (Nikon, Japan) and all images were quantified using Image-ProPlus6.0 software (Media Cybernetics, Silver Spring, Maryland, USA). Percentages of positive stains for F4/80 within the fields were averaged for each mouse and then for each group as described before. Date expressed as mean ± standard deviation.
Western blotting analysis
Total protein was extracted from mouse hearts and the levels were determined using a BCA Protein Assay Kit (Thermo Scientific, Rockford, Illinois, USA). Equal amounts of total protein (50 μg) were loaded and then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Thermo Scientific, Rockford, Illinois, USA). The membranes were then blocked in 5% BSA (YEASEN, Shanghai, China) for 1 h, followed by overnight incubation at 4 °C with the following primary antibodies: IL-1β (cat: mAb #12242, diluted concentration 1:1000, Cell Signaling Technology, Danvers, MA, USA), P62 (cat:ab109012, diluted concentration 1:800, abcam, England), LC3I/II (cat: ab62721, diluted concentration 1:500, abcam, England), DAPDH (cat: ABS16, diluted concentration 1:10000, EMD Millipore Corp, USA). The secondary antibodies were horseradish peroxidase (HRP)-linked-Antibody, anti-rabbit IgG (diluted concentration 1:5000, Cell Signaling Technology, Danvers, MA, USA) and horseradish peroxidase (HRP)-labeled-Antibody, Goat Anti-Mouse IgG (diluted concentration 1:5000, Biosharp Life Sciences, Hefei, China). Blots were processed for enhanced chemifluorescence using a Pierce ECL western blotting substrate (Tanon, Shanghai, China). Images were collected using ChemiDoc™ MP Imaging System (Bio-Rad, Hercules, CA, USA). Bands on immunoblots were quantified by densitometry using Image Lab software (version 6.0, Bio-Rad).
Real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA was purified from mouse hearts with TRIZol ((Thermo Scientific, Rockford, Illinois, USA)) following manufacturer’s protocol. The total RNA was used for reverse-transcription and amplified using a One Step SYBR RT-PCR Kit (TaKaRa, Shiga, Japan). After that, the cDNA was amplified with SYBR Premix Ex Taq (KAPA, Cape, South Africa) by a Real-Time PCR System (Roche, Basel, Swiss). GAPDH was used as the internal control. The sequences of the primers for each gene detected are listed in Supplementary Table 1. The PCR program was as follows:95℃,10 min; 95℃,5s,60℃,34s for a total of 40cycles. The expression levels of target genes were determined using the 2−ΔΔCT method. Table 1. Primers
Annexin V-FITC/PI staining and flow cytometry
H9c2 cells were treated as described above and were stained by using FITC Annexin V/Dead Cell Apoptosis Kit (cat: v13242, Invitrogen,USA) according to the manufacturer's instructions. Then, cells were diluted in the dilution buffer and the apoptotic cells were measured by a BD FACS Calibur flow cytometer (BD Biosciences, Heidelberg, Germany) in a total of 10,000 counted cells.
Transmission electron microscope and observation of autophagosome
Briefly, the cell medium was removed. Then, cells were washed with 0.1 cacodylate buffer and fixed with a solution containing 3% glutaraldehyde and 2% paraformaldehyde in PBS. Later, the rest of the procedure was conducted using the standard protocol. Transmission electron microscope (JEM-1400PLUS) was used to obtain the images.
mRFG-GFP-LC3 assay
H9c2 cardiomyocytes were seeded into the six-well-plate to reach about 60% confluence. Later, they were divided into 4 groups (NC group, HG group, IL-1β group, HG+ IL-1β group) and transfected with adenovirus (20 MOI, HBAD-mRFP-GFP-LC3, Hanbio, Shanghai, China) for another 48 hours. In brief, cells were fixed in 4% paraformaldehyde for 10 mins and were washed by PBS for three times. The, they were sealed by 10% glycerinum to limit fluorescence quenching. Leica DMI6000 was used to detect related fluorescence. The GFP signals were observed in the lysosomal acidic conditions, whereas mRFP fluorescence was relatively stable. Therefore, this tandem-tagged fluorescent protein showed yellow dots in the autophagosomes, while exhibited red only in lysosomes. Quantification of GFP and mRFP fluorescence dots, and overlay between two different signals were recorded and analyzed using ImageJ software.
Statistical analysis
All data are shown as the mean±SEM. Student’s t-test (two-sided), one-way ANOVA or two-way ANOVA followed by Bonferroni's pos-hoc test were used to calculate P value accordingly. Error bars represented standard errors, and numbers of experiments (n) were as indicated. P < 0.05 was regarded as significant. All statistical analyses were performed using SPSS 20.0 software.