Overexpression of GATA4 enhances the antiapoptotic effect of exosomes secreted from cardiac colony-forming unit fibroblasts via miRNA221-mediated targeting of the PTEN/PI3K/AKT signaling pathway

Figures

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  Fig. 1. Characterization of cCFU-Fs transduced with GATA4 (cCFU-FsGATA4) and identification of exosomes from cCFU-Fs. a Immunostaining of cCFU-Fs transduced with GATA4 (cCFU-FsGATA4) or empty vector (cCFU-FsNC, control). GFP was expressed in both cCFU-FsNC and cCFU-FsGATA4. Scale bar: 50 μm. b Western blot of GATA4 in cCFU-FsGATA4 and cCFU-FsNC (n = 3). c TEM image of exosomes (n = 6). Scale bar: 200 nm. d NanoSight analysis of the particle size (n = 4). e Detection of CD9, TSG101, and Alix by Western blotting (n = 3)

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  Fig. 2. The effect of cCFU-FsGATA4 exosomes on hypoxia-induced cardiomyocyte injury. a Cell viability of H9c2 cells after coculture with GATA4-Exo or NC-Exo under hypoxic conditions for 24 h was analyzed by CCK8 assay (n = 4, **p < 0.01 for hypoxia vs normoxia; *p < 0.05 between the indicated groups). b, c Western blot analysis of c-caspase-3 in H9c2 cells after exposure to hypoxia, GATA4-Exo, or NC-Exo (n = 3, **p < 0.01 for hypoxia vs normoxia and hypoxia vs GATA4-Exo; *p < 0.05 for GATA4-Exo vs NC-Exo). d, e Apoptotic H9c2 cells in response to hypoxia and coculture with GATA4-Exo or NC-Exo are determined by flow cytometry (Q1:viable cells, Q2: early apoptotic cells, Q3: late apoptotic cells, Q4: Necrotic cells, n = 3) (c-Cas-3: cleaved-caspase-3). (n = 3, *p < 0.05, **p < 0.01 using two-tailed Student’s t test. Data are represented as mean and SEM)

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  Fig. 3. Enhanced cardiac function and decreased apoptotic cells after myocardial infarction in mice transplanted with GATA4-Exo. a, b Representative M-mode images (a) and quantification (b) of EF% and FS% measured by echocardiography of sham (n = 6), PBS-treated (n = 6), NC-Exo-treated (n = 6), and GATA4-Exo-treated (n = 6) mice at 28 days after MI. (**p < 0.01 for PBS vs SHAM, #p < 0.05 for NC-Exo vs PBS, ##p < 0.01 for GATA4-Exo vs PBS, & p < 0.05 for GATA4-Exo vs NC-Exo). c, d Masson trichome-stained myocardial sections at 28 days after MI in mice treated with PBS, NC-Exo, or GATA4-Exo (n = 6 mice per experimental group, for each sample, 4 to 6 slices were taken according to the size of the heart). Scar tissue and viable myocardium are identified in blue and red, respectively. The percentage of the fibrotic area after AMI was calculated using the software ImageJ. The bar graph on the right quantifies the extent of fibrosis in each treatment group (##p < 0.01 for GATA4-Exo vs PBS, & p < 0.05 for GATA4-Exo vs NC-Exo). Scale bar: 1 mm. EF%: ejection fraction %; FS%: fraction shortening %. e Immunohistochemistry of sham-, PBS-, GATA4-Exo-, or NC-Exo-treated heart sections marking…

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  Fig. 4. MicroRNA (miRNA) expression profiling and the effect of miR221 on hypoxia-induced cardiomyocyte injury. a Heat map and supervised hierarchical clustering of the 10 upregulated and 4 downregulated miRNAs with a ≥ 2-fold difference in expression between GATA4-Exo and NC-Exo (triplicate assays were done for each exosomes replica from different cCFU-F isolates). b Validation of microarray data using real-time PCR, and the relative expression of miRNAs in GATA4-Exo compared to NC-Exo (n = 3). c The cell viability of H9c2 cells after coculture with miR221 mimics or miR221 inhibitor under hypoxic conditions for 24 h was analyzed by CCK8 assay (n = 4, **p < 0.01 for comparisons between groups). d, e Apoptotic H9c2 cells in response to hypoxia and coculture with a miR221 mimics or miR221 inhibitor determined by flow cytometry (n = 3, *p < 0.05 for comparisons between groups)

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  Fig. 5. PTEN is a target gene of miR221. a Luciferase reporter assays were performed using the wild-type PTEN 3′ UTR sequence containing either the seed sequence for miR221 recognition (PTEN-3′ UTRwt) or a mutated 3′ UTR (PTEN-3′ UTRmut). Luciferase activity was determined 48 h after transfection. The ratio of normalized luciferase activities is shown (n = 3). b Real-time PCR analysis of PTEN expression in H9c2 cells after treatment with a miR221 mimics or inhibitor (n = 3). c, d The protein levels of PTEN in H9c2 cells as determined by Western blotting (n = 3, **p < 0.01, *p < 0.05 for comparisons between groups)

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  Fig. 6. The PTEN-PI3K/Akt signaling pathway is involved in the antiapoptotic effect of GATA4-Exo. a–c PTEN mRNA and protein levels after treatment with hypoxia, GATA4-Exo, or miR221 inhibitor were determined by qPCR and Western blotting, respectively (n = 3, **p < 0.01 for comparisons between groups). d The cell viability of H9c2 cells after treatment with hypoxia, GATA4-Exo, miR221 inhibitor, and/or siPTEN was analyzed by CCK8 assay (n = 3, **p < 0.01 for comparisons between groups). e, f Apoptotic H9c2 cells in response to hypoxia, GATA4-Exo, miR221 inhibitor, and siPTEN were determined by flow cytometry (n = 3, *p < 0.05, **p < 0.01 for comparisons between groups). g–i Western blot analysis of p-Akt, Akt, and c-caspase-3 in H9c2 cells after exposure to hypoxia, GATA4-Exo, miR221 inhibitor, and/or siPTEN. Relative expression was determined following normalization to β-actin levels (n = 3, *p < 0.05, **p < 0.01 for comparisons between groups). All data were obtained from 3 independent experiments. j–l The protein expression levels of PTEN and c-caspase-3 within the myocardium. Relative expression was determined following normalization to β-actin levels (*p < 0.05, **p < 0.01 between…