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. 2018 Apr 3;8(9):2565–2582. doi: 10.7150/thno.22878

Figure 4.

Figure 4

Mechanical stress promotes miR-378 secretion from cardiomyocytes in an EVs-dependent manner. (A) Electron micrographs of rat cardiomyocyte-derived EVs, showing sizes of approximately 20 to 100 nm in diameter for typical cells and larger sizes of approximately 80 to >250 nm for stretched cells. Scale bar: 200 nm (n=5). EVs are indicated by arrows. (B) Western blot analysis of marker protein CD63 from purified EVs secreted from equal numbers of cardiomyocytes with or without mechanical stress. (C) GW4869 treatment inhibited EVs secretion induced by mechanical stress. Cardiomyocytes subjected to mechanical stretching were cultured in dimethyl sulfoxide (DMSO) or with 0.1 μM (+), 1 μM (++) or 10 μM (+++) GW4869. The purified exosomes secreted by equal numbers of control or GW-treated cardiomyocytes were analyzed by Western blotting for the presence of the CD63 protein. (D) Secretion of miR-378 was suppressed by the treatment with GW4869. Values represent the mean ± SE from three independent experiments. **, P< 0.01 and *, P< 0.05 vs. control without MS. (E) Schematic representation of the workflow for investigating the mechanism of miR-378 secretion. (F) Western blot analysis of MMP9 expression in CFs and quantitative analysis. Cardiac fibroblasts were resuspended in conditioned medium and were then stretched or not stretched for 24 h. (G) Quantitative real-time PCR analysis of mRNA expression of collagen types I and III in CFs. Values represent the mean ± SE from three independent experiments. **, P< 0.01 and *, P< 0.05. (H) Quantitative polymerase chain reaction (qRT-PCR) analyses of miR-378 expression in the cardiac fibroblasts after treatment with the supernatant from the stretched cardiomyocytes (CM-MS) or with the supernatant from the stretched cardiomyocytes with GW4869 added (CM+GM-MS); the supernatant from the unstretched cardiomyocytes was used as the negative control (CMs). Values represent the mean ± SE, n=3. **, P< 0.01 and *, P< 0.05 vs. CFs treated with supernatant of unstretched cardiomyocytes without MS; ##, P< 0.01 and #, P< 0.05 vs. CFs treated with supernatant of unstretched cardiomyocytes that were subjected to MS. (I) CFs treated with the supernatant of the stretched cardiomyocytes with GW4869 added were transfected with miR-378 and subsequently subjected to mechanical stretching for 24 h. MMP9 expression was detected by Western blotting and quantitatively analyzed. Values represent the mean ± SE from three independent experiments. **, P< 0.01 vs. control CFs without MS and miR-378 mimics transfection; ##, P< 0.01 vs. CFs without miR-378 mimics transfection that were subjected to MS. (J) Schematic representation of the workflow. (K) Western blot analysis of MMP9 expression in CFs and the quantitative analysis. Cardiac fibroblasts were resuspended in conditioned medium and were then stretched or not stretched for 24 h. (L) Quantitative real-time PCR analysis of mRNA expression of collagen types I and III in CFs. Values represent the mean ± SE from three independent experiments. **, P< 0.01 and *, P< 0.05. (M) Cardiac fibroblasts were incubated with PKH67-labeled (green) EVs from cardiomyocytes for 30 min and 24 h independently (n=5-7) and then fixed for confocal imaging. Scale bar: 10 μm. The exosomes were obtained from the supernatant of CMs without MS or with MS for 24 h.