Electrical Stimulation Increases the Secretion of Cardioprotective Extracellular Vesicles from Cardiac Mesenchymal Stem Cells

Figures

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  Figure 1. Characterization of EVs from C-MSCCtrl and C-MSCELSM. (A,B) Transmission electron micrograph images of EVs from C-MSC. Scale bar = 100 nm. (C,D) nanoparticle tracking analysis using ZetaView® Particle Tracking Analyzer to measure the size and number of C-MSC-derived EVs with or without ELSM.

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  Figure 2. HL-1 cardiomyocytes treated with CM from ELSM-treated C-MSC showed reduced hypoxia-induced apoptosis, as determined by TUNEL staining and flow cytometry. (A,B) percentage of TUNEL positivity in HL-1 cells treated with CM from C-MSCCtrl or C-MSCELSM; (C,D) percentage of TUNEL positivity in HL-1 cells treated with EV-depleted CM from C-MSCCtrl or C-MSCELSM; (E) statistical analysis of the percentage of TUNEL-positive cardiomyocytes in the 4 groups (ns not significant; ** p < 0.01, *** p < 0.001, n = 3).

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  Figure 3. nSMase2 protein levels were determined by Western blot after treatment with or without ELSM for the indicated times. (A,B) Western blot analysis of nSMase2 expression levels in C-MSC; β-actin protein levels were used as loading control (**** p < 0.0001, n = 3).

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  Figure 4. Knockdown of nSMase2 inhibits ELSM-induced EV release from C-MSC. (A) qRT-PCR quantification of nSMase2 mRNA levels (normalized to β-actin levels) in C-MSC transfected with nSMase2 siRNA (si-nSMase2) versus si-NT with/without ELSM treatment (ns not significant; * p < 0.05, *** p < 0.001, n = 3). (B) Quantification of relative AChE activity (normalized to protein concentration and then normalizing to the Ctrl si-NC group) in EVs from si-NT and si-nSMase2-transfected C-MSC with/without ELSM treatment (*** p < 0.001, **** p < 0.0001, n = 4).

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  Figure 5. Reduced apoptosis in HL-1 cardiomyocytes treated with CM from ELSM-treated C-MSC is dependent on nSMase2. (A–D) Percentage of TUNEL+ cardiomyocytes treated with CM from si-NT transfected C-MSC without ELSM, si-NT transfected C-MSC with ELSM, si-nSMase2 transfected C-MSC without ELSM, and si-nSMase2 transfected C-MSC with ELSM; (E) statistical analysis of the percentage of TUNEL+ cardiomyocytes in the 4 groups (ns not significant; * p < 0.05, ** p < 0.01, n = 3).

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  Figure 6. Schematic overview of proposed the mechanisms of ELSM-induced cardiomyocyte protection. ELSM increases expression of nSMase2, leading to increased EV secretion, which protects cardiomyocytes from hypoxia-induced apoptosis.