STEMIN- and YAP5SA-induced exosomes prevent cardiomyocyte apoptosis

Figures

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  Figure 1. STEMIN and YAP5SA significantly increase chromatin accessibility to the loci of miRNAs predicted to target key components of the apoptotic pathway, including the caspase and TP53 gene families Heatmaps showing differentially remodeled loci for miRNA genes. Out of 105 miRNA genes considered, STEMIN- and YAP5SA-mRNA enhanced the chromatin accessibility of genes that code for 46 miRNAs. (A–C) that target (A) caspases and ancillary markers, (B) TP53 and ancillary markers, (C) Venn diagram, showing the number of miRNA loci remodeled by STEMIN- and YAP5SA-treated cells, bar charts (D–G) of (D) SRF/control ATAC seq peaks, where 45% of the 105 miRNAs were upregulated by > 1-fold, (E) STEMIN/control ATAC seq PEAKS, where 85% of the 105 miRNAs were upregulated >1-fold, (F) YAP1/control ATAC seq PEAKS, where 67% of the 105 miRNAs were upregulated >1-fold, and (G) YAP5SA/control ATAC seq PEAKS, where 93% of the 105 miRNAs were upregulated >1-fold.

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  Figure 2. STEMIN and YAP5SA upregulate anti-apoptotic miRNAs and suppress the expression of apoptotic genes in cardiomyocytes OMICS analysis shows. (A) heatmap of differentially expressed apoptotic, cell-cycle arrest, and proliferative markers in STEMIN-treated adult human primary cardiomyocytes. Apoptotic markers, including TP53, CASP6, CASP12, and BAK1, were downregulated. (B) Volcano plot shows STEMIN upregulated miRNAs, including hsa-miR-101-3p and hsa-miR-499a-5p. (C and D) GO chord plots of differentially upregulated and downregulated phenotypes (GO-terms), in STEMIN- vs. SRF-treated cardiomyocytes. (E) A heatmap of YAP5SA-treated adult human primary cardiomyocytes, showing downregulated apoptotic markers, including CASP3, CASP6, CASP9, and TP53. (F) Volcano plot shows differentially expressed miRNAs, including the upregulation of hsa-let-7c-5p, and (G and H) GO chord plots of differentially upregulated and downregulated phenotypes (GO-terms), in YAP5SA- vs. YAP1-treated cardiomyocytes.

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  Figure 3. Exosomes isolated from STEMIN and YAP5SA-treated cardiac myocytes show the accumulation of anti-apoptotic miRNAs, which may block the expression of apoptotic markers Nanoparticle tracking analysis of exosomes secreted into culture media from AC16 cardiomyocytes transfected with (A–C), (A) Empty reagent (control), (B) STEMIN-mRNA, (C) YAP5SA-mRNA. Duplicates of ∼10 μg of secreted exosomes/sample were identified by an Orbitrap mass spectrophotometer, and following proteomic analysis, volcano plots of duplicate samples are shown for (D) STEMIN, with preferentially secreted proteins such as KRAS, RAB6B, RHOC, and HNRNPH3. (E) YAP5SA, with preferentially secreted proteins such as UBE2V2, ZEB2, RAP2C, SRM, and THYN1; they both share serpin, CNTLN1, and CTSC. OMICS analysis shows a heatmap from miR-seq of (Fi) control-, STEMIN- and YAP5SA-generated exosomes, and (Fii) differentially expressed anti-apoptotic miRNAs, and their predicted apoptotic targets determined by specific miRDB [https://mirdb.org/] and IMOTA (interactive multi-omics tissue atlas [https://ccb-web.cs.uni-saarland.de/imota/mit/]).

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  Figure 4. STEMIN, YAP5SA, and their activated miRNAs block staurosporine-induced apoptosis in cardiomyocytes (A) Immunofluorescence images of STEMIN- and YAP5SA-treated cardiomyocytes. (B) Quantitative cleaved-CASP3 (FITC)/DAPI relative fluorescence bar chart from STEMIN- and YAP5SA-treated cardiomyocytes. (C) Immunofluorescence images of hsa-let-7c-5p-, miR-101-3p-, and miR-499a-5p-treated cardiomyocytes. (D) Quantitative cleaved-CASP3 (FITC)/DAPI relative fluorescence bar chart from hsa-let-7c-5p-, miR-101-3p-, and miR-499a-5p-treated cardiomyocytes. Results of assays in (A–D) were performed in triplicate (n = 3); data are represented as mean ± SEM, and analyzed by one-way ANOVA. (E–G) Immunohistochemistry images and plot, showing 4× and 10× magnified images of cleaved-CASP3 immuno-stained tissue sections of infarcted mice hearts injected with an empty reagent and an equimolar combination (COMBO) of STEMIN + YAP5SA (white arrows showing point of injection), (F) split channel images and (G) bar chart showing the quantitative cleaved-CASP3/FITC:DAPI mean relative fluorescence. Results of assays in (E–G) were performed on seven animals: three control mice (two males, one female), four…

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  Figure 5. Secreted exosomes from STEMIN and YAP5SA modRNA-transfected cardiac myocytes, transferred to staurosporine-treated cardiomyocytes, hinder the expression of apoptotic markers (A–C) Immunohistochemistry images and plot, showing images of cleaved-CASP3 and TUNEL immuno-staining of fixed and permeabilized AC16 cells previously cultured with exosomes from (A) AC16-only cells, STEMIN-treated AC16 cells, and YAP5SA-treated AC16 cells, after exposure to staurosporine: split channel images of DAPI (blue), cleaved-CASP3 (green), and TUNEL (red), and (B and C) bar chart showing (B) quantitative cleaved-CASP3/FITC:DAPI and (C) TUNEL:DAPI mean relative fluorescence. Data are represented as mean ± SEM and analyzed by one-way ANOVA. Top and bottom vertical error bars and horizontal bars with p-values are included in the bar graphs 5B and 5C.