BMSC-derived EVs alleviate doxorubicin-induced cardiotoxicity via miR-210-3p involving the ACVR1B-SMAD3-PGC-1α signaling axis

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  Fig. 1. BMSC-derived EVs attenuate DIC. A Representative TEM image of BMSC-EVs showing typical cup‑shaped morphology; scale bar = 500 nm. B Representative western blots of the EVs markers CD81, CD9, and β-Tubulin in BMSC and BMSC-derived EVs. C experimental timeline and design of the DOX mice treated with BMSC-EVs. D–F Echocardiographic analysis of LV ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) of mice (n = 5). G Hematoxylin and eosin-stained heart of mice. H Masson trichrome staining, and I Quantification of fibrosis area (n = 5, scale bar = 50 μm). J MTT assay for cell viability in BMSC-EVs vs. Vehicle treated DOX HL-1s (n = 7). K Representative PI-stained images and quantification of cell death levels of HL-1s (n = 6, scale bar = 100 μm). L, representative images of DCFH-DA-stained HL-1s and quantification of oxidative stress levels (n = 6, scale bar = 100 μm). Data are shown as mean ± SD. D through L, two-way ANOVA followed by the Bonferroni multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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  Fig. 2. Identification of miR-210-3p is a potential target of BMSC-EVs in DIC. A Heatmap of differentially expressed microRNA in Vehicle (n = 3) or BMSC-EVs (n = 3)-treated in DIC. B microRNA levels of in Ctrl, DOX and BMSC-EVs treated HL-1s by RT-qPCR (n = 4). C Representative images of FISH staining for miR-210-3p combined with BMSC-EVs labeling-PKH26. Green: miR-210-3p; Red: PKH26; Blue: DAPI. Scale bar = 50 μm. D Enrichment analysis of biological process (BP) of upregulated microRNA in BMSC-EVs vs. Vehicle treated DOX HL-1s by RNA-seq. Data are shown as mean ± SD. A, unpaired Student t-test. B, two-way ANOVA followed by the Bonferroni multiple comparison test. *P < 0.05, **P < 0.01

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  Fig. 3. miR-210-3p overexpression improves DOX induced cell death and mitochondrial dysfunction. A Representative PI-stained images and quantification of cell death levels in NC mimics or miR-210-3p mimics-transfected HL-1s (n = 6, scale bar = 100 μm). B MTT assay for cell viability in NC mimics or miR-210-3p mimics-transfected HL-1s (n = 8). C Representative images of DCFH-DA-stained HL-1s and quantification of oxidative stress levels in NC mimics or miR-210-3p mimics-transfected HL-1s (n = 6, scale bar = 100 μm). D Representative images of mitoSOX-stained HL-1s and quantification of oxidative stress levels in NC mimics or miR-210-3p mimics-transfected HL-1s (n = 6, scale bar = 100 μm). E Representative JC-1-stained HL-1s and quantification of mitochondrial membrane potential levels in NC mimics or miR-210-3p mimics-transfected HL-1s (n = 5, scale bar = 100 μm). F Representative western blotting images, and quantitative analysis of ETC-related proteins in NC mimics or miR-210-3p mimics-transfected HL-1s (n = 5). Data are shown as mean ± SD. A through F, two-way ANOVA followed by the Bonferroni multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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  Fig. 4. miR-210-3p deficiency aggravates DOX induced cell death and mitochondrial dysfunction. A Representative PI-stained images and quantification of cell death levels of NC inhibitor or miR-210-3p inhibitor-transfected HL-1s (n = 6, scale bar = 100 μm). B MTT assay for cell viability in NC inhibitor or miR-210-3p inhibitor-transfected HL-1s (n = 7). C Representative images of DCFH-DA-stained HL-1s and quantification of oxidative stress levels in NC inhibitor or miR-210-3p inhibitor-transfected HL-1s (n = 5, scale bar = 100 μm). D Representative images of mitoSOX-stained HL-1s and quantification of oxidative stress levels in NC inhibitor or miR-210-3p inhibitor-transfected HL-1s (n = 6, scale bar = 100 μm). E Representative JC-1-stained HL-1s and quantification of mitochondrial membrane potential levels in NC inhibitor or miR-210-3p inhibitor-transfected HL-1s (n = 6, scale bar = 100 μm). F Representative western blotting images, and quantitative analysis of ETC-related proteins in NC inhibitor or miR-210-3p inhibitor-transfected HL-1s (n = 5). Data are shown as mean ± SD. A through F, two-way ANOVA followed by the Bonferroni multiple comparison test. *P < 0.05, **P < 0.01, ***P <…

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  Fig. 5. miR-210-3p target gene screening and regulatory mechanism validation. A Venn diagram of three independent miRNA differential analysis databases reveals 6 possible mRNAs directly targeting miR-210-3p. B Sequence of ACVR1B 3’ UTR with miR-210 targeting sites. C Luciferase reporter assay to evaluate the direct binding of miR-210 with the 3’ UTR of ACVR1B (n = 3). D Acvr1b mRNA expression levels in NC mimics or miR-210-3p mimics-transfected HL-1s (n = 4). E Acvr1b mRNA expression levels in NC inhibitor or miR-210-3p inhibitor-transfected HL-1s (n = 4). F Acvr1b mRNA expression levels in BMSC-EVs vs. Vehicle treated DOX HL-1s (n = 4). G Representative western blotting images and quantitative analysis of NC mimics or miR-210-3p mimics-transfected HL-1s (n = 3). H Representative western blotting images and quantitative analysis of NC inhibitor or miR-210-3p inhibitor-transfected HL-1s (n = 3). Data are shown as mean ± SD. A through H, two-way ANOVA followed by the Bonferroni multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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  Fig. 6. miR-210-3p targeting ACVR1B inhibits mitochondrial damage in DIC. A Representative PI-stained images and B MTT assay for cell viability in NC inhibitor, miR-210-3p inhibitor, siNC+ siACVR1B or miR-210-3p inhibitor+siACVR1B-transfected HL-1s (n = 9). C Representative images of mitoSOX-stained and D Quantification of oxidative stress levels in NC inhibitor, miR-210-3p inhibitor, siNC+ siACVR1B or miR-210-3p inhibitor+siACVR1B-transfected HL-1s (n = 5, scale bar = 100 μm). E Representative JC-1-stained HL-1s and F quantification of mitochondrial membrane potential levels (n = 5, scale bar = 100 μm). G Representative western blotting images, H Quantitative analysis of ACVR1B, I–L quantitative analysis of ETC-related proteins, and M, N quantitative analysis of PGC-1α and phosphorylated SMAD3 in HL-1s (n = 3). Data are shown as mean ± SD. A through N, two-way ANOVA followed by the Bonferroni multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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  Fig. 7. miR-210-3p suppresses mitochondrial Dysfunction and cardiac dysfunction in DIC. A Experimental timeline and design of the DOX mice treated with AAV9-NC and AAV9-miR-210-3p. B–D Echocardiographic analysis of LV ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) of mice (n = 5). E Representative images of hematoxylin and eosin-stained mouse heart tissue (scale bar = 50 μm). F Masson trichrome staining, and G Quantification of fibrosis area (n = 5, scale bar = 50 μm). H Representative western blotting images, and I quantitative analysis of ETC-related proteins in mouse heart tissue (n = 3). J Representative western blotting images, and K quantitative analysis of ACVR1B, PGC-1α and phosphorylated SMAD3 in mouse heart tissue (n = 3). Data are shown as mean ± SD. A through J, two-way ANOVA followed by the Bonferroni multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001