Trophoblast Stem-Cell-Derived Exosomes Improve Doxorubicin-Induced Dilated Cardiomyopathy by Modulating the let-7i/YAP Pathway

Figure 1. Characterization of Trophoblast Exosomes (A) Transmission electron microscopic analysis of exosomes. Scale bar, 200 nm. (B) The particle sizes of the extracted exosomes. (C) Western blot analysis of exosomal marker proteins, including CD9, flotillin, and Hsp70. CM, culture medium; EXO, exosomes. (D) Representative pictures of AC16 cell uptake of exosomes. White arrow indicated the cells with exosomes.

Figure 2. Differentially Expressed microRNAs in DCM and Enriched in Trophoblast Exosomes (A) Differentially expressed miRNAs in DCM patients. (B) Highly expressed miRNAs in trophoblast exosomes. (C) The expression level of let-7i-5p in the control and dilated cardiomyopathy groups. (D) The expression level of let-7i-5p in the control and DOX-exposed AC16 cells by qPCR assays. All data are presented as the mean ± SEM, n = 6/group. ∗∗p < 0.01; ∗∗∗p < 0.001, compared to the control group.

Figure 3. Effect of let-7i on the Expression of YAP1 in DCM (A) The relative expression level of YAP1 from the serum of healthy individuals and HF patients from the GEO dataset. (B) Western blot analysis of YAP1 in the myocardial tissue of DOX-induced HF mice. (C) Relative protein levels were analyzed by densitometric analysis. (D) An illustration of the seed sequence of let-7i binding to the wild-type and mutated 3′-UTR of YAP1 mRNA based on bioinformatic prediction. (E) Dual luciferase reporter results of HEK293T cells cotransfected with luciferase reporter vectors (YAP1 3′-UTR-WT or YAP1 3′-UTR-mut) and miRNA (let-7i-5p-NC or let-7i-5p-OE). NC, negative control mimics; OE, overexpression. All data are presented as the mean ± SEM, n = 6/group. ∗∗p < 0.01; ∗∗∗p < 0.001, compared to the control group.

Figure 4. Effect of TSC-exo and let-7i on Apoptosis in DOX-Induced Cardiomyocytes In Vitro via YAP-Hippo Signaling The groups were categorized into the control, DOX, DOX-Exo, and DOX-let-7i-5p groups based on the treatment of DOX or let-7i. (A) Apoptosis results detected by flow cytometry. (B) The percentage of early apoptotic cells (Annexin V+ PI–), late apoptotic cells (Annexin V+ PI+), and overall apoptotic cells (Annexin V+). (C and D) Western blot of Bcl2 and cleaved caspase 3 (C) and (D) western blot of YAP1, CTGF, and TEAD1 (D) in the control group and the groups treated with DOX, DOX-Exos, and DOX-let-7i-5p. And relative protein levels were analyzed by densitometric analysis. (E–G) The mRNA expression levels of (E) YAP1, (F) CTGF, and (G) TEAD1 in the group treated with control, DOX, DOX-Exos, and DOX-let-7i-5p were measured by real-time PCR. All data are presented as the mean ± SEM, n = 6/group. ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group; #p < 0.05, ##p < 0.01, ###p < 0.001 compared to the DOX-treated group.

Figure 5. Effect of Trophoblasts Exosomal let-7i on DOX-Injured Heart Function In Vivo 8-week-old male mice were divided into four groups: control, DOX, DOX-Exo, and DOX-AAVup-let-7i-5p. (A) Left ventricular ejection fraction (LVEF, %). (B) Left ventricular fraction shortening (LVFS, %). (C) Left ventricular internal diameter in diastole (LVIDs, %). (D) Left ventricular end-diastolic diameter (LVEDd, %). (E) The mRNA expression levels of ANP, β-MHC, and type I collagen in myocardial tissue were measured by real-time PCR. (F) Representative micrographs of Masson staining of heart tissue sections. Scale bar, 1,000 μm. All data are presented as the mean ± SEM, n = 6 mice/group. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group; #p < 0.05, compared to the DOX treatment group.

Figure 6. Effect of Exosomes on YAP1/Hippo Signaling Pathway by Delivery of let-7i 8-week-old male mice were divided into four groups: control, DOX, DOX-Exo, and DOX-AAVup-let-7i-5p. (A) The mRNA expression level of let-7i-5p in myocardial tissue. (B) The mRNA expression level of YAP1 in myocardial tissue. (C) The serum level of IL-6. (D) The serum level of IL-1β. (E) Western blot analysis of YAP1, cleaved caspase 3, CTGF, and ANP. (F) Relative protein levels were analyzed by densitometric analysis. All data are presented as the mean ± SEM, n = 6 mice/group. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to the control group; #p < 0.05, compared to the DOX treatment group.