Plasma exosomal miR‐339‐3p promotes myocardial remodeling in chronic heart failure by regulating USP25‐mediated DDX58 deubiquitination

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  FIGURE 1. Exosomes isolated from the healthy controls and HF patients. To confirm the extraction efficiency of plasma exosomes from patients with HF and their uptake ability by cardiomyocytes, plasma samples from 5 HF patients and 5 healthy individuals were used as research objects. (A) Transmission electron microscopy images of exosomes from healthy controls and HF patients, displaying round or cup‐shaped vesicles with a diameter of 30–100 nm, which is consistent with the typical morphological characteristics of exosomes. (B) The expression of exosomal markers (CD9, CD63 and TSG101) by western blot analysis. (C) Confocal microscopy showing PKH26‐labeled exosomes were taken up by AC16 cardiomyocytes. HF, heart failure.

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  FIGURE 2. Plasma exosomes from HF patients promote cardiomyocyte apoptosis and hypertrophy. To investigate the effect of plasma exosomes from HF patients on cardiomyocytes, AC16 cells were divided into four groups: normal control group (NC), angiotensin II‐treated group (Ang II), Ang II + healthy human plasma exosome group (Ang II + NC‐exo), and Ang II + HF‐exo group. (A) CCK‐8 assay for detecting cell viability, showing that compared with the NC group, cell viability in the Ang II group is significantly reduced, and the Ang II + HF‐exo group exhibits a further decrease in cell viability compared to the Ang II and Ang II + NC‐exo groups. (B) Flow‐cytometry for detecting apoptosis, revealing that the apoptosis rate in the Ang II group is higher than that in the NC group, and the Ang II + HF‐exo group has a significantly higher apoptosis rate than the Ang II and Ang II + NC‐exo groups. (C) The surface area of AC16 cells after FITC‐Phalloidin staining, showing that the Ang II group has a significantly increased hypertrophy area compared to the NC group. (D, E) RT‐PCR and Western blot for detecting the expression of hypertrophy‐related markers A‐type natriuretic peptide, B‐type natriuretic…

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  FIGURE 3. Screening of different expressed blood exosomes miRNA between healthy controls and HF patients. To screen for differentially expressed miRNAs in plasma exosomes from HF patients (HF‐exo) and healthy individuals (NC‐exo), miRNA sequencing analysis was performed on the two groups of exosomes. A total of 1129 miRNAs were detected. (A) Volcano plot analysis of miRNA expression differences, identifying 12 differentially expressed miRNAs. (B) Heatmap analysis displays the expression patterns of differential miRNAs in NC‐exo and HF‐exo samples. (C) Reverse transcription‐quantitative polymerase chain reaction validation of differently expressed miRNAs. (D) HF patient‐derived exosomes increase miR‐339‐3p expression in Ang II‐treated cardiomyocytes. *, **, and *** represent p < 0.05, p < 0.01, and p < 0.001 compared to the NC‐exo group, respectively. ### p < 0.0001 versus Ang II group. HF, heart failure; miRNA, microRNA; NC, negative control.

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  FIGURE 4. miR‐339‐3p in plasma exosomes of HF patients promotes cardiomyocyte apoptosis and hypertrophy. To verify the regulatory role of miR‐339‐3p in plasma exosomes from HF patients on cardiomyocytes, AC16 cells were divided into three groups: Ang II group, Ang II + HF‐exo + negative control inhibitor group (Ang II + HF‐exo + NC inhibitor), and Ang II + HF‐exo + miR‐339‐3p inhibitor group (Ang II + HF‐exo + miR‐339‐3p inhibitor). (A) Transfection efficiency detected by RT‐qPCR, confirming that the miR‐339‐3p inhibitor can effectively reduce the expression of miR‐339‐3p in cells. (B) Expression of miR‐339‐3p in AC16 cells detected by RT‐qPCR. (C) Cell viability detected by CCK‐8 assay shows that compared with the Ang II and Ang II + HF‐exo + NC inhibitor groups, the Ang II + HF‐exo + miR‐339‐3p inhibitor group has significantly increased cell viability. (D) Cell death detected by flow cytometry. (E) FITC‐phalloidin staining of surface area of AC16 cells. (F) Expression of pathological hypertrophy markers ANP, BNP, and Myh7 detected by RT‐qPCR. (G) Expression of pathological hypertrophy markers ANP, BNP and Myh7 detected by western blot analysis. ** and *** represent p < 0.01 and p <…

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  FIGURE 5. Identification of downstream target genes of miR‐339‐3p. (A) To screen the downstream target genes of miR‐339‐3p, predictive analysis was conducted using three databases: TargetScan (728 target genes), miRDB (46 target genes), and Starbase (256 target genes). (B) RT‐qPCR detection of miR‐339‐3p expression in cells after transfection with miR‐339‐3p mimic. (C) In order to explore the effect of miR‐339‐3p on NREP, USP25 and IP6K2 genes, we set up four treated groups: the NC mimic group, miR‐339‐3p mimic group, NC inhibitor group and miR‐339‐3p inhibitor group. Expression of target genes NREP, USP25 and IP6K2 in AC16 cells detected by RT‐qPCR analysis. (D) Expression of target gene USP25 in different treated groups accessed by western blot analysis. (E) Luciferase assay used to confirm the miR‐339‐3p targets USP25. (F) RNA immunoprecipitation assay performed to confirm that miR‐339‐3p targets USP25. ** and *** represent p < 0.01 and p < 0.001 compared to the control group (NC mimic group or anti‐IgG group). # and ### represents p < 0.05 and p < 0.001 compared to the miR‐339‐3p mimic group or NC inhibitor group. NC, negative control; RNA, Ribonucleic acid; RT‐qPCR, reverse…

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  FIGURE 6. miR‐339‐3p promotes cardiomyocyte apoptosis and hypertrophy by targeting USP25. To verify whether miR‐339‐3p regulates cardiomyocyte function by targeting USP25, AC16 cells were divided into four groups: Ang II + NC mimic + NC OE group (Ang II + NC mimic + NC OE), Ang II + miR‐339‐3p mimic + NC OE group (Ang II + miR‐339‐3p mimic + NC OE), Ang II + NC mimic + USP25 OE group (Ang II + NC mimic + USP25 OE), and (Ang II + miR‐339‐3p mimic + USP25 OE). (A) Transfection efficiency of USP25 OE. (B) RT‐qPCR results of USP25 relative mRNA expression. (C) Cell viability detected by CCK‐8. (D) Detection of cell apoptosis by flow cytometry. (E) Detection of cardiomyocyte volume by immunofluorescence staining. (F) Detection of pathological hypertrophy markers ANP, BNP, and Myh7 expression by RT‐qPCR. (G) Expression of pathological hypertrophy markers ANP, BNP, and Myh7 detected by western blot analysis. *, **, and *** represent p < 0.05, p < 0.01, and p < 0.001 compared to the control group (Ang II + NC mimic + NC OE group). #, ##, and ### represents p < 0.05, p < 0.01, and p < 0.001 compared to the Ang II + NC mimic + USP25 OE group. ANP, A‐type natriuretic peptide; BNP, B‐type natriuretic…

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  FIGURE 7. USP25 promotes the deubiquitination of DDX58. To explore the interaction between USP25 and DDX58 and its regulatory effect on DDX58 ubiquitination, a series of experiments were conducted. (A) Protein–protein interaction prediction analysis on USP25 using the String website detects DDX58 as an interacting protein of UPS25. (B) Pearson correlation analysis based on the TCGA database shows a positive correlation between USP25 and DDX58 expression in human left ventricular tissues. (C) Relationship between USP25 and DDX58 detected by co‐immunoprecipitation analysis. (D) Expression of DDX58 in negative control OE group and USP25 OE group detected by western blot analysis. (E) Ubiquitination level of DDX58 detected by IP experiment with overexpressed USP25. (F) Endogenous DDX58 ubiquitination level detected by IP with MYC‐tagged antibody. (G) K48 and K63 ubiquitin chains of DDX58 detected by western blot analysis. OE, overexpression; TCGA, the Cancer Genome Atlas; USP25, ubiquitin‐specific protease 25.

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  FIGURE 8. USP25 inhibits cardiomyocyte apoptosis and hypertrophy by stabilizing DDX58. To clarify whether USP25 exerts its regulatory effect on cardiomyocytes by stabilizing DDX58, AC16 cells were divided into four groups: Ang II + NC OE + sh‐NC group, Ang II + USP25 OE + sh‐NC group, Ang II + NC OE + sh‐DDX58 group, and Ang II + USP25 OE + sh‐DDX58 group. (A) Transfection efficiency of sh‐DDX58 shows that the sh‐DDX58‐1 has the highest transfection efficiency, so we chose the sh‐DDX58‐1 for the next experiments. (B) Cell viability detected by CCK‐8 assay. (C) Cell apoptosis detected by flow cytometry. (D) Cardiomyocyte volume detected by immunofluorescence staining. (E) Expression of pathological hypertrophy markers ANP, BNP and Myh7 detected by RT‐qPCR. (F) Expression of pathological hypertrophy markers ANP, BNP and Myh7 detected by western blot analysis. *, **, and *** represent p < 0.05, p < 0.01, and p < 0.001 compared to the Ang II + NC OE + sh‐NC group. #, ##, and ### represents p < 0.05, p < 0.01, and p < 0.001 compared to the Ang II + USP25 OE + sh‐NC group. ANP, A‐type natriuretic peptide; BNP, B‐type natriuretic peptide; NC, negative control; OE, overexpression; USP25,…

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  FIGURE 9. miR‐339‐3p inhibition alleviates TAC‐induced ventricular remodeling in in vivo experiments. To verify the regulatory effect of miR‐339‐3p on ventricular remodeling in vivo, a TAC‐induced mouse model of ventricular remodeling was established, and mice were divided into four groups: Sham + anti‐NC group, Sham + anti‐miR‐339‐3p group, TAC + anti‐NC group, and TAC + anti‐miR‐339‐3p group. (A) HW to TL ratio, showing that compared with the Sham + anti‐NC group, the TAC + anti‐NC group has a significantly increased HW/TL ratio. (B) Gross heart images of mice and quantification of gross heart sectional area, demonstrating that the TAC + anti‐NC group has an enlarged heart and increased sectional area compared to the Sham + anti‐NC group, while anti‐miR‐339‐3p reversed this change. (C) Echocardiographic images, revealing that the TAC + anti‐NC group exhibits abnormal cardiac structure and function compared to the Sham + anti‐NC group, while anti‐miR‐339‐3p makes these indicators return to normal. (D) EF and FS of mice detected by echocardiography, revealing that the TAC + anti‐NC group reduced EF and FS compared to the Sham + anti‐NC group, while anti‐miR‐339‐3p makes these indicators…

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  FIGURE 10. miR‐339‐3p inhibition suppressed TAC‐induced ventricular remodeling in in vivo experiments. To further confirm the effect of miR‐339‐3p on TAC‐induced ventricular remodeling, pathological staining and molecular detection were performed on the four groups of mice: Sham + anti‐NC, Sham + anti‐miR‐339‐3p, TAC + anti‐NC, TAC + anti‐miR‐339‐3p. (A) Hematoxylin‐eosin staining of heart tissue of different mice treated groups, the upper panels show low‐magnification images of heart sections, and lower panels show high‐magnification views of the corresponding areas to display detailed histological changes. (B) Myocardial fibrosis was assessed by Masson's trichrome staining. (C) Cardiomyocyte hypertrophy was evaluated by wheat germ agglutinin staining. (D) Cardiomyocyte apoptosis was detected by TUNEL staining. (E) mRNA expressions of ANP, BNP and Myh7 in different treated groups detected by RT‐qPCR. (F) Western blot results and quantification of protein expression of ANP, BNP and Myh7 in different treated groups detected by western blot analysis. *** represents p < 0.001 compared to the Sham + anti‐NC group. ## and ### represent p < 0.05 and p < 0.001 compared to the TAC + anti‐NC group.…