LncRNA ZNF593-AS alleviates diabetic cardiomyopathy via suppressing IRF3 signaling pathway

Figures

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  Figure 1. LncRNA ZNF593-AS expression is decreased in diabetic hearts (A) ZNF593-AS expression in the hearts of db/db mice and high-fat-diet mice using RT-qPCR. (B) Co-localization of ZNF593-AS with cardiomyocyte-specific marker using FISH assay. (C) RT-qPCR analysis of ZNF593-AS in AC16 and HL-1 cells under the stimulation of PA. (D) FISH of ZNF593-AS in AC16 and HL-1 cells under the stimulation of PA. Comparisons were performed by Student’s t test for (A) and (C).

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  Figure 2. ZNF593-AS attenuates diabetes-induced cardiac dysfunction (A) Schematic of the strategy used for ZNF593-AS deletion. (B, C, and D) Echocardiographic analysis and hemodynamic parameters of ZNF593-AS KO and WT mice fed with high-fat or normal diet: EF value, FS value (B), E/A ratio (C), dp/dtmax and dp/dtmin (D). (E and F) Immunostaining of TUNEL staining in the hearts of ZNF593-AS KO and WT mice fed with high-fat or normal diet. (G) Schematic of the strategy used for ZNF593-AS cardiomyocyte-specific Tg mice construction. (H, I, and J) Echocardiographic analysis and hemodynamic parameters of ZNF593-AS Tg and WT mice fed with high-fat or normal diet: EF value, FS value (H), E/A ratio (I), dp/dtmax, and dp/dtmin (J). (K and L) Immunostaining of TUNEL staining in the hearts of ZNF593-AS Tg and WT mice fed with high-fat or normal diet. One-way ANOVA with the Tukey post-test was used for comparisons.

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  Figure 3. ZNF593-AS interrupts IRF3-dependent signaling via suppressing IRF3 phosphorylation (A) Flow cytometry analysis showed ZNF593-AS knockdown accelerated PA-induced apoptosis of HL-1 cells. (B) Flow cytometry analysis showed ZNF593-AS overexpression antagonized PA-induced apoptosis of HL-1 cells. (C) Scatterplot diagram of DEGs between control and ZNF593-AS overexpression groups in PA-treated HL-1 cells. Blue points represent downregulated genes, red points represent upregulated genes, gray points represent non-DEGs, and black points represent Irf3 (fold change >2 or <0.5, p < 0.05). (D) RT-qPCR validation of the expression of representative IRF3-dependent genes. (E) Western blotting analysis of phosphorylated and total IRF3 in HL-1 lysates of control and ZNF593-AS overexpression under PA treatment. Student’s t test was used for comparisons in (D) and (E). One-way ANOVA with the Tukey post-test was used in (A) and (B).

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  Figure 4. IRF3 signaling pathway is activated in diabetic heart (A) qPCR analysis of mtDNA in cytosolic fraction. Nuclear DNA,18S rDNA served as loading control. (B) Western blotting analysis of phosphorylated and total IRF3 in the hearts of HFD and db/db mice (mouse IRF3 band was observed in approximately 50 kDa). (C) Western blotting analysis of phosphorylated and total IRF3 in the hearts of heart failure patients with or without diabetes (human IRF3 band was observed in approximately 55 kDa). (D) RT-qPCR analysis of ZNF593-AS levels in the hearts of heart failure patients with or without diabetes. (E) Correlation analysis between ZNF593-AS expression and IRF3 phosphorylation level. Student’s t test was used for comparisons in (A), (B), (C), and (D). Pearson correlation analysis was used in (E).

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  Figure 5. ZNF593-AS inhibits diabetes-induced activation of IRF3 signaling pathway (A and B) Western blotting analysis of IRF3 in cytoplasmic and nuclear extracts of HL-1 (A) and AC16 cells (B) transfected with ZNF593-AS under the stimulation of PA. GAPDH and Lamin B1 served as loading control. (C, D, and E) Western blotting analysis of phosphorylated and total IRF3 (C), western blotting analysis of cytoplasmic and nuclear IRF3 (D), and RT-qPCR analysis of the expression of Cxcl10, Ccl2, and Ccl5 mRNA levels (E) in ZNF593-AS KO and WT mice hearts. (F, G, and H) Western blotting analysis of phosphorylated and total IRF3 (F), western blotting analysis of cytoplasmic and nuclear IRF3 (G), and RT-qPCR analysis of the expression of Cxcl10, Ccl2, and Ccl5 mRNA levels (H) in ZNF593-AS Tg and WT mice hearts. One-way ANOVA with the Tukey post-test was used for comparisons.

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  Figure 6. ZNF593-AS directly binds IRF3 (A and B) Anti-apoptotic effect of ZNF593-AS overexpression was blocked by IRF3 knockdown (A) and BX795 treatment (4 μM) (B). (C) RNA pull-down assay followed by western blotting of IRF3. HL-1 and AC16 cell lysates were incubated with biotin-labeled sense or antisense (negative control) of ZNF593-AS. (D) RIP assay to determine the binding of IRF3 and ZNF593-AS by using IRF3 antibody or IgG (negative control) in HL-1 and AC16 cells. (E) Schematic diagram of IRF3 full-length and truncations deleting different domain with FLAG tag at C-terminal (upper panel). Western blotting analysis of HL-1 cells transfected with IRF3 full-length and different truncations (lower panel). (F) RIP assay with FLAG antibody in HL-1 cells transfected with IRF3 full-length and different truncations. (G) Schematic diagram of the function of ZNF593-AS in diabetic cardiomyopathy. Student’s t test was used for comparisons in (D). One-way ANOVA with the Tukey post-test was used (A) and (B).