IFI-16 inhibition attenuates myocardial remodeling following myocardial infarction

Figures

• 

  Figure 1. Elevation of IFI-16/IFI-204 inflammasome following myocardial infarction (A and B) Western blot analysis depicting the expression levels of IFI-16, ASC, pro-caspase-1, cleaved-caspase-1, GSDMD, and cleaved-GSDMD in AC16 myocytes exposed to hypoxia and nutrient deprivation for 0–6 h (control, H2, H4, and H6) with sample collection at the specified time points. (C) Timeline outlining the collection of heart samples at 3 hours (3 h), 3 days (3 d), and 2 weeks (2 w) after ligation to establish a myocardial infarction (MI) mouse model. Sham heart samples were obtained at 2 weeks after surgery. Hematoxylin and eosin (H&E) staining, along with IFI204 immunostaining, was performed in a mouse MI model at different time points. (D and E) Western blot analysis shows the expression of IFI-204, ASC, pro-caspase-1, cleaved-caspase-1, GSDMD, and cleaved-GSDMD in mouse heart tissue collected at different time points after coronary ligation and in sham-operated hearts. The western blot experiments were repeated three times, and the data are presented as mean ± SEM.

• 

  Figure 2. IFI-16 recognition of mitochondrial released DNA (A) Quantification of released mitochondrial DNA copy number in AC16 myocytes exposed to hypoxia and nutrient deprivation for 4 h (H4) compared to the normal control (Con) using qPCR with D loop1, a mitochondrial DNA-specific primer. (B) Measurement of mitochondrial DNA leakage in hypoxia and nutrient deprivation for 4 h (H4) with or without VBIT4 treatment (5 μM). (C and D) Western blot analysis illustrating the expression levels of IFI-16, cleaved-caspase-1, and pro-caspase-1 in AC16 myocytes in hypoxia and nutrient deprivation for 4 h (H4) with or without VBIT4 treatment. (E) Immunofluorescence staining displaying the localization of IFI-16 (labeled with FITC-tagged secondary antibody) and mitochondria (labeled with mito-tracker-red) in hypoxia and nutrient deprivation for 4 h and normal control in AC16 cells. Western blot experiments were repeated three times, and data are presented as mean ± SEM.

• 

  Figure 3. IFI-16/IFI-204 knockdown suppress inflammasome-associated proteins activation following myocardial infarction (A and B) Myocytes were transfected with the lentivirus, ifi16-shRNA, to knock down IFI-16 expression, followed by exposure to hypoxia and nutrient deprivation for 4 h (H4). Scramble-shRNA was used as control. Western blot analysis was performed to assess the levels of IFI-16, ASC, pro-caspase-1, cleaved-caspase-1, GSDMD, and cleaved-GSDMD. (C and D) Injection of adeno-associated virus (AAVs) to knock down ifi204 expression in mice was used, with AAV-Ctrl injection serving as the normal control. Two weeks after surgery, hearts were harvested for further examination after ultrasound echocardiography. Western blot analysis of IFI-16, ASC, pro-caspase-1, cleaved-caspase-1, GSDMD, and cleaved-GSDMD expression in heart tissue obtained from mice in the different treatment groups. (E) The assessment of IFI-204 expression within tissues was conducted through immunofluorescence analysis. In this analysis, green fluorescence signified wheat germ agglutinin (WGA), blue fluorescence represented DAPI staining, and red fluorescence indicated the presence of Cy3-labeled IFI-204. Scale…

• 

  Figure 4. Knockdown of IFI-16/IFI-204 reduces apoptosis in tissues and cells after myocardial infarction (A) TUNEL detection in myocytes. Lentiviral transfection was utilized to knock down IFI-16 in AC16 cells. Subsequently, TUNEL detection was performed after 4 h of hypoxia and nutrient deprivation (H4). Images captured via laser confocal microscopy depict blue fluorescence for DAPI and red fluorescence for TUNEL-positive cells. Scale bar: 100 μm. Statistical analysis was conducted using ImageJ. (B) Western blot analysis was performed to evaluate the expression of p53 and cleaved caspase-9 in myocytes in both the scramble and shRNA groups. (C) TUNEL staining was conducted on cardiac slices from the sham group, AAV-Ctrl group, and AAV-IFI204 group. Laser confocal microscopy captured images, and statistical analysis was performed using ImageJ. Scale bar: 50 μm. (D) Western blot detection and statistical analysis were employed to assess the expression of p53 and cleaved-caspase-9 in the sham group, AAV-Ctrl group, and AAV-IFI204 group. The western blot experiments were replicated three times, and data are presented as mean ± SEM.

• 

  Figure 5. IFI-204 knockdown improves heart function and attenuates remodeling post myocardial infarction (A) Two weeks post-surgery, mice were anesthetized with pentobarbital (50 mg/kg) for echocardiography. Parasternal short axis view and parasternal long axis view images were obtained. Statistical analysis of echocardiography data was performed in the sham, AAV-Ctrl, and AAV-IFI204 groups (n = 5 in each group). (B and C) Images of Masson staining in the sham, AAV-Ctrl, and AAV-IFI204 groups (n = 5) were captured. Statistical analysis of fibrosis proportion in Masson-stained samples was conducted. (D and E) Images of H&E staining in the sham, AAV-Ctrl, and AAV-IFI204 groups (n = 5) were obtained, and statistical analysis of the number of cells within the field of view was conducted. (F) Western blot analysis was performed to assess TNF-α and α-SMA expression in heart tissue obtained from mice in the Sham, AAV-Ctrl, and AAV-IFI204 groups. Data are presented as mean ± SEM.

• 

  Figure 6. Consistent expression trends of IFI-16 and galectin-3 (A) Pearson correlation heatmap illustrates the repeatability of samples within and between groups in myocytes overexpressing (OV) IFI-16 with the IFI16-pcdh-GFP+PURO plasmid compared to the control group using the pcdh-GFP+PURO plasmid (n = 3), all samples were subjected to 4-h hypoxia and nutrient deprivation (H4). (B) Volcano plots depict the differentially expressed genes between the OV-H4 and H4 groups. Red and green dots represent up-regulated (189 dots) and down-regulated (21 dots) transcripts, respectively (p < 0.05, [log2fc] > 1). (C) An analysis of disease association was performed on the different genes using disgenet. (D) Heatmap displays myocardial ischemia-related genes between the OV-H4 and Vector-H4 groups. (E and I) Western blot analysis of galectin-3 expression in AC16 myocytes during the in vitro hypoxia and nutrient deprivation for 0–6 h. (F and J) Western blot analysis of galectin-3 expression in IFI-16 knockdown AC16 cells in 4 h of hypoxia and nutrient deprivation in vitro model (H4). (G and K) Western blot analysis of galectin-3 expression in the hearts of the post-infarction myocardial remodeling mouse…

• 

  Figure 7. IFI-16 combines to galectin-3 directly (A and B) Western blot analysis of co-immunoprecipitated products. (A) The eluted products obtained from co-immunoprecipitation (coIP) were subjected to western blot detection for GAPDH and galectin-3. (B) The eluted products were subjected to western blot detection for IFI-16 and galectin-3. (C) Fluorescence double staining analysis in AC16 cells. Galectin-3 was labeled using a Cy3 secondary antibody, while IFI16 was labeled with an FITC secondary antibody. DAPI staining was additionally employed. The images were captured using laser confocal microscopy. (D) Fluorescence double staining analysis in heart slices. Galectin-3 was labeled using an FITC secondary antibody, while IFI16 was labeled with a Cy3 secondary antibody. DAPI staining was additionally employed. The images were magnified by a factor of 63×. (E) Overexpressing (OV) IFI-16 with the IFI16-pcdh-GFP+PURO plasmid and siRNA-mediated interference with galectin-3 expression. Con-siRNA and Con-OV served as the negative control group. Western blot analysis was performed to assess the levels of IFI-16, galectin3, ASC, pro-caspase-1, cleaved-caspase-1, GSDMD, and cleaved-GSDMD. ∗…