BIRC3-CASP8 axis orchestrates the PANoptosis spectrum: taming the inflammatory storm to prevent post-ischemic heart failure

Figures

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  Figure 1. Study workflow. The workflow summarizes the experimental and analytical strategy used to investigate PANoptosis in myocardial ischemia-reperfusion injury, including validation of PANoptosis occurrence, assessment of single-pathway PCD inhibition, screening of core PANoptosis-related pathways, and experimental validation of the BIRC3-CASP8 axis. Abbreviations: I/R, ischemia/reperfusion; PANoptosis, an integrated programmed cell death process involving apoptosis, pyroptosis, and necroptosis; PCD, programmed cell death; TEM, transmission electron microscopy; H9c2, rat cardiomyoblast cell line H9c2; OGD/R, oxygen-glucose deprivation/reperfusion; si-ZBP1, small interfering RNA targeting Z-DNA binding protein 1; PI, propidium iodide; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; EthD-III, ethidium homodimer III; IF, immunofluorescence; BIRC3, baculoviral IAP repeat containing 3; CASP8, caspase-8.

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  Figure 2. PANoptosis activation in myocardial ischemia-reperfusion injury. (A) Immunofluorescence images of C-CASP3, N-GSDMD, and P-MLKL. (B) Overlap in the executive proteins of the three PCD types, indicated by white arrows. (C) Transmission electron microscopy images. (D) Western blot images and quantification of programmed cell death–related biomarkers after silencing PANoptosis-related ZBP1 (n=3). (E) Western blot images and quantification of PCD-related biomarkers after treatment with apoptosis, pyroptosis, and necroptosis inhibitors (n=3). *P<0.05, **P<0.01, ***P<0.001.

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  Figure 3. Mortality rates of different PCD types. (A–C): Representative images and quantitative results of TUNEL staining, EthD-III staining, and PI staining, respectively, following treatment with apoptosis, pyroptosis, and necroptosis inhibitors (n=5).

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  Figure 4. Protein–protein interaction and pathway analysis of PANoptosis-related proteins. (A) PPI network of PANoptosis-related proteins within the corresponding programmed cell death pathways. (B) Reactome pathway enrichment analysis. (C) Bayesian regulatory network of PANoptosis-related genes.

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  Figure 5. Transcriptomic and single-cell sequencing analyses of PANoptosis in heart failure. (A) Key PANoptosis-related genes identified using combined machine learning algorithms. (B) Immune infiltration analysis, cell death scoring, and correlation heatmap of key genes and scoring metrics in the training set using CIBERSORT and ssGSEA algorithms. (C) Cell type identification in the GSE247468 dataset. (D) Cell death scores of cardiomyocytes. (E) Pseudotime expression analysis. (F) Gene expression changes after virtual knockout of BIRC3. ****P<0.0001.

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  Figure 6. Experimental validation of BIRC3 and CASP8 in PANoptosis regulation. (A) Western blot analysis of the effects of BIRC3 modulation on cell death patterns in OGD/R-treated H9C2 cells (n=3). (B) Staining analysis of changes in apoptosis, pyroptosis, and necroptosis after BIRC3 up- or downregulation (n=5). (C) Immunofluorescence analysis of the effects of BIRC3-CASP8 on the expression of PCD executive proteins (n=5). *P<0.05, **P<0.01, ***P<0.001.

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  Figure 7. In vivo validation of BIRC3 in PANoptosis regulation. (A) Western blot analysis of the effects of BIRC3 knockdown and apoptosis inhibition on PANoptosis in heart tissue (n=3). (B) Echocardiographic assessment of LVEF improvement following combined BIRC3 silencing and Z-VAD-FMK treatment (n=5). (C) Immunofluorescence analysis of reduced PANoptosis occurrence after the combined intervention. *P<0.05, **P<0.01, ***P<0.001.

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  Figure 8. BIRC3-CASP8 axis regulates PANoptosis in myocardial ischemia-reperfusion injury. BIRC3 promotes PANoptosis by inhibiting CASP8 activation, leading to inflammatory cell death and myocardial damage. BIRC3 knockdown or inhibition restores CASP8 activity, shifts cell death toward restrained apoptosis, and preserves cardiac function.