GABARAPL1 Exerts Regulatory Effects on Hypoxia‐Induced Pyroptosis in the Pathogenesis of Myocardial Infarction

FIGURE 1. Identification of DEGs. (A) The Volcano plots of DEGs in the GSE66360 dataset. (B) The Venn diagram of DEGs in the GSE66360 and autophagy and pyroptosis‐related genes. (C) Cumulative residual distribution of each machine learning model. (D) Boxplots showing the residuals of each machine learning model. Red dot represented the RMSE values. (E) The important features in KNN, RF, SVM, GLM and XGB machine models. (F) ROC analysis of five machine learning models based on 5‐fold cross‐validation. (G) Box plots showing differential expressions of GABARAPL1, IL1β, FN1, NLRP3 and GZMB in MI and Control samples. ***p < 0.001 vs. Control group. (H) PPI network of the five important genes. (I) GO analysis of the genes in the PPI network. (J) KEGG analysis of the genes in the PPI network.

FIGURE 2. GSEA analysis and Immune infiltration analysis. (A) The heatmap of chemokine, Immunostimulator, Immunoinhibitor, MHC and receptor in the GSE66360 dataset. (B) Correlation analysis of FN1, GABARAPL1, GZMB, NLRP3 and IL1β with chemokine in the GSE66360 dataset. (C) The Violin plot of the immune cells in the GSE66360 dataset. (D–H) Correlation analysis of FN1, GABARAPL1, GZMB, NLRP3 and IL1β with immune cells in the GSE66360 dataset.

FIGURE 3. ROC curve and Nomogram analysis. (A‐E) The ROC curve for evaluating the diagnostic efficacy of GABARAPL1, IL1β, FN1, NLRP3, and GZMB. (F) Construction of a nomogram for predicting the risk of the five pivotal genes based on the SVM model.

FIGURE 4. GABARAPL1 expression is increased in the myocardium of post‐MI mice. (A–C) qRT‐PCR and Western blotting analyses of GABARAPL1 expression in mouse heart tissues at the indicated time points post‐MI. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Sham group; n = 3. (D–F) qRT‐PCR and Western blotting analyses of GABARAPL1 expression in C166 cells with or without CoCl2. **p < 0.01 vs. Con group; n = 3. (G–I) qRT‐PCR and Western blotting analyses of GABARAPL1 expression in HUVEC cells with or without CoCl2; HIF1a was used as a marker of hypoxia. **p < 0.01 vs. Con group; n = 3. (J, K) Western blotting analyses of GABARAPL1 expression in C166 cells with or without hypoxia. **p < 0.01 vs. Con group; n = 3. Data were presented as mean ± SD. Statistical significance was assessed by one‐way ANOVA and Student's t‐test.

FIGURE 5. GABARAPL1 modulates hypoxic‐induced pyroptosis in endothelial cells. (A,B) Western blotting analyses of LC3B, NLRP3, and IL1β expression in endothelial cells with or without CoCl2. **p < 0.01 vs. Con group; n = 3. (C–F) Western blotting analyses showing that the expression of pyroptosis factors was significantly increased under hypoxia, which was markedly aggravated by siGABARAPL1. **p < 0.01, ***p < 0.001 vs. siCtrl group; ## p < 0.01 vs. siCtrl + CoCl2 group; n = 3. Data were presented as mean ± SD. Statistical significance was assessed by one‐way ANOVA and Student's t‐test.

FIGURE 6. PIK90 treatment significantly alleviates cardiac dysfunction and myocardial fibrosis induced by MI. (A) The timeline of the experimental design for PIK90 treatment. (B) Survival rate analysis. (C) Representative echocardiographic images on the 28 days post‐MI. (D) Echocardiographic analysis of Ejection Fraction (EF). ***p < 0.001 vs. Sham group; # p < 0.05 vs. MI group; ns (no significance) vs. MI group; n = 4–6. (E) Echocardiographic analysis of Fractional Shortening (FS). ***p < 0.001 vs. Sham group; # p < 0.05 vs. MI group; ns (no significance) vs. MI group; n = 4–6. (F) The analysis of heart weight (HW) and body weight (BW) ratio. **p < 0.01 vs. Sham group; # p < 0.05 vs. MI group; ns (no significance) vs. MI group; n = 4–6. (G) The Hematoxylin and Eosin (H&E) staining, Masson staining, and Wheat Germ Agglutinin (WGA) staining of adult mouse hearts at 28 days post MI, scale bar = 1 mm or 20 μm. (H–J) Quantitative analysis of infarction area (H), fibrosis area (I) and Cardiomyocyte size (J). ***p < 0.001 vs. Sham group; ## p < 0.01 vs. MI group; ns (no significance) vs. MI group; n = 3. Data were presented as mean ± SD. Statistical significance was assessed by one‐way ANOVA.

FIGURE 7. PIK90 upregulates GABARAPL1 expression and mitigates myocardial pyroptosis induced by MI. (A, B) Western blotting analysis showing that the expression of NLRP3 under the treatment of PIK90. **p < 0.01 vs. Sham group; # p < 0.05 vs. MI group; n = 3. (C, D) Western blotting analysis showing that PIK90 inhibited the expression of GSDMD, IL18, and NLRP3. **p < 0.01, ***p < 0.001 vs. Con group; n = 3. (E–H) Western blotting analysis showing that the expression of IL1β, Caspase1, and NLRP3 under hypoxia was significantly decreased with the treatment of PIK90. ***p < 0.001 vs. Con group; # p < 0.05 vs. Con+Hypoxia group; n = 3. Data were presented as mean ± SD. Statistical significance was assessed by one‐way ANOVA and Student's t‐test.