PCSK9 alleviates doxorubicin‑induced pyroptosis of cardiomyocytes

Figures

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  Figure 1.. Toxicity and biogenic analysis of DOX in cardiomyocytes. (A) Cell morphology of AC-16 cells under a light microscope after treatment with DOX for 48 h. (B) The cell viability of AC-16 cells after treatment with increasing concentrations of DOX for 24 and 28 h was determined using the Cell Counting Kit-8 assay. (C) LDH release in AC-16 cells treated with different concentrations of DOX for 48 h. (D) PCA revealed a significant difference between the DOX and control group. (E) The heatmap shows the cluster analysis of differentially expressed genes, with different colors representing the level of gene expression. (F) Key genes regulating cholesterol metabolism and lipid synthesis, such as PCSK9, SREBF2 and SREBF1, as well as the important proinflammatory cytokine IL-1β, showed significant changes in abundance (outlined). Red represents upregulated proteins and green represents downregulated proteins and black represents the proteins with no significant changes in expression. The differentially abundant protein volcano plot uses the fold change (log2) value as the X-axis and the P-value (−log10) as the Y-axis. **P<0.01; n=3. DOX, doxorubicin; PCA, principal component analysis; LDH,…

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  Figure 2.. Decreased expression levels of PCSK9 in DOX-induced cardiomyocyte pyroptosis. (A) The expression of PCSK9 mRNA was detected using qPCR. Immunofluorescence labeling of PCSK9 (B) subcellular localization and (D) quantitative analysis of fluorescence intensity (scale bar, 100 µm). (C) Representative western blot images and (E) quantification of PCSK9 protein expression levels. The expression levels of (F) IL-1β, (G) caspase-1, (H) GSDMD and (I) NLRP3 mRNA were detected by qPCR. (J) Representative western blot images and quantification of (K) IL-1β and (L) caspase-1 protein expression levels. *P<0.05; **P<0.01. n=5. DOX, doxorubicin; PCSK9, Proprotein convertase subtilisin/kexin type 9; GSDMD, gasdermin D, NLRP3, NOD-, LRR- and pyrin domain-containing protein 3.

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  Figure 3.. Effects of PCSK9 on DOX-induced AC-16 cell pyroptosis. (A) mRNA expression of PCSK9. (B) Representative immunofluorescence images of PCSK9 subcellular localization (scale bar, 100 µm) and (D) Quantitative analysis of fluorescence intensity. (C) Representative western blot images and (E) quantification of PCSK9 protein expression levels. The mRNA expression levels of (F) IL-1β, (G) caspase-1, (H) GSDMD and (I) NLRP3 were detected using qPCR. (J) Western blotting was used to measure the expression and (K) quantification of IL-1β in AC-16 cells with PCSK9 knockdown. *P<0.05; **P<0.01. n=5. DOX, doxorubicin; PCSK9, Proprotein convertase subtilisin/kexin type 9; GSDMD, gasdermin D, NLRP3, NOD-, LRR- and pyrin domain-containing protein 3.

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  Figure 4.. Reduced tolerance to DOX in PCSK9−/− mice. (A) Mouse tail genotype identification of PCSK9−/− mice and WT mice. (B) Changes in the survival rate of mice from the initiation of treatment to Day 30. n=8. (C) Changes in weight of the mice during drug administration. n=8. (D) Masson's trichrome staining was used to evaluate the degree of cardiac fibrosis in the mice (scale bar, 200 µm). (E) The cardiac function of the mice was assessed using echocardiography 25 days after DOX administration. n=5. Assessment of cardiac function in mice: (F) EF and (G) FS. (H) Representative immunofluorescence images of GSDMD and caspase-1 subcellular localization (scale bar, 100 µm). *P<0.05. DOX, doxorubicin; PCSK9, Proprotein convertase subtilisin/kexin type 9; EF, ejection fraction; FS, fractional shortening; GSDMD, gasdermin D.