Usp31 Promotes Cardiomyocyte Injury Under Ischemic Stress and Is Inhibited by Sodium Tanshinone IIA Sulfonate

Figures

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  Figure 1. Transcriptomic profiling identifies stress-response genes persistently induced during OGD/R in cardiomyocytes. (A) Representative morphological images of HL-1 cells under different treatment conditions using the conventional 20% O2-based OGD/R protocol. N, normal culture condition under 20% O2 with 25 mM glucose; OGD, oxygen-glucose deprivation under 1% O2 in glucose-free medium for 6 h; OGD/R, OGD for 6 h followed by reoxygenation under 20% O2 with glucose replenishment for 6 h (scale bar 50 µm). (B) Immunoblot analysis of the indicated markers in HL-1 cells under normal, OGD, and OGD/R conditions using the conventional 20% O2-based OGD/R protocol. HIF1α was used as a marker of hypoxic stress, and p-AMPK was used as a marker of metabolic stress. (C,D) Volcano plot analysis of differentially expressed genes (DEGs) between OGD and normal conditions (C) and between OGD/R and normal conditions (D). Genes with absolute fold change ≥ 2 and FDR-adjusted p value ≤ 0.05 were considered significantly differentially expressed. Red dots indicate significantly upregulated genes, and green dots indicate significantly downregulated genes. (E) Venn diagram and heatmap illustrating the overlap…

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  Figure 2. Usp31 is persistently induced during OGD/R under conventional and physiologic oxygen-based culture conditions. (A) qRT-PCR validation of representative genes persistently upregulated under both OGD and OGD/R conditions in HL-1 cells using the conventional 20% O2-based OGD/R protocol. Data are presented as mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001. (B) Immunoblot analysis of the indicated protein levels in HL-1 cells under normal, OGD, and OGD/R conditions using the conventional 20% O2-based OGD/R protocol. β-actin was used as the loading control. (C) Immunoblot analysis of the indicated markers in HL-1 cells using the physiologic 5% O2-based OGD/R protocol. Cells were maintained under 5% O2 with 25 mM glucose, subjected to OGD under 1% O2 in glucose-free medium for 6 h, and then reoxygenated under 5% O2 with glucose replenishment for 6 h. HIF1α was used as a marker of hypoxic stress, and p-AMPK was used as a marker of metabolic stress. (D) qRT-PCR validation of representative genes persistently upregulated under OGD and OGD/R conditions using the physiologic 5% O2-based protocol. Data are presented as mean ± SEM. ***, p < 0.001. (E) Immunoblot analysis of Usp31 and…

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  Figure 3. Usp31 deficiency attenuates OGD/R-induced cardiomyocyte injury by suppressing NF-κB activation. (A) Immunoblot analysis showing the knockout efficiency of two sgRNAs targeting Usp31 in HL-1 cells. U31#1 and U31#2 indicate two independent Usp31-targeting sgRNAs/clones. U31#1 showed more efficient Usp31 depletion than U31#2 and was selected for subsequent functional experiments. (B) CCK-8 assay showing cell viability in control and Usp31-deficient HL-1 cells under basal and OGD/R conditions. Experiments were performed using the conventional 20% O2-based OGD/R protocol. **, p < 0.01; ***, p < 0.001. (C) Quantification of LDH release in culture supernatants showing reduced membrane damage in Usp31-deficient HL-1 cells after OGD/R treatment. Experiments were performed using the conventional 20% O2-based OGD/R protocol. **, p < 0.01; ***, p < 0.001. (D) NF-κB luciferase reporter assay showing that Usp31 deficiency suppresses OGD/R-induced NF-κB transcriptional activation in HL-1 cells. Experiments were performed using the conventional 20% O2-based OGD/R protocol. **, p < 0.01; ***, p < 0.001. (E) Relative mRNA expression of NF-κB target genes, including Tnf, Il6, Il1b, and Ccl2,…

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  Figure 4. Usp31 regulates p65 ubiquitination, and STS inhibits recombinant Usp31 enzymatic activity in vitro. (A) Co-immunoprecipitation assay showing the interaction between Flag-tagged Usp31 and endogenous TRAF2 and p65 in HL-1 cells following OGD/R treatment. (B) Immunoprecipitation of p65 followed by immunoblot analysis of the indicated proteins in control and Usp31-deficient HL-1 cells under OGD/R conditions. p50 was used as a binding control for p65. (C) Schematic diagram of the Ub-Rho110-based high-throughput screening assay used to identify compounds that inhibit Usp31 deubiquitinase activity. (D) Coomassie brilliant blue staining of purified recombinant Usp31 protein expressed in E. coli and the chemical structure of sodium tanshinone IIA sulfonate (STS). (E) Ub-Rho110-based fluorogenic assay showing that STS inhibits the enzymatic activity of recombinant Usp31 in vitro. N-ethylmaleimide (NEM) was used as a positive control for inhibition of deubiquitinase activity. (F) Immunoprecipitation of p65 followed by immunoblotting for ubiquitin in HL-1 cells under OGD/R conditions, with or without Usp31 overexpression. Usp31-overexpressing cells were treated with either vehicle or STS…

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  Figure 5. STS attenuates Usp31-associated NF-κB activation and cardiomyocyte injury during OGD/R. (A) CCK-8 assay showing that STS treatment improves cell viability in Usp31-overexpressing HL-1 cells under OGD/R conditions. Experiments were performed using the conventional 20% O2-based OGD/R protocol. *, p < 0.05; **, p < 0.01. (B) LDH release assay showing that STS treatment reduces membrane damage in Usp31-overexpressing HL-1 cells under OGD/R conditions. **, p < 0.01; ***, p < 0.001. (C) NF-κB luciferase reporter assay demonstrating that STS attenuates NF-κB activation induced by Usp31 overexpression during OGD/R. **, p < 0.01; ***, p < 0.001. (D) qRT-PCR analysis showing that STS reduces the mRNA expression of NF-κB target genes, including Tnf, Il6, Il1b, and Ccl2, in Usp31-overexpressing HL-1 cells under OGD/R conditions. Data are presented as mean ± SEM. **, p < 0.01; ***, p < 0.001.