Arachidonate lipoxygenase 5 metabolism axis promoting ferroptosis: a potential druggable target for doxorubicin-induced cardiomyopathy

Figures

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  Fig. 1. ALOX5 is upregulated in human HF hearts and in vivo and in vitro models of DIC. a The principal component analysis showed that the gene expression patterns were consistent within each group but distinct between groups. b Expression of Alox5 in NRCMs treated with Dox or DMSO for 6 h or 24 h in the GSE166957 dataset. c Experimental design for assessing the ALOX5 expression in human HF hearts. d The mRNA level of Nppb and Alox5 (n = 3). e, f Representative WB and IF images, and quantitative analysis of ALOX5 protein level (n = 3). g Experimental design for exploring the ALOX5 expression in the in vivo DIC model. h The Alox5 mRNA level in murine heart tissues (n = 4). i–k Representative WB, IF, and IHC images, and the quantitative analysis of ALOX5 protein level in murine heart tissues (n = 4). l Experimental design for exploring the expression of ALOX5 in the in vitro DIC model. m The Alox5 mRNA level in H9C2 cells (n = 3). n WB analysis of ALOX5 protein level in H9C2 cells (n = 4). o Representative IF staining images and quantitative analysis of ALOX5 protein level in H9C2 cells (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. HF heart failure, DIC doxorubicin-induced…

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  Fig. 2. Cardiomyocyte-specific overexpression of Alox5 aggravates DIC by ‌promoting ferroptosis in vivo. a Experimental design for investigating the effect of cardiomyocyte-specific Alox5 overexpression on DIC in vivo. b 5-HETE content in murine heart tissues (n = 4). c, d Body weight and HW/TL of mice (n = 5). e–g Representative cardiac echocardiography images and quantitative analysis of LVEF and LVFS in mice (n = 5). h Serum LDH levels (n = 4). i Representative HE staining images of the heart tissues (n = 3). j, k GSH content and MDA levels in the heart tissues (n = 4). l WB analysis of the expressions of SLC7A11 and GPX4 in heart tissues (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. DIC doxorubicin-induced cardiomyopathy, Fer-1 Ferrostatin-1, Dox doxorubicin, 5-HETE, 5-hydroxyicosatetraenoic acid, HW/TL heart weight/tibia length, LVEF left ventricular ejection fraction, LVFS left ventricular fractional shortening, LDH lactic dehydrogenase, HE Hematoxylin and Eosin, GSH glutathione, MDA malondialdehyde, WB Western Blot.

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  Fig. 3. ALOX5 inhibition ameliorates DIC by suppressing ferroptosis in vivo. a Experimental design for investigating the effect of inhibiting ALOX5 on DIC in vivo. b 5-HETE content in murine heart tissues (n = 4). c, d Body weight and HW/TL of mice (n = 6). e–g Representative cardiac echocardiography images and quantitative analysis of LVEF and LVFS in mice (n = 6). h Serum LDH levels (n = 4). i Representative HE staining images of heart tissues (n = 3). j, k GSH content and MDA levels in heart tissues (n = 4). l WB analysis of the expressions of SLC7A11 and GPX4 in heart tissues (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. DIC doxorubicin-induced cardiomyopathy, Zil Zileuton, Dox doxorubicin, 5-HETE 5-hydroxyicosatetraenoic acid, HW/TL heart weight/tibia length, LVEF left ventricular ejection fraction, LVFS left ventricular fractional shortening; LDH lactic dehydrogenase, HE Hematoxylin and Eosin, GSH glutathione, MDA malondialdehyde, WB Western Blot.

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  Fig. 4. Upregulation of the ALOX5 metabolism axis contributes to Dox-induced cardiomyocyte ferroptosis. a The schematic diagram of the Mendelian randomization study design, which is based on three assumptions: (1) The IVs should be robustly associated with the exposure (AA). (2) IVs should not be associated with any confounding factors. (3) IVs exert effects on the outcome (HF) only through exposure and not through other pathways. b Mendelian randomization analysis of the association between plasma AA level and HF. c Scatterplots of the effect size for each SNP on AA and HF risk. d 5-HETE content in H9C2 cells (n = 3). e CCK8 analysis of the cell viability (n = 3). f–h LDH release, GSH content, and MDA levels in H9C2 cells (n = 3). i DHE staining of H9C2 cells (n = 3). j Flow cytometer analysis with BODIPY™ 581/591 C11 staining (n = 3). k, l GSH content and MDA levels in H9C2 cells (n = 3). m Flow cytometer analysis with BODIPY™ 581/591 C11 staining (n = 3). n WB analysis of the expressions of SLC7A11 and GPX4 in H9C2 cells (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Dox doxorubicin, IVs instrumental variables, AA arachidonic acid, HF heart failure, SNP…

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  Fig. 5. ALOX5 metabolism axis regulates ferroptosis in an NRF2-dependent manner. a, c WB analysis of NRF2 expression in H9C2 cells (n = 4). b, d Nrf2 mRNA level in H9C2 cells (n = 3). e, f GSH content and MDA levels in H9C2 cells (n = 3). g Flow cytometer analysis with BODIPY™ 581/591 C11 staining (n = 3). h WB analysis of the expressions of NRF2, SLC7A11, and GPX4 in H9C2 cells (n = 4). ns no significance; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. WB Western Blot, Dox doxorubicin, Zil Zileuton, 5-HETE 5-hydroxyicosatetraenoic acid, GSH glutathione, MDA malondialdehyde.

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  Fig. 6. ALOX5 metabolism axis facilitates NRF2 ubiquitination-dependent degradation via the PI3K/AKT/GSK-3β pathway. a, b WB analysis of NRF2 protein levels in H9C2 cells with CHX (100 μg/ml) incubation at different times (n = 3). c, d Protein levels of NRF2 in Dox or 5-HETE-treated H9C2 cells with or without MG132 (10 μM) treatment for 6 h (n = 3). e Immunoprecipitation assay of cell lysates from Dox-treated H9C2 cells exposed to MG132 with or without Zil treatment, precipitated with anti-NRF2 antibody and then immunoblotted with anti-ubiquitin antibody (n = 3). f Immunoprecipitation assay of cell lysates from ethanol or 5-HETE-treated H9C2 cells exposed to MG132, precipitated with anti-NRF2 antibody and then immunoblotted with anti-ubiquitin antibody (n = 3). g WB analysis of protein levels of p-PI3K, PI3K, p-AKT, AKT, p-GSK-3β, and GSK-3β (n = 4). h WB analysis of protein levels of p-AKT, AKT, p-GSK-3β, GSK-3β, NRF2, SLC7A11, and GPX4 (n = 4). i Upregulation of the ALOX5 metabolism axis promotes cardiomyocyte ferroptosis in DIC via PI3K/AKT/GSK-3β mediated NRF2 ubiquitination-dependent degradation. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. WB Western Blot, CHX…