IKKα-STAT3-S727 axis: a novel mechanism in DOX-induced cardiomyopathy

Figures

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  Fig. 1. Increased expression of IKKα in mononuclear cells and macrophages in DCM. A Mononuclear cells from the peripheral blood of patients with tumours treated with DOX and healthy controls were purified. Representative flow cytometry plot of the F4/80 and CD11c levels in mononuclear cells; n = 5 per group. B mRNA levels of IKKα in mononuclear cells from patients with tumours treated with DOX and healthy controls. The transcripts levels were normalized to GAPDH. C Representative immunostaining images for macrophages (CD68, red), IKKα (green), and DAPI (blue) in the heart tissues of dilated cardiomyopathy patients. Magnification 600X. D Representative Western blot analysis of IKKα levels in the heart tissue of mice treated with or without DOX was performed with GAPDH as a loading control; N = 6 in each group. E Representative immunostaining images for macrophages (CD68, green), IKKα (red), and DAPI (blue) in the heart tissues of C57BL/6 mice treated with or without DOX. Magnification 600X. *P < 0.05, **P < 0.01. P values were calculated using the unpaired Student’s t-test. Quantification of macrophages colocalized with IKKα

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  Fig. 2. IKKα deficiency aggravated cardiac dysfunction and pathological ventricular remodeling induced by DOX. A Changes in mouse survival at different time points in different groups; N = 40 in each group; *: IKKαflox/flox mice with DOX vs. mIKKα−/− mice with DOX. B and C Representative echocardiography images of murine hearts and left ventricular ejection fraction (EF), left ventricular fractional shortening (FS) and left ventricular volume (LV vol) at end-diastole and systole determined by echocardiography. N = 6 in each group. D Representative morphological changes in murine hearts and haematoxylin and eosin (HE), Masson and picrosirius red staining of myocardial sections. Magnification 400X. E Quantification of HE and interstitial fibrotic area (%) as determined by Masson trichome staining and Sirius red staining of heart sections. F Representative images of heart tissues from IKKαflox/flox and mIKKα−/− mice imaged using [68Ga]Ga-DOTA-FAPI-04. G Representative images of heart tissues from IKKαflox/flox and mIKKα−/− mice stained with collagen I (Col 1) and fibronectin (Fn). Magnification 400X. *P < 0.05, **P < 0.01, ***P < 0.001. P values were calculated by unpaired Student’s t-test

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  Fig. 3. IKKα influences DOX-induced cardiac function in mice after bone marrow transplantation through macrophages. A Bone marrow transplant procedure. B mRNA levels of IKKα in the mice; n = 5 per group. C and D Representative echocardiography images of murine hearts and left ventricular ejection fraction (EF), left ventricular fractional shortening (FS) and left ventricular volume (LV vol) at end-diastole and systole determined by echocardiography; N = 5 in each group. *P < 0.05, **P < 0.01, ns: not significant. P values were calculated using an unpaired Student’s t-test

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  Fig. 4. IKKα deficiency aggravated cardiac oxidative stress and macrophage reprogramming induced by DOX. A Representative immunostaining images for macrophages (CD68, green), proinflammatory macrophages (iNOS, red), and DAPI (blue) in the heart tissues of IKKαflox/flox and mIKKα−/− mice. Magnification 400X. B mRNA levels of TNF-α, IL-6 and IL-1β in the heart tissues of IKKαflox/flox and mIKKα−/− mice. The transcript levels were normalized to those of GAPDH. C Representative immunostaining images for DHE and TUNEL in the heart tissues of IKKαflox/flox and mIKKα−/− mice. Magnification 100X. D Representative images of heart tissues from IKKαflox/flox and mIKKα−/− mice stained with 4-HNE. Magnification 400X. E Serum levels of LDH, MDA, GSH and SOD in IKKαflox/flox and mIKKα−/− mice; N = 6 in each group. F Metabolism analysis of the OCR and ECAR in macrophages purified from IKKαflox/flox and mIKKα−/− mice treated with DOX. Quantification of macrophage oxidative phosphorylation and glycolysis as determined by the OCR and ECAR. **P < 0.05, **P < 0.01, **P < 0.001, ns: not significant. P values were calculated using an unpaired Student’s t-test

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  Fig. 5. IKKα deficiency aggravated macrophage mitochondrial damage and metabolic recombination induced by LPS. A Representative immunostaining images for DHE in primary macrophages from IKKαflox/flox and mIKKα−/− mice. Magnification 100X. B Representative immunostaining images for mitochondrial ROS in primary macrophages from IKKαflox/flox and mIKKα−/− mice. Magnification 1000X (oil immersion lens). C Representative immunostaining images for JC-1 in primary macrophages from IKKαflox/flox and mIKKα−/− mice. Magnification 1000X (oil immersion lens). *P < 0.05. D Metabolism analysis of the OCR and ECAR in primary macrophages from IKKαflox/flox and mIKKα−/− mice stimulated with LPS. Quantification of macrophage oxidative phosphorylation and glycolysis as determined by the OCR and ECAR. E mRNA levels of HK1, HK2, PGK1 and Slc2a in primary macrophages from IKKαflox/flox and mIKKα−/− mice; N = 6 in each group. The transcripts were normalized to those of 18 S rRNA. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant. P values were calculated using an unpaired Student’s t-test

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  Fig. 6. STAT3 is a marker of a DOX-related proinflammatory macrophage cluster. IKKαflox/flox and macrophage IKKα knockout (mIKKα−/−) mice were treated with or without DOX for 2 weeks. A Signature of the proinflammatory process for each cluster. The signature score was calculated on the basis of proinflammatory factor score (STAT3, IL6Ra, CCL5, TNFAIP3, TGFB1, IL-1β, TNF-α, HMGB1, CCL2, Hmox1, CCL4, NFKB1, TLR2, CD14, NFE2L2, PLA2G7, NLRP3, CXCL2, JUN, and CCL3). B and C GO analysis (B) and KEGG analysis (C) of macrophage cluster 3. D Heatmap of genes associated with macrophage cluster 3 in IKKαflox/flox and mIKKα−/− mice. E Pearson correlation coefficients between the mRNA levels of STAT3 and representative cytokines, and chemokines. F GO analysis of STAT3hi macrophages in cluster 3

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  Fig. 7. IKKα colocalized with STAT3 and increased STAT3-S727 phosphorylation. A. Representative immunostaining images for macrophages (CD68, red), IKKα (red), and STAT3 (purple) in the heart tissues of IKKαflox/flox and mIKKα−/− mice. Magnification 600X. Quantification of STAT3 colocalized with IKKα and macrophages. B.Co-IP analysis of IKKα and STAT3 in primary macrophages from IKKαflox/flox mice stimulated with LPS. C and D Western blotting analysis was used to determine the levels of STAT3, STAT3-Y705 and STAT3-S727 in primary macrophages from IKKαflox/flox and mIKKα−/− mice stimulated with LPS; N = 6 in each group. *P < 0.05, **P < 0.01, ns: not significant. P values were calculated using an unpaired Student’s t-test

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  Fig. 8. Inhibition of STAT3-S727 alleviated IKKα deficiency-induced cardiac injury and oxidative stress in cardiomyocytes. A and B IKKαflox/flox and macrophage IKKα knockout (mIKKα−/−) mice induced with DOX were treated with or without niclosamide for 2 weeks. Representative echocardiography images of murine hearts and left ventricular ejection fraction (EF), left ventricular fractional shortening (FS) and left ventricular volume (LV vol) at end-diastole and systole determined by echocardiography; N = 6 in each group. C Western blotting analysis was used to determine the levels of STAT3-S727 in primary macrophages from IKKαflox/flox mice treated with or without niclosamide for 4 h after LPS stimulation. D Representative immunostaining images for DHE in HiPSC-CMs cocultured with primary macrophages stimulated with LPS from IKKαflox/flox and mIKKα−/− mice treated with or without niclosamide for 4 h. Magnification 100X. E Representative immunostaining images for TUNEL in HiPSC-CMs cocultured with primary macrophages stimulated with LPS from IKKαflox/flox and mIKKα−/− mice treated with or without niclosamidefor 4 h. Magnification 400X. *P < 0.05, **P < 0.01. P values were calculated using an…