The function of PCSK9 in doxorubicin-induced cardiotoxicity and its underlying mechanism

Figures

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  Fig. 1. DIC in mice. A Show the survival rate over 5 days (n = 10). B Bar graph of survival rate (n = 10). C Body weight of mice in both groups over 5 days (n = 10). D Images of the heart size of both groups of mice (n = 10). E Representative echocardiography images in the both groups of mice (n = 7). F–M The Statistical results are IVSd, IVSs, LVIDd, LVIDs, LVPWd, LVPWs, LVEF and LVFS respectively (n = 7). N HE staining of myocardial tissue from both groups of mice (n = 6), scale bar = 50 μm. Data are presented as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns. No significant vs. Control group.

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  Fig. 2. The expression of PCSK9 protein in myocardial tissue of mice was up-regulated after DOX treatment, and PCSK9 inhibitor improved DIC. A WB images of PCSK9 protein expression in cardiac tissue of both groups of mice (n = 8). B Gray scale scanning analysis of PCSK9/GAPDH protein expression (n = 8). C Show the survival rate over 12 days (n = 10). D Bar graph of survival rate (n = 10). E Body weight of the three groups of mice over 12 days (n = 10). F Images of the heart size of three groups of mice (n = 10). G Heart weight (n = 10). H Heart weight to body weight ratio (n = 10). Data are presented as mean ± SD, *P < 0.05, ***P < 0.001, vs. Control group, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. DOX group.

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  Fig. 3. PCSK9 inhibitor improved DOX-induced cardiac dysfunction, myocardial injury and myocardial fibrosis in mice. A Representative echocardiography images in the three groups of mice (n = 7). B–I The Statistical results are IVSd, IVSs, LVIDd, LVIDs, LVPWd, LVPWs, LVEF and LVFS respectively (n = 7). J HE staining and Masson staining of myocardial tissue from the three groups of mice (n = 6), scale bar = 50 μm, myocardial fibers are shown in red, collagen fibers are shown in blue. Data are presented as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control group, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. DOX group.

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  Fig. 4. PCSK9 inhibitor improved DOX-induced myocardial apoptosis. A TUNEL staining images of myocardial tissues of the three groups of mice (n = 6), scale bar = 100 μm. B The percentage of TUNEL positive in the three groups of mice (n = 6). C Immumohis tochemical staining images of PCSK9 and Caspase-3 (n = 8), scale bar = 50 μm. D WB images of PCSK9 and apoptosis-related protein expression in myocardial tissue of the three groups of mice (n = 5). E–G Results of gray-scale scanning analysis of relative protein expression of PCSK9/GAPDH, Bax/Bcl-2 and Cleaved Caspase-3/Pro Caspase-3 (n = 5). Data are presented as mean ± SD, **P < 0.01, ***P < 0.001 vs. Control group, ##P < 0.01, ###P < 0.001 vs. DOX group.

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  Fig. 5. The changes of PCSK9 gene and protein and the localization of PCSK9 in H9C2 cardiomyocytes after DOX treatment. A The cells were treated with DOX for 12 h, 24 h and 48 h (n = 6). B Changes in relative expression of PCSK9 mRNA after DOX treatment (n = 6). C WB images of PCSK9 protein expression after DOX treatment (n = 6). D Results of gray-scale scanning analysis of relative protein expression of PCSK9/GAPDH after DOX treatment (n = 6). E Ultra-high resolution images of changes in PCSK9 localization after 24 h and 48 h of DOX treatment (n = 5), scale = 10 μm. Data are presented as mean ± SD, **P < 0.01, ***P < 0.001 vs. 0µM group, #P < 0.05, ##P < 0.01 vs. 24 h control group, &&P < 0.01, &&&P < 0.001 vs. 48 h control group.

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  Fig. 6. Interfering with PCSK9 improved DOX-induced apoptosis of H9C2 cardiomyocytes. A WB images of apoptosis-related protein expression after DOX treatment (n = 6). B Results of gray-scale scanning analysis of relative protein expression of Cleaved Caspase-3/Pro Caspase-3 after DOX treatment (n = 6). C Results of gray-scale scanning analysis of relative protein expression of Bax/Bcl-2 after DOX treatment (n = 6). D WB images of PCSK9 gene interference effect after DOX treatment (n = 6). E Results of gray-scale scanning analysis of relative protein expression interference PCSK9 gene after DOX treatment (n = 6). F TUNEL staining images of cells treated with DOX at different times (n = 6), scale bar = 100 μm. G Percentage of TUNEL positive cells treated with DOX at different times (n = 6). H TUNEL staining showed that PCSK9 interference improved DOX-induced apoptosis in H9C2 cardiomyocytes (n = 6), scale bar = 50 μm. I Percentage statistics of apoptosis after PCSK9 interference (n = 6). Data are presented as mean ± SD, *P < 0.05 vs. 12 h control group, **P < 0.01, ***P < 0.001 vs. si-NC group, ##P < 0.01, ###P < 0.001 vs. 24 h control group, &&&P < 0.001 vs. 48 h control group.

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  Fig. 7. The mechanism of PCSK9 in DOX-induced apoptosis of H9C2 cardiomyocytes. A CO-IP forward verification (n = 5). B CO-IP reverse verification (n = 5), whole protein sample of H9C2 cardiomyocytes in Input group (positive control), Nonspecific IgG-binding proteins in IgG group (negative control). C WB images of KPNB1 protein expression after DOX treatment (n = 6). D Results of gray-scale scanning analysis of relative protein expression of KPNB1 after DOX treatment (n = 6). E WB images of KPNB1 and apoptosis-related proteins after interfering with PCSK9 (n = 5). F Results of gray-scale scanning analysis of relative protein expression of PCSK9, KPNB1, Bax/Bcl-2 and Cleaved Caspase-3/Pro Caspase-3 after interfering PCSK9 gene (n = 5). Data are presented as mean ± SD, *P < 0.05 vs. 12 h control group, **P < 0.01, ***P < 0.001 vs. si-NC group, ##P < 0.01 vs. 24 h control group, &&P < 0.01 vs. 48 h control group.

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  Fig. 8. Research mechanism diagram. PCSK9 may interact with KPNB1 to induce apoptosis in cardiomyocyte and PCSK9 inhibitor may be a potential drug for improving DOX-induced cardiotoxicity.