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

Abstract

Doxorubicin (DOX) is an anthracycline class of chemotherapy drug, the application of which is limited due to its cardiotoxic effects. Recombinant Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9), is a serine protease pivotal in lipid metabolism and has a profound correlation with the onset of cardiovascular diseases. This study uncovers a connection between PCSK9 and DOX-induced cardiotoxicity (DIC). This research found that injection of DOX in mice caused cardiac toxicity. DOX treatment up-regulated the expression of PCSK9 protein in myocardial tissue. Evolocumab (PCSK9 inhibitors) improved cardiac function, myocardial injury, and fibrosis in DOX-treated mice, indicating a protective effect against DIC. The mechanism involved modulation of cardiomyocyte apoptosis and regulation of apoptosis-related proteins, including Bax/Bcl-2 ratio and Cleaved Caspase-3/Pro Caspase-3 ratio. DOX exhibited concentration- and time-dependent cytotoxic effects on H9C2 cardiomyocytes, promoting apoptosis. PCSK9 nuclear aggregation occurred in H9C2 cardiomyocytes after DOX treatment, and PCSK9 interacted with the Importin subunit beta-1 (KPNB1) protein. Interference with PCSK9 up-regulated KPNB1 expression, affecting apoptosis-related proteins and improving DOX-induced H9C2 cardiomyocyte apoptosis. In short, the elucidation of this mechanism is helpful involve that PCSK9 inhibitor may be a potential drug for improving DIC.

Keywords: Apoptosis; Cardiotoxicity; Doxorubicin; KPNB1; PCSK9.

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.

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.

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.

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.

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.

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.

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.

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.