Venetoclax Induces Cardiotoxicity through Modulation of Oxidative-Stress-Mediated Cardiac Inflammation and Apoptosis via NF-κB and BCL-2 Pathway

Figures

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  Figure 1. Serum levels of cardiac enzymes. Sera from different groups were analyzed to measure CK-MB and cTnI levels (A,B, respectively). Data are presented as mean ± SD (n = 5). * p < 0.05. Cont, control; LDV, low-dose venetoclax (50 mg/kg); HDV, high-dose venetoclax (100 mg/kg); CK-MB, creatine kinase-MB; cTn-I, troponin I cardiac muscle.

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  Figure 2. VTX-induced cardiac hypertrophy. (A) Body weight; (B) heart weight; and (C) heart weight to body weight ratio. Data are presented as mean ± SD. * p < 0.05; n.s., no significant changes were observed (p > 0.05). (D–G) mRNA levels of (D) α-Mhc, (E) β-Mhc, (F) β-Mhc to α-Mhc ratio, and (G) Bnp were measured using RT-PCR. Data are presented as mean ± SD (n = 5). * p < 0.05, ** p < 0.01; n.s., no significant changes were observed (p > 0.05). Data were normalized to β-actin as a housekeeping gene and one-way analysis of variance (ANOVA), followed by Tukey–Kramer multiple-comparisons tests. Cont, control; LD, low-dose venetoclax (50 mg/kg); HD, high-dose venetoclax (100 mg/kg); α-Mhc, alpha myosin heavy chain; α-Mhc, beta myosin heavy chain; Bnp, brain natriuretic peptide.

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  Figure 3. Light micrographs of cardiac tissues using H&E stain. (A,D) Represents the regular, parallel, and branching striated myocardial fibers with cross-striation and regular nuclei with no evidence of inflammation or necrosis. (B,E) Myocardium section obtained from low-dose VTX shows normal appearance of the myocardial fibers, but with slightly nuclear enlargement. (C,F) Represents heart section from rats treated with a high dose of VTX, and shows the presence of a focus of myocardial damage associated with chronic inflammatory reaction (arrowhead). Images (A–C) obtained at 400× magnification, while images (D–F) were obtained at 600× magnification (scale bar = 50 µm and 20 µm, respectively). (G) Histopathological score. (H) Cardiomyocytes’ cross-sectional area. Data are presented as mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001. Cont, control; LD, low-dose venetoclax (50 mg/kg); HD, high-dose venetoclax (100 mg/kg).

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  Figure 4. VTX-induced apoptosis in the heart. The mRNA levels of Bax (A) and Bcl-2 (B) were measured using RT-PCR. Data were normalized to β-actin as a housekeeping gene and one-way analysis of variance (ANOVA), followed by Tukey–Kramer multiple-comparisons tests. (C) Representative Western blot analysis of Bcl-2 protein levels. (D) Representative Western blot analysis of Cleaved Cas-3 protein levels. Data are presented as mean ± SD (n = 5). * p < 0.05, ** p < 0.01; n.s., no significant changes were observed (p > 0.05). Cont, control; LD, low-dose venetoclax (50 mg/kg); HD, high-dose venetoclax (100 mg/kg); Bax, Bcl-2 associated X; Bcl-2, B-cell lymphoma 2; Cleaved Cas-3, cystinyl aspartate-specific proteases 3; β-actin, beta actin.

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  Figure 5. VTX-induced oxidative stress and inflammation in the heart. (A–E) The mRNA levels of Ifn-γ, Tgf-β, Nf-κb-p-65, Tnf-α, and Il-6 were measured using RT-PCR. Data were normalized to β-actin as a housekeeping gene and one-way analysis of variance (ANOVA), followed by Tukey–Kramer multiple-comparisons tests. (F–I) Representative Western blot analysis of protein levels of Nf-κb-p-65, Tnf-α, Il-6, and Sod-2. Data are presented as mean ± SD (n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001; n.s., no significant changes were observed (p > 0.05). Cont, control; LD, low-dose venetoclax (50 mg/kg); HD, high-dose venetoclax (100 mg/kg); Ifn-γ, interferon gamma; Tgf-β, transforming growth factor beta; Nf-κb-p-65, nuclear factor kappa-B; Tnf-α, tumor necrosis factor alpha; Il-6, interleukin-6; Sod-2, superoxide dismutase-2; β-actin, beta actin.

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  Figure 6. Effects of VTX on oxidative stress status. Biochemical analysis of MDA (A), CAT (B), and GSH (C). Data are presented as mean ± SD (n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001; n.s., no significant changes were observed (p > 0.05). Cont, control; LD, low-dose venetoclax (50 mg/kg); HD, high-dose venetoclax (100 mg/kg); MDA, malondialdehyde; CAT, catalase; GSH, glutathione.

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  Figure 7. Schematic representation of cardiotoxicity mechanism of venetoclax (VTX).