Dasatinib targets c-Src kinase in cardiotoxicity

Figures

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  Fig. 1. Toxicity of dasatinib on resident cardiac cells A. Neonatal rat ventricular cardiomyocytes were treated with different concentrations of dasatinib for 24 h. Shown are analysis for cardiomyocyte viability by ATP assay (left panel) and analysis for cell death by measuring the release of adenylate kinase from ruptured cardiomyocytes (right panel). N = 6. B. Human umbilical vein endothelial cells treated with dasatinib for 24 h. Shown is analysis for cell viability by ATP assay and analysis for cell death by measuring the release of adenylate kinase. N = 6. C. Human cardiac fibroblasts treated with dasatinib for 24 h. Shown is analysis for cell viability by ATP assay and analysis for cell death by measuring the release of adenylate kinase. N = 6. *p < 0.05, * *p < 0.01, * **p < 0.001 vs Vehicle. Statistics were calculated by one-way analysis of variance followed by Dunnett’s post hoc test.

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  Fig. 2. Analysis for target of dasatinib in cardiomyocytes A. Neonatal rat cardiomyocytes were treated with 100 nM and 300 nM dasatinib for 24 h and co-treated with fibroblast growth factor (FGF) where indicated. Shown is Western blot analysis for phosphorylated STAT3, Akt and ERK. GAPHD was used as a loading control. B. Dose response curve of the cardiomyocyte toxicity of dasatinib. Cardiomyocytes were treated with 10, 30, 100, 300 and 1000 nM dasatinib for 24 h and data from ATP assay were plotted against dasatinib concentration. C. Profiling data of kinases targeted by dasatinib.

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  Fig. 3. The role of c-Src in regulation of cardiomyocyte viability A-B. Neonatal rat cardiomyocytes were transfected with control siRNA, 10 nM of c-Src siRNA or 30 nM of c-Src siRNA as indicated in the figure. A. Shown is Western blot analysis for c-Src and GAPDH. B. Shown is analysis for cardiomyocyte viability by ATP assay and analysis for cell death by measuring the release of adenylate kinase 5 days after transfection (N = 4). *p < 0.05, * *p < 0.01 vs control siRNA. Statistics were calculated by one-way analysis of variance followed by Dunnett’s post hoc test. C. Cardiomyocytes were transfected with control siRNA, 25 nM of c-Src siRNA or 80 nM of c-Src siRNA as indicated. Cell death was monitored by adenylate kinase assay 4, 5 and 6 days after transfection. Data are shown as relative to adenylate kinase release from control siRNA treated cells at 4 days after transfection. * *p < 0.01 vs control siRNA; #p < 0.05 vs 25 nM c-Src siRNA. Statistics were calculated by repeated measures ANOVA.

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  Fig. 4. Gatekeeper mutation in c-Src rescues the cardiomyocytes from dasatinib-induced toxicity Neonatal rat ventricular cardiomyocytes were transfected with wild type c-Src or with c-Src T338I. A. Shown is Western blot analysis for c-Src and GAPDH. B. Shown is analysis for cardiomyocyte viability by ATP assay after treatment of cells with 100 nM or 300 nM dasatinib for 24 h. N = 4. * *p < 0.01, * **p < 0.001 vs Vehicle. Statistics were calculated by one-way analysis of variance followed by Dunnett’s post hoc test. C. Data shown as a comparison of toxicity of dasatinib on control cardiomyocytes and cardiomyocytes transfected with wild type c-Src or c-Src T338I. N = 4. #p < 0.05 vs control cells treated with indicated dose of dasatinib. Statistics were calculated by one-way analysis of variance followed by Tukey’s post hoc test.