Diacylglycerol Kinase ζ Attenuates Doxorubicin‐Induced Cardiotoxicity Through p53 Degradation

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  Figure 1. Dgkζ protects cardiac atrophy, cardiac function, and survival rate in doxorubicin‐induced cardiotoxicity. A, Representative Western blot of Dgkζ, E6ap, Hsp70, and Chip in Dgkζ‐Tg mice myocardium lysates. Quantification of Dgkζ, E6ap, Hsp70, and Chip protein levels in Dgkζ‐Tg mice (n=6). Results are presented as mean±SE (*P<0.05, **P<0.01, ***P<0.001, vs WT mice, using Student t test). B, Immunoprecipitation showing interactions of Dgkζ with E6ap in Dgkζ‐Tg mice myocardium lysates. Western blots after immunoprecipitation with anti‐Dgkζ showing interactions of Dgkζ with E6ap in Dgkζ‐Tg mice myocardium lysates. C, Immunoprecipitation showing interactions of Dgkζ with Hsp70 in Dgkζ‐Tg mice myocardium lysates. Western blots after immunoprecipitation with anti‐Dgkζ showing interactions of Dgkζ with Hsp70, but not Chip, in Dgkζ‐Tg mice myocardium lysates. D, Survival curves up to 14 days after Dox injection in WT and Dgkζ‐Tg mice. E, Representative images of cardiac function by M‐mode echocardiograms from WT and Dgkζ‐Tg mice 7 days after Dox injection. Comparison of left ventricular ejection fraction between WT and Dgkζ‐Tg mice 7 days after Dox injection. Results are presented as…

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  Figure 2. Dgkζ inhibits p53 expression and subsequent apoptosis in doxorubicin‐induced cardiotoxicity. A, Representative Western blot of Dgkζ in Dgkζ‐Tg mice myocardium nucleus and cytosolic lysates 7 days after Dox injection. Representative Western blot of p53 in Dgkζ‐Tg mice myocardium lysates 7 days after Dox injection. B, Quantitative analyses of nuclear‐to‐cytosolic Dgkζ ratio (n=4, per group). C, p53 protein levels in Dgkζ‐Tg mice myocardium lysates 7 days after Dox injection (n=6, per group). D, TUNEL staining showing decreased TUNEL‐positive cells in Dgkζ‐Tg mice myocardium 7 days after Dox stimulation (n=4, per group). Results are present as the mean±SE (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 using Tukey–Kramer test). E, Heat map of differentially expressed genes between WT and Dgkζ‐Tg mice 5 days after Dox administration. F, Top upregulated and downregulated genes. G, Enriched pathways in differential expressed genes. H, Gene Ontology RNA sequence analysis. Top 10 upregulated gene sets. DAPI indicates 4′,6‐diamidino‐2‐phenylindole; DEGs, differential expressed genes; Dgkζ, diacylglycerol kinase ζ; Dgkζ‐Tg, cardiac‐specific overexpression of Dgkζ transgenic mouse; Dox,…

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  Figure 3. Protein and mRNA expression of Dgkζ, E6ap, Hsp70, Chip, Mdm2, and p53 in doxorubicin‐induced cardiotoxicity. A, Time course of Dgkζ, E6ap, Hsp70, Chip, Mdm2, and p53 protein expression levels in H9C2 cardiomyocytes after Dox stimulation (0.5 μmol/L). Quantification of Dgkζ, E6ap, Hsp70, Chip, Mdm2, and p53 protein levels in H9C2 cells after Dox stimulation (n=6, per group). B, Dgkζ, E6ap, Hsp70, Chip, Mdm2, and p53 mRNA levels in H9C2 cardiomyocytes after Dox stimulation (0.5 μmol/L). Quantitative analysis of Dgkζ, E6ap, Hsp70, Chip, Mdm2, and p53 mRNA expression (n=6, per group). Results are presented as mean±SE (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs 0 hour using Dunnett test). Chip indicates C‐terminus of Hsp70‐interacting protein; Dgkζ, diacylglycerol kinase ζ; Dox, doxorubicin; E6ap, E6‐associated protein; Hsp70, heat shock protein 70; and Mdm2, mouse double mutant 2 homolog.

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  Figure 4. Effect of Dgkζ overexpression on p53 expression and cardiomyocyte apoptosis in doxorubicin‐induced cardiotoxicity. A, Representative Western blot of Dgkζ, p53, and cleaved caspase‐3 in Flag‐Dgkζ‐transfected H9C2 cardiomyocytes after Dox stimulation (0.5 μmol/L, 12 h). Quantitative analyses of p53 and cleaved caspase‐3 protein levels in Flag‐Dgkζ‐transfected H9C2 cardiomyocytes after Dox stimulation (0.5 μmol/L, 12 hour) (n=6, per group). B, Representative Western blot of Dgkζ, p53, and cleaved caspase‐3 in Flag‐Dgkζ‐transfected neonatal rat cardiomyocytes after Dox stimulation (0.5 μmol/L, 12 hour). Quantitative analyses of p53 and cleaved caspase‐3 protein levels in Flag‐Dgkζ‐transfected neonatal rat cardiomyocytes after Dox stimulation (0.5 μmol/L, 12 hour) (n=6, per group). C, Fluorescence images of JC‐1 aggregates (red) and JC‐1 monomers (green) indicating the mitochondrial membrane potential in each group (n=6, per group). D, Inhibition of TUNEL‐positive cells by overexpression of Flag‐Dgkζ after Dox stimulation (0.5 μmol/L, 12 hour) (n=4, per group). Results are presented as the mean±SE (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 using Tukey–Kramer test). Dgkζ indicates…

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  Figure 5. Dgkζ induces p53 degradation through the interaction with E6ap in doxorubicin‐induced cardiotoxicity. A, Immunoprecipitation showing the interactions of p53 with Dgkζ and E6ap. Western blots after immunoprecipitation with anti‐p53 antibody showing interactions of p53 with Dgkζ and E6ap in Flag‐Dgkζ‐transfected H9C2 cardiomyocytes after Dox stimulation (0.5 μmol/L, 12 h). B, Ubiquitylated p53 was assessed by immunoprecipitation. Flag‐Dgkζ‐transfected or empty vector‐transfected H9C2 lysates were immunoprecipitated with mouse anti‐p53 antibody and immunoblotted with goat anti‐Dgkζ antibody and mouse anti‐ubiquitin antibody. MG132, a proteasome inhibitor, was administered to inhibit p53 degradation. C, Representative Western blot of E6ap and p53 in H9C2 cardiomyocytes transfected with siRNA E6ap, Flag‐Dgkζ vector, or both after Dox stimulation (0.5 μmol/L, 12 hour). D and E, Quantitative analyses of E6ap and p53 protein levels in H9C2 cardiomyocytes transfected with siRNA E6ap, Flag‐Dgkζ, or both after Dox stimulation (0.5 μmol/L, 12 hour) (n=6, per group). Results are presented as mean±SE (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 using Tukey–Kramer test). F, Ubiquitylated E6ap…

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  Figure 6. Effects of Chip and Mdm2 on Dgkζ‐regulated p53 suppression in doxorubicin‐induced cardiotoxicity. A, Representative Western blot of Dgkζ and p53 in H9C2 cardiomyocytes transfected with siRNA Chip after Dox stimulation (0.5 μmol/L, 12 hour). B and C, Quantitative analyses of Dgkζ and p53 protein levels in H9C2 cardiomyocytes transfected with siRNA Chip after Dox stimulation (0.5 μmol/L, 12 hour) (n=6, per group). D, Representative Western blot of p53 and cleaved caspase‐3 in H9C2 cardiomyocytes transfected with siRNA Chip, Flag‐Dgkζ vector, or both after Dox stimulation (0.5 μmol/L, 8 hour). E and F, Quantitative analyses of p53 and cleaved caspase‐3 protein levels in H9C2 cardiomyocytes transfected with siRNA Chip, Flag‐Dgkζ, or both after Dox stimulation (0.5 μmol/L, 8 hour) (n=6, per group). G, Representative Western blot of p53 and cleaved caspase‐3 in H9C2 cardiomyocytes transfected with siRNA Mdm2, Flag‐Dgkζ vector, or both after Dox stimulation (0.5 μmol/L, 16 h). H and I, Quantitative analyses of p53 and cleaved caspase‐3 protein levels in H9C2 cardiomyocytes transfected with siRNA Mdm2, Flag‐Dgkζ, or both after Dox stimulation (0.5 μmol/L, 16 h) (n=6, per group). Results…

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  Figure 7. A simplified model depicting Dgkζ function in Dox‐induced cardiotoxicity. Top: Cardiac‐specific overexpression of Dgkζ transgenic mice preserve cardiac function and improve survival in Dox‐induced cardiotoxicity. Bottom: Dgkζ augments p53 ubiquitin proteasomal degradation through the interaction with E6ap and Hsp70 with resultant inhibition of cardiomyocyte apoptosis in Dox‐induced cardiotoxicity. Chip and Mdm2 also play a role in Dgkζ‐mediated p53 ubiquitylation. Chip indicates C‐terminus of Hsp70‐interacting protein; Dgkζ‐Tg, diacylglycerol kinase ζ transgenic; Dox, doxorubicin; E6ap, E6‐associated protein; Hsp70, heat shock protein 70; Mdm2, mouse double mutant 2 homolog; and WT, wild‐type.