Protein Kinase C Epsilon Overexpression Protects the Heart Against Doxorubicin-Induced Cardiotoxicity Via Activating SIRT1

Figures

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  Fig. 1. DOX-induced cardiac injury and dysfunction in mice. A Serum CK-MB levels. B Serum LDH levels. C Mice body weight. D Ratio of heart weight to tibial length in mice. E–H Ultrasonic examination of mouse heart: EF% and FS value of systolic function and E/A ratio of diastolic function. I Masson staining and WGA staining; scale bar: 20 μm. J Quantitative analysis of cardiac fibrosis area by Masson staining. K Quantitative analysis of myocyte cross-sectional area by WGA staining. HW/TL: Ratio of heart weight to tibial length. All data are expressed as mean ± S.E.M (n = 4–6). *P < 0.05 vs Ctrl group, **P < 0.01

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  Fig. 2. DOX-induced heart oxidative stress and apoptosis in mice. A The expressions of PKC-ɛ, SIRT1, Bcl-2, Bax, and Cleaved Caspase3 in myocardial tissue were detected by Western blotting. B The expression level of PKC-ɛ protein. C The expression level of SIRT1 protein. D The expression level of Bcl-2 protein. E The expression level of Bax protein. F The ratio of Bax to Bcl-2. G The expression level of Cleaved Caspase3 protein. H, I Analysis of ROS production by DHE staining; scale bar: 100 μm. J Determination of MDA content. All data are expressed as mean ± S.E.M (n = 4–6). *P < 0.05 vs Ctrl group, **P < 0.01

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  Fig. 3. DOX decreased the expressions of PKC-ɛ and SIRT1 in NRVMs, promoted oxidative stress and apoptosis. A Detection of Cell viability. B Detection of LDH. C The expressions of PKC-ɛ, SIRT1, Bcl-2, Bax, and Cleaved Caspase3 in NRVMs were detected by Western blotting. D The expression level of PKC-ɛ protein. E The expression level of SIRT1 protein. F The expression level of Bcl-2 protein. G The expression level of Bax protein. H The ratio of Bax to Bcl-2. I The expression level of Cleaved Caspase3 protein. J, K Analysis of apoptosis by TUNEL staining; scale bar: 100 μm. L, M Analysis of ROS production by DHE staining; scale bar: 100 μm. N Determination of MDA content. O, P The cell skeleton and nucleus were stained using the Phalloidin-iFluor 488 reagent or DAPI. Red fluorescence indicates actin filaments, whereas blue fluorescence indicates the nuclei; scale bar: 50 μm. All data are expressed as mean ± S.E.M (n = 3–6). *P < 0.05 vs NC group, **P < 0.01

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  Fig. 4. PKC-ɛ may be a key upstream factor of SIRT1. A, B Co-IP PKC-ε and SIRT1 in primary cardiomyocytes. The relative density of NC + DOX group was normalized against the control group (NC). C Western blotting detected the expression of PKC-ε and SIRT1 in NRVMs. D Expression level of PKC-ε protein in NRVM cells after transfecting adenovirus-overexpressed PKC-ε gene. E Expression level of SIRT1 protein in NRVM cells after transfecting adenovirus-overexpressed PKC-ε gene. The relative density of NC + Adv-PKC-ε group was normalized against the NC + Adv-Ctrl group (NC + Adv-Ctrl). All data are expressed as mean ± S.E.M (n = 6). *P < 0.05 vs NC + Adv-Ctrl group, **P < 0.01

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  Fig. 5. Overexpression of PKC-ε decreased the cardiomyocyte injury induced by DOX. A Detection of Cell viability. B Detection of LDH. C The expressions of PKC-ɛ, SIRT1, Bcl-2, Bax, and Cleaved Caspase3 were detected by Western blotting. D The expression level of PKC-ɛ protein. E The expression level of SIRT1 protein. F The expression level of Bcl-2 protein. G The expression level of Bax protein. H The ratio of Bax to Bcl-2. I The expression level of Cleaved Caspase3 protein. J, K Analysis of apoptosis by TUNEL staining; scale bar: 100 μm. L, M Analysis of ROS production by DHE staining; scale bar: 100 μm. N Determination of MDA content. O, P The cell skeleton and nucleus were stained using the Phalloidin-iFluor 488 reagent or DAPI. Red fluorescence indicates actin filaments, whereas blue fluorescence indicates the nuclei; scale bar: 50 μm. The relative densities of all other groups were normalized against the NC + Adv-Ctrl group (NC + Adv-Ctrl). All data are expressed as mean ± S.E.M (n = 5–6). *P < 0.05 vs. NC + Adv-Ctrl group, &P < 0.05 vs. Adv-Ctrl + DOX group, #P < 0.05 vs. NC + Adv-PKC-ε group, **P < 0.01 vs. NC + Adv-Ctrl group

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  Fig. 6. PKC-ε attenuated DOX-induced oxidative stress and apoptosis by activating SIRT1. A Detection of Cell viability. B Detection of LDH. C The expressions of PKC-ɛ, SIRT1, Bcl-2, Bax, and Cleaved Caspase3 were detected by Western blotting. D The expression level of PKC-ɛ protein. E The expression level of SIRT1 protein. F The expression level of Bcl-2 protein. G The expression level of Bax protein. H The ratio of Bax to Bcl-2. I The expression level of Cleaved Caspase3 protein. J, K Analysis of apoptosis by TUNEL staining; scale bar: 100 μm. L, M Analysis of ROS production by DHE staining; scale bar: 100 μm. N Determination of MDA content. O, P The cell skeleton and nucleus were stained using the Phalloidin-iFluor 488 reagent or DAPI. Red fluorescence indicates actin filaments, whereas blue fluorescence indicates the nuclei; scale bar: 50 μm. The relative densities of all other groups were normalized against the Adv-Ctrl + DOX group (Adv-Ctrl + DOX). All data are expressed as mean ± S.E.M (n = 5–6). &P < 0.05 vs. Adv-Ctrl + DOX group; $P < 0.05 vs. Adv-PKC-ε + EX527 + DOX group; ns not significant