Fig 5. Enhanced oxidative stress and impaired antioxidant systems in cardiomyocytes lacking Mdm2.
(A) Immunoblot analysis of key genes selected from top-ranked gene sets identified in Fig 3C involved in detoxification processes of reactive oxygen species (ROS). One representative immunoblot of 3 independent experiments is shown. Left ventricular samples from Mdm2f/f;mcm mice were analyzed at 7 days post-Tam. (B) Densitometric analysis of Western blot results shown in Fig 5A employing ImageJ software. Numbers at the bottom of each panel indicate fold-change. n = 3 (C) Isolated cardiac mitochondria from Tam-treated Mdm2f/f;mcm have a membrane potential (MMP) that is susceptible to ROS-induced depolarization. Mitochondria were mechanically isolated, incubated with JC-1 (5.0 ug/mL), and were then treated with antimycin (50 uM). JC-1 emission at 535/595 nm was recorded at 1 reading/min for 30 min using a fluorescence spectrophotometer. The rate between two time points (emission at 595 nm/min) was calculated in the most linear range of decline for JC-1 fluorescence intensity. n = 4. (D) DCFAD (dichlorofluorescin diacetate) emission data in isolated cardiac mitochondria from Tam-treated Mdm2f/f;mcm mice. n = 4. (E) JC-1 green emission data in isolated cardiac mitochondria from Tam-treated Mdm2f/f;mcm mice. RLU, relative light units. n = 4. (F) Higher oxidative genomic DNA damage in Mdm2f/f;mcm mice post-Tam when compared to vehicle-injected controls. Concentrations of 8-hydroxy-2’-deoxyguanosine (8-OHdG), a biomarker for oxidative DNA damage in the indicated strains was determined by a competitive enzyme-linked ELISA employing 8-OHdG antibodies. n = 4. (G) Significantly enhanced levels of 4-hydroxyalkenals (4-HAE), an indicator of ROS-dependent lipid peroxidation in Mdm2f/f;mcm mice post-Tam compared with control injected animals. n = 4. (H and I) Markedly reduced glutathione/oxidized glutathione (GSH/GSSG) ratios, an indicator of cardiac oxidative stress, in Mdm2f/f;mcm mice post-Tam compared to controls. n = 4. (J) Significantly downregulated mitochondrial superoxide dismutase (Sod2) and catalase (Cat) activities as determined spectrophotometrically in Mdm2f/f;mcm mice post-Tam. n = 4. Fig 5A to J: Left ventricular samples from 12-week-old mice were analyzed at 7 days post-Tam. Fig 5 data are means±s.e.m.