Catalase Knockdown Reverses The Activation Of Akt And The Increase In Mitochondrial Complex I Induced By MnSOD Overexpression In Mouse Heart Endothelial Cells

Introduction: We have recently showed that endothelial cell (EC)-specific overexpression of mitochondrial antioxidant MnSOD (MnSOD-OE) induced Akt and ERK activation, mitochondrial complex I (C-I) biogenesis, supercomplex assembly, and a shift from glycolysis to mitochondrial respiration in mouse heart ECs (MHEC). In vivo, MnSOD-OE induced coronary angiogenesis and improved cardiac function in a transgenic animal model of myocardial infarction. Hypothesis: We hypothesize that antioxidant catalase mediated neutralization of MnSOD-induced conversion of superoxide to H 2 O 2 within mitochondria (mito) is crucial for C-I biogenesis, ATP synthesis, and Akt and ERK activation in MnSOD-OE MHEC. Methods: We used an EC-specific, tetracycline (Tet)-controlled, transgenic MnSOD-OE model. Isolated MHECs (Tet-ON=Control, Tet-OFF=MnSOD-OE) were reverse transfected with either negative control (NC)- or catalase-siRNA (Tet-ON NC or CAT siRNA, Tet-OFF NC or CAT siRNA) and evaluated by western blot and ATP luminescence. Data were analyzed by Two-Way ANOVA and Tukey’s post-hoc. A p -value ≤0.05 was considered statistically significant. Results: Catalase-siRNA resulted in 54% decrease in catalase expression (2.01±0.05 in Tet-OFF NC vs. 0.93±0.05 in Tet-OFF CAT siRNA) in MHEC, resulting in 69% increase in mito-ROS (17.33±1.71 in Tet-OFF NC vs. 29.33±2.42 in Tet-OFF CAT siRNA). Catalase knockdown and increased mito-ROS resulted in 30% decrease in p-44/42 ERK (1.92±0.08 in Tet-OFF NC vs 1.35±0.17 in Tet-OFF CAT siRNA) and 17% decrease in p-Akt ser473 /t-Akt ratio in MnSOD-OE MHECs (1.57±0.07 in Tet-OFF NC vs 1.30±0.08 in Tet-OFF CAT siRNA). MnSOD-OE MHECs also showed decreased expression of mitochondrial C-l (by 31%; 1.43±0.06 in Tet-OFF NC vs 0.99±0.03 in Tet-OFF CAT siRNA) and C-III (by 29%; 0.90±0.06 in Tet-OFF NC vs 0.64±0.03 in Tet-OFF CAT siRNA), and decreased ATP luminescence when compared with the control group (by 27%; 12836±568.6 in Tet-ON CAT siRNA vs 9319±650.8 in Tet-OFF CAT siRNA). Conclusions: Catalase appears to play crucial role in reducing mito-ROS and inducing C-I biogenesis, ATP synthesis, and activation of Akt and ERK in MnSOD-OE MHECs.