Role of NADPH oxidase in H9c2 cardiac muscle cells exposed to a simulated ischemia-reperfusion.

Several cardiovascular pathologies, including hypertension, cardiac hypertrophy and heart failure, are characterized by increased oxidative stress due to an augment in Reactive Oxygen Species (ROS) formation. This phenomenon is principally attributable to NADPH oxidase, a superoxide producing enzyme. Oxidative stress is also enhanced after ischemia-reperfusion (I/R), but little is known about the role and the activation mechanisms, in cardiac myocytes under these conditions, of NADPH oxidase. We found that H9c2 rat cardiac muscle cells subjected to an “in vitro” simulated ischemia (substrate-free medium plus hypoxia) followed by reperfusion, displayed an increase in ROS levels attributable to NADPH oxidase activity. The mechanism responsible for NADPH oxidase activation involved both an enhance in NOX2 membrane expression (1.285 fold vs control, p<0.05) and in p47phox membrane translocation (1.990 fold vs control, p<0.01). The increased NADPH oxidase activity was associated with higher levels of lipid peroxidation, activation of the redox-sensitive kinases ERK and JNK, and with cell death. Treatment with diphenyleneiodonium (DPI), a NADPH oxidase inhibitor, prevented I/R-induced ROS formation, together with the related lipoperoxidative damage and JNK activation, without affecting ERK phosphorylation. The treatment finally provided a significant protection against cell death. Our findings provide an insight in NADPH oxidase activation mechanisms under I/R and suggest this enzyme as a possible target in cardioprotective strategies against I/R injury, a condition of great importance in human pathology.