Role of NADPH oxidase in H9c2 cardiac  muscle cells exposed to simulated ischaemia-reperfusion.
Oxidative stress is associated with several cardiovascular pathologies, including hypertension, cardiac hypertrophy and heart failure. Although oxidative stress is also increased after ischaemia-reperfusion (I/R), little is known about the role and the activation mechanisms, in cardiac myocytes under these conditions, of NADPH oxidase, a superoxide-producing enzyme. We found that rat cardiac muscle cells (H9c2) subjected to an in vitro simulated ischaemia (substrate-free medium plus hypoxia) followed by 'reperfusion', displayed increased reactive oxygen species (ROS) production attributable to a parallel increase of NADPH oxidase activity. Our investigation on mechanisms responsible for NADPH oxidase activation showed a contribution of both the increase of NOX2 expression and p47(phox) translocation to the membrane. We also found that the increase of NADPH oxidase activity was associated with higher levels of lipid peroxidation, the activation of redox-sensitive kinases, in particular ERK and JNK, and with cell death. Diphenyleneiodonium (DPI), a flavoprotein inhibitor used as NADPH oxidase inhibitor, prevented I/R-induced ROS formation in treated cells, together with the related lipoperoxidative damage, and JNK phosphorylation without affecting ERK activation, resulting in protection against cell death. Our results provide evidence that NADPH oxidase is a key enzyme involved in I/R-induced oxidant generation and suggest it can be a possible target in cardioprotective strategies against I/R injury, a condition of great importance in human pathology.
Borchi
Elisabetta
E
Department of Biochemical Sciences, University of Florence, Florence, Italy.
Parri
Matteo
M
Department of Biochemical Sciences, University of Florence, Florence, Italy.
Papucci
Laura
L
Department of Experimental Pathology and Oncology, University of Florence, Florence, Italy.
Becatti
Matteo
M
Department of Biochemical Sciences, University of Florence, Florence, Italy.
Nassi
Niccolò
N
Department of Pediatrics, University of Florence, Florence, Italy.
Nassi
Paolo
P
Department of Biochemical Sciences, University of Florence, Florence, Italy.
Nediani
Chiara
C
Department of Biochemical Sciences, University of Florence, Florence, Italy.