Role of NADPH oxidases in cardiomyocite survival

NADPH oxidases are membrane-associated multi-unit enzymes differently expressed and regulated in all mammalian tissues which generate reactive oxygen species (superoxide and hydrogen peroxide) involved in several pathophysiological processes. Among the seven isoforms identified, in the cardiovascular system NOX1 is expressed in vascular smooth muscle cells, NOX2 and NOX4 in endothelial and smooth muscle cells, cardiomyocytes and fibroblasts and NOX5 in endothelial cells. The different effects arisen from the various isoforms can be explained by specific activation of a particular NOX, the reactive oxygen species produced, the modulation of different downstream pathways and/or by the intracellular localization of each isoform. Recently it has been shown that NOX2 and NOX4, the major isoforms expressed in cardiomyocytes, contribute to determine cell survival/death in HEK293 cells, mediating different cellular signaling pathways in response to various agonists. Moreover, NOX4 overexpression was shown to protect the myocardium against pressure overload induced remodeling, while NOX2 is involved in the development of cardiac hypertrophy, pressure overload and infarct. In cardiomyocytes, however, the role of the activation and the downstream pathways triggered by these enzymes are still largely unknown. The proposed work, therefore, aims to study for the first time NADPH oxidases regulation and activity in isolated components of the cardiovascular system to identify the pathways triggering cell survival/death, unveiling the early molecular mechanisms involved in cardiac failure. Cell biology techniques (isolation and cultivation of cardiomyocytes, smooth muscle cells, fibroblasts and endothelial cells) and biochemical techniques (Western blot analysis, determination of reactive oxygen species and cell death markers) will be applied. In the course of the project, double transgenic and knock-out NOX2/NOX4 mouse models will also be generated. During the return phase, the interaction between NADPH oxidases and aldehyde dehydrogenase 2 in cardiomyocytes and in smooth muscle cells will be studied, mainly focusing on NOX-dependent regulation of aldehyde dehydrogenase 2 and on impairment of vasorelaxation to nitroglycerin.