Reactive oxygen species (ROS) are second messengers in signalling cascades regulating cardiovascular differentiation of mouse embryonic stem (ES) cells. Previously NAD(P)H oxidases were identified as the most important enzymes generating ROS in differentiating ES cells. However, their impact on cardiomyocyte and blood vessel differentiation has not yet been investigated in detail.

According to our working hypothesis specific members of the NAD(P)H oxidase family (NOX) regulate cardiac differentiation of ES cells by initiating ROS-mediated signalling pathways. We used permanent lentiviral transfection with shRNA targets to silence the expression of specific NOX enzymes in ES cells. It was assured that the down regulation of individual members of the NOX family did not interfere with other NOX enzymes. Real Time-PCR, Western blot, immunohistochemistry, confocal laser scanning microscopy, dichlorodihydrofluorescein (DCF) and dihydroethidine (DHE) microfluorometric measurements for ROS detection as well as functional studies were used to analyse the effect of specific NOX silencing in clones of transfected ES cells on cardiovascular differentiation and ROS generation.

It is demonstrated that various subunits of NAD(P)H oxidases i.e. NOX1, NOX2, NOX4, p47phox, p67phox, NOXO1, and NOXA1 were expressed during cardiovascular differentiation of ES cells. Gene silencing of NOX1, NOX2 and NOX4 in murine embryonic stem cells resulted in inhibition of cardiomyogenesis in all shRNA transfected ES cell clones as evaluated by Real Time-PCR, Western blot and functional studies. The number of beating areas in EBs was reduced in all transfected clones. The expression of the cardiac markers alphaMHC and alpha-actinin was decreased. The shRNA transfected clones revealed a reduced ROS generation, indicating an involvement of ROS in signalling pathways regulating cardiomyogenesis of ES cells.

Our results reveal that NOX1, NOX2 and NOX4 are key regulators in cardiomyogenesis of ES cells.