ES-cell fate is determined by intra- and extracellular factors. Reactive oxygen species (ROS) are involved in cardiac differentiation and outlined NAD(P)H oxidases (NOX enzymes) as key mediators of ROS generation during early embryonic stem (ES) cell differentiation. Furthermore, nitric oxide (NO) is serving as paracrine factor during ES cell differentiation into cardiomyocytes. However, the interplay between NO and ROS during cardiomyogenesis is not well understood.

We postulate that superoxide and peroxynitrite at low concentrations stimulate cardiomyogenesis. Under conditions where NO is generated at low levels it stimulates cardiomyogenesis indirectly via generation of peroxynitrite; if NO is robustly generated it acts as superoxide scavenger and becomes less important as a stimulus for cardiomyogenesis.

NO was measured by microfluorometric DAF- fluorescence, superoxide by DHE-fluorescence and peroxynitrite by DCF-fluorescence and confocal laser scanning microscopy. NOX enzyme expression, NO-synthetase (NOS) expression and the expression of cardiac specific markers were assessed by Real Time-PCR and Western blot. cGMP measurements for determination of NO generation were performed using the radioimmunoassay.

Differentiating ES cells displayed a distinct pattern of NOX enzyme and e-NOS expression. Active NAD(P)H oxidases were expressed in early stages of ES cell differentiation, whereas the expression of e-NOS was continuously upregulated, with maximum expression at day 8 and 10 of cell culture. In parallel endogenous superoxide generation decreased from day 4 to day 12, and NO generation increased continuously with the time of ES cell culture. The mesodermal marker Brachyury showed a time dependent upregulation at day 3. During the time course of cardiomyogenesis the early cardiogenic marker Nkx2.5 was upregulated from day 4 on, followed by alpha-MHC and MLC2v at day 9 and 12, respectively. The impact of ROS and NO for cardiomyogenesis was investigated by incubation of ES cell-derived embryoid bodies from day 3 to day 10 with ROS scavengers and e-NOS inhibitors. DPI, an inhibitor of both NAD(P)H oxidases and NOS, and the peroxynitrite scavenger N-(2-mercapto-propionyl)-glycine (NMPG) inhibited cardiomyocyte differentiation. In contrast, the e-NOS inhibitor L-NAA was without any significant effect on cardiomyogenesis. The mitochondrial respiratory chain complex I inhibitor rotenone did not affect the expression of cardiac specific genes, indicating that ROS generated within the respiratory chain are not involved in cardiomyogenesis. In contrast incubation of differentiating embryoid bodies with low concentrations of either H₂O₂ or the peroxynitrite donor 3-morpholino-sidnonimine (SIN-1) stimulated cardiomyogenesis.

In summary, ROS, including superoxide, H₂O₂ and peroxynitrite at low concentrations stimulate cardiomyogenesis of ES cells. NO alone is not sufficient to induce cardiomyocyte differentiation from ES-cells in vitro.