Peroxisome proliferator-activated receptors (PPARalpa, -beta and -gamma) are nuclear receptors involved in transcriptional regulation of lipid and energy metabolism. Since the energy demand increases when cardiac progenitor cells develop rhythmic contractile activity, we hypothesized that PPAR activation may play a critical role during cardiomyogenesis of embryonic stem (ES) cells.We show that ES cells express PPARalpa, -beta, and -gamma mRNA during differentiation of ES cells towards cardiac cells. Treatment of embryoid bodies with PPARalpha agonists (WY14643, GW7647 and ciprofibrate) significantly increased cardiomyogenesis and expression of the cardiac genes ANP, MHCalpha, MHCbeta, MLC2a, MLC2v, and cardiac alpha-actin. Furthermore, WY14643 increased PPARalpha gene expression and the expression of the cardiogenic transcriptionfactors GATA4, Nkx2.5, DTEF1 and MEF2c. In contrast, the PPARalpha antagonist MK886 decreased cardiomyogenesis, whereas the PPARbeta agonist L-165,041 as well as the PPARgamma agonist GW1929 were without effects. Treatment with PPARa but not PPARb, and PPARg agonists and MK886 resulted in generation of reactive oxygen species (ROS), which was inhibited in the presence of the NADPH oxidase inhibitors diphenylen iodonium (DPI) and apocynin and the free radical scavengers vitamin E and N-(2-mercapto-propionyl)-glycine (NMPG), whereas the mitochondrial complex I inhibitor rotenone was without effects. The effectof PPARalpha agonists on cardiomyogenesis of ES cells was abolished upon preincubation with free radical scavengers and NADPH oxidase inhibitors, indicating involvement of ROS in PPARa-mediated cardiac differentiation.In summary our data indicate, that stimulation of PPARalpha but not PPARbeta and -gamma enhances cardiomyogenesis in ES cells using a pathway that involves ROS and NADPH oxidase activity.
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