Anticonvulsant valproic acid inhibits cardiomyocyte differentiation of embryonic stem cells by increasing intracellular levels of reactive oxygen species

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Abstract
BACKGROUND The anticonvulsant valproic acid (VPA) exerts teratogenic properties and has been demonstrated to cause neural tube defects and malformations of the heart. The effect of VPA on the differentiation of cardiomyocytes from pluripotent murine embryonic stem cells (ES cells) was investigated. METHODS Embryoid bodies derived from ES cells were treated with different concentrations of VPA and the differentiation of cardiomyocytes was monitored by immunohistochemical staining for sarcomeric α‐actinin. Cytotoxicity was evaluated by the use of the dead cell stain SYTOX green. Intracellular levels of reactive oxygen species (ROS) within the tissue were evaluated by the use of the redox‐sensitive dye dichlorodihydrofluorescein diacetate (H2DCFDA). RESULTS VPA retarded the growth of ES cell‐derived embryoid bodies but did not exert cytotoxic effects. The compound dose‐dependently inhibited the development of spontaneously beating clusters of cardiomyocytes within embryoid bodies grown from ES cells and reduced the extension of beating areas of cardiac cells. Furthermore, VPA significantly increased ROS levels, indicating that VPA altered the intracellular redox balance. To investigate whether the inhibition of cardiomyocyte differentiation by VPA was owing to increased ROS overwhelming the intracellular antioxidative defense, the compound was coadministered with the free radical scavenger vitamin E. CONCLUSIONS This treatment significantly restored cardiomyogenic differentiation, indicating that VPA inhibits cardiomyogenesis of ES cells by increasing intracellular ROS levels. Birth Defects Research (Part A) 67:174–180, 2003.