The data is represented as mean S.E from three independent experiments

important role. Accumulating evidence TSU68 indicates interaction between GR and proteins involved in methylation, such as DNMT3b and MeCP2, leading to promoter methylation of genes. In a recent study, Crudo and colleagues found that prenatal exposure to synthetic glucocorticoids resulted in an altered global DNA methylation pattern in an organ and development dependent manner, and these changes were also observed in the next generation, indicating dexamethasone-caused methylation as a long-term manipulation in the gene regulation. Many mammalian genes possesses glucocorticoid-response element, and the expression is subjected to regulation by GR activation. GR binding to targets genes leads to change of methylation status on the gene promoters. In the present study, inhibition of DNA methylation by 5-AZA blocked the dexamethasone-induced changes in cardiomyocyte proliferation and reversed the dexamethasone-induced decrease of cardiomyocyte number, providing novel evidence of DNA methylation in dexamethasone-induced change of gene regulation and heart development. Of importance, these findings suggest a potential strategy to abrogate or reverse the dexamethasone-induced adverse effects, considering the routine administration of dexamethasone in clinical practice to deal with preterm risks. Despite the effect of dexamethasone, the finding that 5-AZA alone promoted cardiomyocyte 16 / 20 Dexamethasone and Heart Development proliferation at P7 pups resulting in a significant increase in cardiomyocyte number in P14 hearts is intriguing and suggests an important role of DNA methylation in the heart development. This is in agreement with the findings in a study by Kou et al., which showed that DNA synthesis was increased in cardiomyocytes treated with PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19776696 5-AZA. Evidence from recent studies indicates that DNA methylation may be an important mechanism in cell cycle regulation. In Hela cells, multiple DNA sequences are found differentially methylated between G0 and S phase, suggesting PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19777456 dynamic methylation is involved in control of cell cycle. Indeed, cell cycle regulating proteins are targets of epigenetic regulation and one example is the CDI protein P16, which inhibits CDK4/CDK6 activity and leads to G1-cell cycle arrest. Extensive methylation of the p16 promoter results in inactivation of P16 and cell proliferation. In the present study, we found that the dexamethasone treatment had no significant effect on global methylation levels in the hearts of P4 and P7 pups. Given the finding that inhibition of methylation by 5-AZA abrogated dexamethasone-induced effects on cardiomyocyte proliferation and binucleation, it is possible that instead of a genome-wide effect, GR-mediated methylation may be gene specific in the developing heart. It has been shown that dexamethasone has differential effects in the regulation of methylation that is organ-dependent and gene-dependent in an organ. Indeed, the present study demonstrated that dexamethasone significantly decreased cyclin D2, but not p27, in the heart of P4 pups, which was blocked by 5-AZA. This suggests that methylationdependent down-regulation of cyclin D2 may play a role in dexamethasone-induced decrease in cardiomyocyte proliferation. The finding of a development-dependent decrease in global methylation from P4 to P7 hearts is intriguing given that a study conducted by Kou and colleagues reported that global methylation increased progressively with age in cardiomyocytes. A possible reason of this inconsiste