treatment. Cardiomyocytes isolated from day 4, day 7 and day 14 neonatal hearts were counted and normalized to per gram of heart weight. Data are mean SEM, n = 520. p<0.05, DEX vs. Saline; # p<0.05, +5-AZA vs. -5-AZA; p<0.05, P7 vs. P4. doi:10.1371/journal.pone.0125033.g007 12 / 20 Dexamethasone and Heart Development Fig 8. 5-AZA blocks dexamethasone -induced down-regulation of cyclin D2 in the heart. Newborn rats were treated with tapered dose of dexamethasone in the absence or presence of 5-AZA during the first three days of postnatal life. 5-AZA was administered 30 minutes prior to the DEX treatment. Protein was isolated from day 4 neonatal hearts and protein abundance of cyclin D2 and p27 was determined by Western blot. Data are mean SEM, n = 56 p<0.05, DEX vs. Saline. doi:10.1371/journal.pone.0125033.g008 13 / 20 Dexamethasone and Heart Development Fig 9. 5-AZA decreases DNA methylation levels in neonatal hearts. Newborn rats were treated with tapered dose of dexamethasone in the absence or presence of 5-AZA during the first three days of postnatal life. 5-AZA was administered 30 minutes prior to the DEX treatment. Genomic DNA was extracted from day 4 and day 7 neonatal hearts, and methylation levels were measured using a 5-mC ELISA kit. Data are mean SEM, n = 56. # p<0.05, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19776382 +5-AZA vs. -5-AZA; p<0.05, P7 vs. P4. doi:10.1371/journal.pone.0125033.g009 Discussion The synthetic glucocorticoid dexamethasone is commonly used to reduce the morbidity of respiratory complications in preterm infants. Yet, the potential adverse effects of dexamethasone therapy on the developing heart remain unknown. In the present study, we examined the impact of clinically relevant neonatal doses of dexamethasone on cardiomyocyte proliferation and binucleation in the developing heart. The results provided evidence of glucocorticoid-mediated stimulation of premature cardiomyocyte binucleation, inhibition of myocyte proliferation, and reduction in total cardiomyocyte number during the critical window of the heart development. We demonstrated that the dexamethasone-induced effects were abrogated by a GR antagonist Ru486, and PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19777456 thus revealed the GR-mediated effect on premature heart development in newborns. In addition, we provide novel evidence of a potential mechanism of DNA methylation in GR-mediated effects in the developing heart. The results provided insights in the regulation of cardiomyocyte maturation by endogenous glucocorticoids and the underlying mechanisms that may be involved. Dexamethasone has been widely used in clinic to prevent the morbidity of chronic lung disease in preterm infants. In addition to its effect on the lung, glucocorticoids are also essential regulators of the development of other organs such as the 
brain and heart. Given that the order LY341495 developmental stage of hearts and brains in newborn rats is somewhat equivalent to that of the fetal development in the third trimester of human gestation, they provide a model in studying the effect of dexamethasone therapy in preterm infants on the heart and brain development. Recently, Chang and colleagues uncovered that neonatal dexamethasone treatment altered the susceptibility of the immature brain to hypoxic-ischemic brain injury. Studies 14 / 20 Dexamethasone and Heart Development also have provided evidence for negative occurrences with the dexamethasone treatment including myocardial hypertrophy and premature death. De Vries and colleagues investigated the long-term effect of neonatal dexamethas