Fig 1.
Effect of dexamethasone (DEX) on heart development in neonatal rats.
Newborn rats were treated with tapered dose of DEX in the absence or presence of Ru486 during the first three days of postnatal life. Ru486 was administered 30 minutes prior to the DEX treatment. Heart and body weights were determined in day 4 (P4), day 7 (P7) and day 14 (P14) neonatal rats. Data are mean ± SEM, n = 10–21. * p<0.05, DEX vs. Saline; # p<0.05, +Ru486 vs.-Ru486.
Fig 2.
Effect of dexamethasone (DEX) on cardiomyocyte binucleation in neonatal rats.
Newborn rats were treated with tapered dose of DEX in the absence or presence of Ru486 during the first three days of postnatal life. Ru486 was administered 30 minutes prior to the DEX treatment. Cardiomyocytes isolated from day 4 (P4) and day 7 (P7) neonatal hearts were stained with α-actinin, and nuclei stained with Hoechst. Representative staining of mononucleated and binucleated cells were shown in the upper panel. Data are mean ± SEM, n = 6–14. * p<0.05, DEX vs. Saline; # p<0.05, +Ru486 vs.-Ru486; † p<0.05, P7 vs. P4.
Fig 3.
Effect of dexamethasone (DEX) on cardiomyocyte proliferation in neonatal rats.
Newborn rats were treated with tapered dose of DEX in the absence or presence of Ru486 during the first three days of postnatal life. Ru486 was administered 30 minutes prior to the DEX treatment. Panel A: Cardiomyocytes isolated from day 4 (P4) and day 7 (P7) neonatal hearts were double stained with α-actinin and Ki67, and nuclei were stained with Hoechst. Representative staining of α-actinin and Ki67 co-localization was shown in the upper panel. Panel B: Cardiomyocytes isolated from P7 neonatal hearts were examined for BrdU incorporation. Data are mean ± SEM, n = 4–14. * p<0.05, DEX vs. Saline.
Fig 4.
Effect of dexamethasone (DEX) on cardiomyocyte number in neonatal rats.
Newborn rats were treated with tapered dose of DEX in the absence or presence of Ru486 during the first three days of postnatal life. Ru486 was administered 30 minutes prior to the DEX treatment. Cardiomyocytes isolated from day 4 (P4), day 7 (P7) and day 14 (P14) neonatal hearts were counted and normalized to per gram of heart weight. Data are mean ± SEM, n = 6–20. * p<0.05, DEX vs. Saline; † p<0.05, P7 vs. P4.
Fig 5.
5-AZA inhibits dexamethasone (DEX)-mediated effects on heart development in neonatal rats.
Newborn rats were treated with tapered dose of DEX 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. Heart and body weights were determined in day 4 (P4), day 7 (P7) and day 14 (P14) neonatal rats. Data are mean ± SEM, n = 5–21. * p<0.05, DEX vs. Saline; # p<0.05, +5-AZA vs. -5-AZA.
Fig 6.
5-AZA blocks dexamethasone (DEX)-induced effects on cardiomyocyte proliferation and binucleation in neonatal rats.
Newborn rats were treated with tapered dose of DEX 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. Panel A: Cardiomyocytes isolated from day 4 (P4) and day 7 (P7) neonatal hearts were double stained with α-actinin and Ki67, nuclei were stained with Hoechst. Panel B: Cardiomyocytes isolated from P7 neonatal hearts were examined for BrdU incorporation. Panel C: Cardiomyocytes isolated from P4 and P7 neonatal hearts were stained with α-actinin and Hoechst, and mononucleated and binucleated cells were determined. Data are mean ± SEM, n = 4–14. * p<0.05, DEX vs. Saline; # p<0.05, +5-AZA vs. -5-AZA; † p<0.05, P7 vs. P4.
Fig 7.
5-AZA abrogates dexamethasone (DEX)-mediated effects on cardiomyocyte number in neonatal rats.
Newborn rats were treated with tapered dose of DEX 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. Cardiomyocytes isolated from day 4 (P4), day 7 (P7) and day 14 (P14) neonatal hearts were counted and normalized to per gram of heart weight. Data are mean ± SEM, n = 5–20. * p<0.05, DEX vs. Saline; # p<0.05, +5-AZA vs. -5-AZA; † p<0.05, P7 vs. P4.
Fig 8.
5-AZA blocks dexamethasone (DEX)-induced down-regulation of cyclin D2 in the heart.
Newborn rats were treated with tapered dose of dexamethasone (DEX) 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 (P4) neonatal hearts and protein abundance of cyclin D2 (A) and p27 (B) was determined by Western blot. Data are mean ± SEM, n = 5–6 * p<0.05, DEX vs. Saline.
Fig 9.
5-AZA decreases DNA methylation levels in neonatal hearts.
Newborn rats were treated with tapered dose of dexamethasone (DEX) 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 (P4) and day 7 (P7) neonatal hearts, and methylation levels were measured using a 5-mC ELISA kit. Data are mean ± SEM, n = 5–6. # p<0.05, +5-AZA vs. -5-AZA; † p<0.05, P7 vs. P4.