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Dexamethasone Treatment of Newborn Rats Decreases Cardiomyocyte Endowment in the Developing Heart through Epigenetic Modifications.

Gay MS, Li Y, Xiong F, Lin T, Zhang L - PLoS ONE (2015)

Bottom Line: Dexamethasone treatment significantly increased the percentage of binucleated cardiomyocytes in the hearts of P4 pups, decreased myocyte proliferation in P4 and P7 pups, reduced cardiomyocyte number and increased the heart to body weight ratio in P14 pups.Ru486 abrogated the effects of dexamethasone.In addition, 5-aza-2'-deoxycytidine (5-AZA) blocked the effects of dexamethasone on binucleation in P4 animals and proliferation at P7, leading to recovered cardiomyocyte number in P14 hearts. 5-AZA alone promoted cardiomyocyte proliferation at P7 and resulted in a higher number of cardiomyocytes in P14 hearts.

View Article: PubMed Central - PubMed

Affiliation: Center for Perinatal Biology, Division of Pharmacology, Department of Basic Sciences, Loma Linda, California, 92350, United States of America.

ABSTRACT
The potential adverse effect of synthetic glucocorticoid, dexamethasone therapy on the developing heart remains unknown. The present study investigated the effects of dexamethasone on cardiomyocyte proliferation and binucleation in the developing heart of newborn rats and evaluated DNA methylation as a potential mechanism. Dexamethasone was administered intraperitoneally in a three day tapered dose on postnatal day 1 (P1), 2 and 3 to rat pups in the absence or presence of a glucocorticoid receptor antagonist Ru486, given 30 minutes prior to dexamethasone. Cardiomyocytes from P4, P7 or P14 animals were analyzed for proliferation, binucleation and cell number. Dexamethasone treatment significantly increased the percentage of binucleated cardiomyocytes in the hearts of P4 pups, decreased myocyte proliferation in P4 and P7 pups, reduced cardiomyocyte number and increased the heart to body weight ratio in P14 pups. Ru486 abrogated the effects of dexamethasone. In addition, 5-aza-2'-deoxycytidine (5-AZA) blocked the effects of dexamethasone on binucleation in P4 animals and proliferation at P7, leading to recovered cardiomyocyte number in P14 hearts. 5-AZA alone promoted cardiomyocyte proliferation at P7 and resulted in a higher number of cardiomyocytes in P14 hearts. Dexamethasone significantly decreased cyclin D2, but not p27 expression in P4 hearts. 5-AZA inhibited global DNA methylation and blocked dexamethasone-mediated down-regulation of cyclin D2 in the heart of P4 pups. The findings suggest that dexamethasone acting on glucocorticoid receptors inhibits proliferation and stimulates premature terminal differentiation of cardiomyocytes in the developing heart via increased DNA methylation in a gene specific manner.

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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.
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pone.0125033.g006: 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.

Mentions: The effects of 5-AZA on dexamethasone-induced premature cardiomyocyte binucleation and inhibition of proliferation were further studied. As shown in Fig 6A, the dexamethasone-induced decrease of Ki67-positive cardiomyocytes in P7 pups was blocked by 5-AZA. Similarly, dexamethasone-mediated suppression of BrdU incorporation was inhibited by 5-AZA (Fig 6B), suggesting a methylation-dependent mechanism in dexamethasone-induced decease in cardiomyoctye proliferation. Of interest, 5-AZA alone significantly increased Ki67-positive cardiomyocytes, despite the effects of dexamethasone (Fig 6A). Although 5-AZA alone had no significant effect on cardiomyocyte binucleation, it inhibited the dexamethasone-induced increase of percent binucleated cells in the hearts of P4 pups (Fig 6C).


Dexamethasone Treatment of Newborn Rats Decreases Cardiomyocyte Endowment in the Developing Heart through Epigenetic Modifications.

Gay MS, Li Y, Xiong F, Lin T, Zhang L - PLoS ONE (2015)

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.
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pone.0125033.g006: 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.
Mentions: The effects of 5-AZA on dexamethasone-induced premature cardiomyocyte binucleation and inhibition of proliferation were further studied. As shown in Fig 6A, the dexamethasone-induced decrease of Ki67-positive cardiomyocytes in P7 pups was blocked by 5-AZA. Similarly, dexamethasone-mediated suppression of BrdU incorporation was inhibited by 5-AZA (Fig 6B), suggesting a methylation-dependent mechanism in dexamethasone-induced decease in cardiomyoctye proliferation. Of interest, 5-AZA alone significantly increased Ki67-positive cardiomyocytes, despite the effects of dexamethasone (Fig 6A). Although 5-AZA alone had no significant effect on cardiomyocyte binucleation, it inhibited the dexamethasone-induced increase of percent binucleated cells in the hearts of P4 pups (Fig 6C).

Bottom Line: Dexamethasone treatment significantly increased the percentage of binucleated cardiomyocytes in the hearts of P4 pups, decreased myocyte proliferation in P4 and P7 pups, reduced cardiomyocyte number and increased the heart to body weight ratio in P14 pups.Ru486 abrogated the effects of dexamethasone.In addition, 5-aza-2'-deoxycytidine (5-AZA) blocked the effects of dexamethasone on binucleation in P4 animals and proliferation at P7, leading to recovered cardiomyocyte number in P14 hearts. 5-AZA alone promoted cardiomyocyte proliferation at P7 and resulted in a higher number of cardiomyocytes in P14 hearts.

View Article: PubMed Central - PubMed

Affiliation: Center for Perinatal Biology, Division of Pharmacology, Department of Basic Sciences, Loma Linda, California, 92350, United States of America.

ABSTRACT
The potential adverse effect of synthetic glucocorticoid, dexamethasone therapy on the developing heart remains unknown. The present study investigated the effects of dexamethasone on cardiomyocyte proliferation and binucleation in the developing heart of newborn rats and evaluated DNA methylation as a potential mechanism. Dexamethasone was administered intraperitoneally in a three day tapered dose on postnatal day 1 (P1), 2 and 3 to rat pups in the absence or presence of a glucocorticoid receptor antagonist Ru486, given 30 minutes prior to dexamethasone. Cardiomyocytes from P4, P7 or P14 animals were analyzed for proliferation, binucleation and cell number. Dexamethasone treatment significantly increased the percentage of binucleated cardiomyocytes in the hearts of P4 pups, decreased myocyte proliferation in P4 and P7 pups, reduced cardiomyocyte number and increased the heart to body weight ratio in P14 pups. Ru486 abrogated the effects of dexamethasone. In addition, 5-aza-2'-deoxycytidine (5-AZA) blocked the effects of dexamethasone on binucleation in P4 animals and proliferation at P7, leading to recovered cardiomyocyte number in P14 hearts. 5-AZA alone promoted cardiomyocyte proliferation at P7 and resulted in a higher number of cardiomyocytes in P14 hearts. Dexamethasone significantly decreased cyclin D2, but not p27 expression in P4 hearts. 5-AZA inhibited global DNA methylation and blocked dexamethasone-mediated down-regulation of cyclin D2 in the heart of P4 pups. The findings suggest that dexamethasone acting on glucocorticoid receptors inhibits proliferation and stimulates premature terminal differentiation of cardiomyocytes in the developing heart via increased DNA methylation in a gene specific manner.

Show MeSH
Related in: MedlinePlus