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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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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.
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pone.0125033.g002: 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.

Mentions: Binucleation is one of the characteristics of cardiomyocyte maturation and terminal differentiation. Thus, we determined whether dexamethasone treatment influenced cardiomyocyte binucleation in the developing heart. Cardiomyocytes isolated from P4 and P7 pups were stained with a cardiomyocyte marker α-actinin and nuclei were labeled with Hoechst. The percentage of binucleation was scored by counting the number of mononucleated and binucleated cells using microscopy. As shown in Fig 2, in control animals, there was a development-dependent increase in percent binucleated cells in the hearts of P4 and P7 pups. The dexamethasone treatment resulted in a significant increase of percent binucleated cells in the hearts of early developmental age of P4 pups, which was blocked by Ru486 (Fig 2). Ru486 treatment alone, while it had no significant effect on cardiomyocyte binucleation in P4 pups, significantly decreased percent binucleated cells in the hearts of P7 pups (Fig 2). Because binucleation of cardiomyocytes is an early indicator showing the transformation of hyperplasia to hypertrophy growth and suggesting cell destiny from proliferation to differentiation, the proliferation of cardiomyocytes was determined by double staining of α-actinin and Ki67, a marker for cellular proliferation. The dexamethasone treatment showed a tendency in decreasing percentage of Ki67-positive cardiomyocytes in P4 pups, and it significantly decreased Ki67-positive cardiomyocytes in P7 pups (Fig 3A). Ru486 treatment alone had no significant effect on cardiomyocyte proliferation in either P4 or P7 pups, but abrogated the dexamethasone-induced inhibitory effect on myocyte proliferation (Fig 3A). To confirm the proliferation data observed with Ki67, BrdU incorporation was also examined in P7 cardiomyocytes. As shown in Fig 3B, the dexamethasone treatment significantly decreased BrdU incorporation in P7 cardiomyocytes, which was blocked by Ru486.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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pone.0125033.g002: 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.
Mentions: Binucleation is one of the characteristics of cardiomyocyte maturation and terminal differentiation. Thus, we determined whether dexamethasone treatment influenced cardiomyocyte binucleation in the developing heart. Cardiomyocytes isolated from P4 and P7 pups were stained with a cardiomyocyte marker α-actinin and nuclei were labeled with Hoechst. The percentage of binucleation was scored by counting the number of mononucleated and binucleated cells using microscopy. As shown in Fig 2, in control animals, there was a development-dependent increase in percent binucleated cells in the hearts of P4 and P7 pups. The dexamethasone treatment resulted in a significant increase of percent binucleated cells in the hearts of early developmental age of P4 pups, which was blocked by Ru486 (Fig 2). Ru486 treatment alone, while it had no significant effect on cardiomyocyte binucleation in P4 pups, significantly decreased percent binucleated cells in the hearts of P7 pups (Fig 2). Because binucleation of cardiomyocytes is an early indicator showing the transformation of hyperplasia to hypertrophy growth and suggesting cell destiny from proliferation to differentiation, the proliferation of cardiomyocytes was determined by double staining of α-actinin and Ki67, a marker for cellular proliferation. The dexamethasone treatment showed a tendency in decreasing percentage of Ki67-positive cardiomyocytes in P4 pups, and it significantly decreased Ki67-positive cardiomyocytes in P7 pups (Fig 3A). Ru486 treatment alone had no significant effect on cardiomyocyte proliferation in either P4 or P7 pups, but abrogated the dexamethasone-induced inhibitory effect on myocyte proliferation (Fig 3A). To confirm the proliferation data observed with Ki67, BrdU incorporation was also examined in P7 cardiomyocytes. As shown in Fig 3B, the dexamethasone treatment significantly decreased BrdU incorporation in P7 cardiomyocytes, which was blocked by Ru486.

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