Dexamethasone Induces Cardiomyocyte Terminal Differentiation via Epigenetic Repression of Cyclin D2 Gene

Abstract

Dexamethasone treatment of newborn rats inhibited cardiomyocyte proliferation and stimulated premature terminal differentiation of cardiomyocytes in the developing heart. Yet mechanisms remain undetermined. The present study tested the hypothesis that the direct effect of glucocorticoid receptor-mediated epigenetic repression of cyclin D2 gene in the cardiomyocyte plays a key role in the dexamethasone-mediated effects in the developing heart. Cardiomyocytes were isolated from 2-day-old rats. Cells were stained with a cardiomyocyte marker a-actinin and a proliferation marker Ki67. Cyclin D2 expression was evaluated by Western blot and quantitative real-time polymerase chain reaction. Promoter methylation of CcnD2 was determined by methylated DNA immunoprecipitation (MeDIP). Overexpression of Cyclin D2 was conducted by transfection of FlexiCcnD2 (1CcnD2) construct. Treatment of cardiomyocytes isolated from newborn rats with dexamethasone for 48 hours significantly inhibited cardiomyocyte proliferation with increased binucleation and decreased cyclin D2 protein abundance. These effects were blocked with Ru486 (mifepristone). In addition, the dexamethasone treatment significantly increased cyclin D2 gene promoter methylation in newborn rat cardiomyocytes. 5-Aza-2'-deoxycytidine inhibited dexamethasone-mediated promoter methylation, recovered dexamethasone-induced cyclin D2 gene repression, and blocked the dexamethasone-elicited effects on cardiomyocyte proliferation and binucleation. In addition, the overexpression of cyclin D2 restored the dexamethasone-mediated inhibition of proliferation and increase in binucleation in newborn rat cardiomyocytes. The results demonstrate that dexamethasone acting on glucocorticoid receptors has a direct effect and inhibits proliferation and stimulates premature terminal differentiation of cardiomyocytes in the developing heart via epigenetic repression of cyclin D2 gene.

In vitro and in vivo roles of glucocorticoid and vitamin D receptors in the control of cardiomyocyte proliferative potential

“…The intrinsic and extrinsic molecular drivers of this process have been under intensive studies 4,[36][37][38][39][40] . Our observation that both glucocorticoids and vitamin D inhibit proliferation of primary neonatal mouse CMs in vitro is consistent with previous reports in culture 21,22,28,29,41 . GR activation inhibits neonatal rat CM proliferation and increases CM binucleation 21,22 .…”

“…Our observation that both glucocorticoids and vitamin D inhibit proliferation of primary neonatal mouse CMs in vitro is consistent with previous reports in culture 21,22,28,29,41 . GR activation inhibits neonatal rat CM proliferation and increases CM binucleation 21,22 . Vitamin D treatment reduces expression of c-myc and proliferating cell nuclear antigen, and blocks cell proliferation in rat CM culture 41 .…”

“…Specifically, glucocorticoids like dexamethasone are established cell cycle regulators known to repress the cell cycle primarily through the GR, which acts as a transcription factor after glucocorticoid binding 19,20 . It has been reported that GR activation inhibits neonatal rat CM proliferation and increases CM binucleation through epigenetic repression of Cyclin D2 gene 21,22 . Furthermore, adult zebrafish heart regeneration is impaired by either GR agonist exposure 23 or crowding-induced stress through GR activation 24 .…”

DNA methylation and chromatin dynamics during postnatal cardiomyocyte maturation

“…Furthermore, another recent study has reported that inhibition of neonatal rat cardiomyocyte proliferation following treatment with dexamethasone is due to promoter hypermethylation and transcriptional repression of cell cycle genes, including Ccnd2 (Gay et al, 2016). Gay et al also demonstrated that 5aza-dC reactivated cell proliferation in dexamethasone-treated cardiomyocytes through restoration of Ccnd2 expression, providing further support for a direct role for DNA methylation in regulating transcription of cell cycle genes (Gay et al, 2016).…”

“…Gay et al also demonstrated that 5aza-dC reactivated cell proliferation in dexamethasone-treated cardiomyocytes through restoration of Ccnd2 expression, providing further support for a direct role for DNA methylation in regulating transcription of cell cycle genes (Gay et al, 2016). Therefore, identification of the direct targets of DNA methylation at specific cell cycle genes is critical to understand the mechanism of postnatal cardiomyocyte cell cycle arrest.…”