RESUMO
We previously found that in utero caffeine exposure causes down-regulation of DNA methyltransferases (DNMTs) in embryonic heart and results in impaired cardiac function in adulthood. To assess the role of DNMTs in these events, we investigated the effects of reduced DNMT expression on embryonic cardiomyocytes. siRNAs were used to knock down individual DNMT expression in primary cultures of mouse embryonic cardiomyocytes. Immunofluorescence staining was conducted to evaluate cell morphology. A video-based imaging assay and multielectrode array were used to assess cardiomyocyte contractility and electrophysiology, respectively. RNA-Seq and multiplex bisulfite sequencing were performed to examine gene expression and promoter methylation, respectively. At 72 h after transfection, reduced DNMT3a expression, but not DNMT1 or -3b, disrupted sarcomere assembly and decreased beating frequency, contractile movement, amplitude of field action potential, and cytosolic calcium signaling of cardiomyocytes. RNA-Seq analysis revealed that the DNMT3a-deficient cells had deactivated gene networks involved in calcium, endothelin-1, renin-angiotensin, and cardiac ß-adrenergic receptor signaling, which were not inhibited by DNMT3b siRNA. Moreover, decreased methylation levels were found in the promoters of Myh7, Myh7b, Tnni3, and Tnnt2, consistent with the up-regulation of these genes by DNMT3a siRNA. These data show that DNMT3a plays an important role in regulating embryonic cardiomyocyte gene expression, morphology and function.-Fang, X., Poulsen, R. R., Wang-Hu, J., Shi, O., Calvo, N. S., Simmons, C. S., Rivkees, S. A., Wendler, C. C. Knockdown of DNA methyltransferase 3a alters gene expression and inhibits function of embryonic cardiomyocytes.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Embrião de Mamíferos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Miócitos Cardíacos/enzimologia , Potenciais de Ação/fisiologia , Animais , Apoptose , Sinalização do Cálcio/fisiologia , Sobrevivência Celular , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Regulação para Baixo , Técnicas de Silenciamento de Genes , Camundongos , Sarcômeros , DNA Metiltransferase 3BRESUMO
Periventricular white matter injury (PWMI) is the most common cause of brain injury in preterm infants. It is believed that loss of late oligodendrocyte progenitor cells (OPCs) and disrupted maturation of oligodendrocytes contributes to defective myelination in PWMI. At present, no clinically approved drugs are available for treating PWMI. Previously, we found that diazoxide promotes myelination and attenuates brain injury in the chronic sublethal hypoxia model of PWMI. In this study, we investigated the mechanisms by which diazoxide promotes myelination. We observed that diazoxide increases the ratio of differentiated oligodendrocytes in the cerebral white matter, promotes the expression of differentiation-associated transcriptional factors Nkx2.2 and Sox10, and increases the expression of myelin genes CNP and MBP. These results show that diazoxide promotes oligodendrocyte differentiation in the developing brain.