Knockdown of DNA methyltransferase 3a alters gene expression and inhibits function of embryonic cardiomyocytes.
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 β-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.
Child Health Research Institute, Department of Pediatrics, College of Medicine, University of Florida, Gainesville, Florida, USA; and.
Xiefan
X
Fang
Child Health Research Institute, Department of Pediatrics, College of Medicine, University of Florida, Gainesville, Florida, USA; and.
Ryan R
RR
Poulsen
Child Health Research Institute, Department of Pediatrics, College of Medicine, University of Florida, Gainesville, Florida, USA; and.
John
J
Wang-Hu
Child Health Research Institute, Department of Pediatrics, College of Medicine, University of Florida, Gainesville, Florida, USA; and.
Olivia
O
Shi
Department of Mechanical and Aerospace Engineering, College of Engineering, University of Florida, Gainesville, Florida, USA.
Nicholas S
NS
Calvo
Department of Mechanical and Aerospace Engineering, College of Engineering, University of Florida, Gainesville, Florida, USA.
Chelsey S
CS
Simmons
Child Health Research Institute, Department of Pediatrics, College of Medicine, University of Florida, Gainesville, Florida, USA; and.
Scott A
SA
Rivkees
Child Health Research Institute, Department of Pediatrics, College of Medicine, University of Florida, Gainesville, Florida, USA; and cwendler@ufl.edu.
Christopher C
CC
Wendler
D000200:Action Potentials / Q000502:physiology; D000818:Animals; D017209:Apoptosis; D020013:Calcium Signaling / Q000502:physiology; D002470:Cell Survival; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism*; D000090205:DNA Methyltransferase 3A; D015536:Down-Regulation; D004622:Embryo, Mammalian / Q000502:physiology*; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D055785:Gene Knockdown Techniques; D051379:Mice; D032383:Myocytes, Cardiac / Q000201:enzymology*; D012518:Sarcomeres; D000097584:DNA Methyltransferase 3B
D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D000097584:DNA Methyltransferase 3B; C000715535:Dnmt3a protein, mouse; C000619381:Dnmt1 protein, mouse
DNA methylation; cellular morphology; contractility; transcriptome