Knockdown of DNA methyltransferase 1 reduces DNA methylation and alters expression patterns of cardiac genes in embryonic cardiomyocytes.

We previously found that DNA methyltransferase 3a (DNMT3a) plays an important role in regulating embryonic cardiomyocyte gene expression, morphology, and function. In this study, we investigated the role of the most abundant DNMT in mammalian cells, DNMT1, in these processes. It is known that DNMT1 is essential for embryonic development, during which it is involved in regulating cardiomyocyte DNA methylation and gene expression. We used siRNA to knock down DNMT1 expression in primary cultures of mouse embryonic cardiomyocytes. Immunofluorescence staining and multielectrode array were, respectively, utilized to evaluate cardiomyocyte growth and electrophysiology. RNA sequencing (RNA-Seq) and multiplex bisulfite sequencing were, respectively, performed to examine gene expression and promoter methylation. At 72 h post-transfection, reduction of DNMT1 expression decreased the number and increased the size of embryonic cardiomyocytes. Beat frequency and the amplitude of field action potentials were decreased by DNMT1 siRNA. RNA-Seq analysis identified 801 up-regulated genes and 494 down-regulated genes in the DNMT1 knockdown cells when compared to controls. Pathway analysis of the differentially expressed genes revealed pathways that were associated with cell death and survival, cell morphology, cardiac function, and cardiac disease. Alternative splicing analysis identified 929 differentially expressed exons, including 583 up-regulated exons and 308 down-regulated exons. Moreover, decreased methylation levels were found in the promoters of cardiac genes Myh6, Myh7, Myh7b, Tnnc1, Tnni3, Tnnt2, Nppa, Nppb, mef2c, mef2d, Camta2, Cdkn1A, and Cdkn1C. Of these 13 genes, 6 (Myh6, Tnnc1, Tnni3, Tnnt2, Nppa, Nppb) and 1 (Cdkn1C) had increased or decreased gene expression, respectively. Altogether, these data show that DNMT1 is important in embryonic cardiomyocytes by regulating DNA methylation, gene expression, gene splicing, and cell function.

Opposing Effects of Maternal Hypo- and Hyperthyroidism on the Stability of Thalamocortical Synapses in the Visual Cortex of Adult Offspring.

Insufficient or excessive thyroid hormone (TH) levels during fetal development can cause long-term neurological and cognitive problems. Studies in animal models of perinatal hypo- and hyperthyroidism suggest that these problems may be a consequence of the formation of maladaptive circuitry in the cerebral cortex, which can persist into adulthood. Here we used mouse models of maternal hypo- and hyperthyroidism to investigate the long-term effects of altering thyroxine (T4) levels during pregnancy (corresponding to embryonic days 6.5-18.5) on thalamocortical (TC) axon dynamics in adult offspring. Because perinatal hypothyroidism has been linked to visual processing deficits in humans, we performed chronic two-photon imaging of TC axons and boutons in primary visual cortex (V1). We found that a decrease or increase in maternal serum T4 levels was associated with atypical steady-state dynamics of TC axons and boutons in V1 of adult offspring. Hypothyroid offspring exhibited axonal branch and bouton dynamics indicative of an abnormal increase in TC connectivity, whereas changes in hyperthyroid offspring were indicative of an abnormal decrease in TC connectivity. Collectively, our data suggest that alterations to prenatal T4 levels can cause long-term synaptic instability in TC circuits, which could impair early stages of visual processing.

In Utero Caffeine Exposure Induces Transgenerational Effects on the Adult Heart.

Each year millions of pregnant woman are exposed to caffeine, which acts to antagonize adenosine action. The long-term consequences of this exposure on the developing fetus are largely unknown, although in animal models we have found adverse effects on cardiac function. To assess if these effects are transmitted transgenerationally, we exposed pregnant mice to caffeine equivalent to 2-4 cups of coffee at two embryonic stages. Embryos (F1 generation) exposed to caffeine early from embryonic (E) day 6.5-9.5 developed a phenotype similar to dilated cardiomyopathy by 1 year of age. Embryos exposed to caffeine later (E10.5-13.5) were not affected. We next examined the F2 generation and F3 generation of mice exposed to caffeine from E10.5-13.5, as this coincides with germ cell development. These F2 generation adult mice developed a cardiac phenotype similar to hypertrophic cardiomyopathy. The F3 generation exhibited morphological changes in adult hearts, including increased mass. This report shows that in utero caffeine exposure has long-term effects into adulthood and that prenatal caffeine exposure can exert adverse transgenerational effects on adult cardiac function.

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 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.

Evaluation of developmental toxicity of propylthiouracil and methimazole.

BACKGROUND: Propylthiouracil (PTU) and methimazole (MMI) are antithyroid drugs used to treat hyperthyroidism. Despite the widespread use of PTU and MMI during pregnancy, modest clinical data and less animal data are available on the teratogenic potential of these drugs. METHODS: We evaluated the teratogenicity of in utero exposure to PTU or MMI in mice and rats. First, pregnant C57Bl/6 mice were treated daily with PTU (10 or 100 mg/kg), MMI (2 or 20 mg/kg), or vehicle from gestation day (GD) 6 to 16. GD 18 fetuses were evaluated for gross and histopathological abnormalities. Next, pregnant Sprague-Dawley rats were treated daily with PTU (50 or 100 mg/kg), MMI (10 or 20 mg/kg), or vehicle from GD 6 to 19, followed by evaluation for gross and histopathological abnormalities at GD 20. RESULTS: In mice treated with PTU or MMI, no significant histopathological abnormalities or external gross malformations, and no adverse effects on placental weight, litter size, resorption rates, or fetal weight were observed at GD 18. In rats, no adverse effects on litter size, placental weights, or maternal body weights were observed with either PTU or MMI treatment. PTU treatment (50 and 100 mg/kg) and MMI (10 mg/kg) treatment resulted in a decrease in crown-rump length in rat fetuses but no external gross malformations or histopathological abnormalities were observed. CONCLUSION: We did not observe either gross external malformations or histopathological malformations in mice or rats treated long-term with high doses of PTU or MMI during pregnancy.

The Sphingosine-1-phospate receptor 1 mediates S1P action during cardiac development.

BACKGROUND: Sphingosine-1-phosophate (S1P) is a biologically active sphingolipid metabolite that influences cellular events including differentiation, proliferation, and migration. S1P acts through five distinct cell surface receptors designated S1P1-5R, with S1P1R having the highest expression level in the developing heart. S1P1R is critical for vascular maturation, with its loss leading to embryonic death by E14.5; however, its function during early cardiac development is not well known. Our previous studies demonstrated that altered S1P levels adversely affects atrioventricular (AV) canal development in vitro, with reduced levels leading to cell death and elevated levels inhibiting cell migration and endothelial to mesenchymal cell transformation (EMT). RESULTS: We determined, by real-time PCR analysis, that S1P1R was expressed at least 10-fold higher than other S1P receptors in the developing heart. Immunohistochemical analysis revealed S1P1R protein expression in both endothelial and myocardial cells in the developing atrium and ventricle. Using AV canal cultures, we observed that treatment with either FTY720 (an S1P1,3,4,5R agonist) or KRP203 (an S1P1R-specific agonist) caused similar effects on AV canal cultures as S1P treatment, including induction of cell rounding, inhibition of cell migration, and inhibition of EMT. In vivo, morphological analysis of embryonic hearts at E10.5 revealed that S1P1R-/- hearts were malformed with reduced myocardial tissue. In addition to reduced myocardial tissue, E12.5 S1P1R-/- hearts had disrupted morphology of the heart wall and trabeculae, with thickened and disorganized outer compact layer and reduced fibronectin (FN) deposition compared to S1P1R+/+ littermates. The reduced myocardium was accompanied by a decrease in cell proliferation but not an increase in apoptosis. CONCLUSIONS: These data indicate that S1P1R is the primary mediator of S1P action in AV canal cultures and that loss of S1P1R expression in vivo leads to malformed embryonic hearts, in part due to reduced fibronectin expression and reduced cell proliferation.

Identification of the heart as the critical site of adenosine mediated embryo protection.

BACKGROUND: Our understanding of the mechanisms that protect the developing embryo from intrauterine stress is limited. Recently, adenosine has been demonstrated to play a critical role in protecting the embryo against hypoxia via adenosine A1 receptors (A1ARs), which are expressed in the heart, nervous system, and other sites during development. However, the sites of A1AR action that mediate embryo protection are not known. To determine if the heart is a key site of adenosine-mediated embryo protection, A1ARs were selectively deleted in the embryonic heart using a Cre-LoxP system in which the alpha-myosin heavy chain promoter drives Cre-recombinase expression and excision of the A1AR gene from cardiomyocytes. RESULTS: With increasing exposure of maternal hypoxia (10% O2) from 48-96 hours beginning at embryonic day (E) 8.5, embryo viability decreased in the cardiac-A1AR deleted embryos. 48 hours of hypoxia reduced embryonic viability by 49% in embryos exposed from E10.5-12.5 but no effect on viability was observed in younger embryos exposed to hypoxia from E8.5-10.5. After 72 hours of hypoxia, 57.8% of the cardiac-A1AR deleted embryos were either dead or re-absorbed compared to 13.7% of control littermates and after 96 hours 81.6% of cardiac-A1AR deleted embryos were dead or re-absorbed. After 72 hours of hypoxia, cardiac size was reduced significantly more in the cardiac-A1AR deleted hearts compared to controls. Gene expression analysis revealed clusters of genes that are regulated by both hypoxia and A1AR expression. CONCLUSIONS: These data identify the embryonic heart as the critical site where adenosine acts to protect the embryo against hypoxia. As such these studies identify a previously unrecognized mechanism of embryo protection.