Embryos can be exposed to environmental factors that induce hypoxia. Currently, our understanding of the effects of hypoxia on early mammalian development is modest. Potential mediators of hypoxia action include the nucleoside adenosine, which acts through A 1 adenosine receptors (A1ARs) and mediates adverse effects of hypoxia on the neonatal brain. We hypothesized that A 1ARs may also play a role in mediating effects of hypoxia on the embryo. When pregnant dams were exposed to hypoxia (10% O2) beginning at embryonic day (E) 7.5 or 8.5 and continued for 24 -96 h, A 1AR؉/؉ embryos manifested growth inhibition and a disproportionate reduction in heart size, including thinner ventricular walls. Yet, when dams were exposed to hypoxia, embryos lacking A 1ARs (A1AR؊/؊) had much more severe growth retardation than A 1AR؉/؉ or ؉/؊ embryos. When levels of hypoxia-inducible factor 1␣ (HIF1␣) were examined, A1AR؊/؊ embryos had less stabilized HIF1␣ protein than A1AR؉/؊ littermates. Normal patterns of cardiac gene expression were also disturbed in A1AR؊/؊ embryos exposed to hypoxia. These results show that short periods of hypoxia during early embryogenesis can result in intrauterine growth retardation. We identify adenosine and A1ARs as playing an essential role in protecting the embryo from hypoxia.cardiac ͉ heart development ͉ hypoxia-inducible factor ͉ intrauterine growth retardation

Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid metabolite that exerts important effects on numerous cellular events via cell surface receptors, S1P(1-5). S1P influences differentiation, proliferation, and migration during vascular development. However, the effects of S1P signaling on early cardiac development are not well understood. To address this issue, we examined the expression of S1P regulatory enzymes and S1P receptors during cardiac development. We observed that enzymes that regulate S1P levels, sphingosine kinase and sphingosine-1-phosphate phosphatase, are expressed in the developing heart. In addition, RT-PCR revealed that four of the five known S1P receptors (S1P(1-4)) are also expressed in the developing heart. Next, effects of altered S1P levels on whole embryo and atrioventricular (AV) canal cultures were investigated. We demonstrate that inactivation of the S1P producing enzyme, sphingosine kinase, leads to cell death in cardiac tissue which is rescued by exogenous S1P treatment. Other experiments reveal that increased S1P concentration prevents alterations in cell morphology that are required for cell migration. This effect results in reduced cell migration and inhibited mesenchymal cell formation in AV canal cushion tissue. These data indicate that S1P, locally maintained within a specific concentration range, is an important and necessary component of early heart development.

The purpose of this study was to determine both the short-term effects on cardiac development and embryo growth and the long-term effects on cardiac function and body composition of in utero caffeine exposure. Pregnant mice (C57BL/6) were exposed to hypoxia (10% O(2)) or room air from embryonic days (E) 8.5-10.5, and treated with caffeine (20 mg/kg, i.p.) or vehicle (normal saline, 0.9% NaCl). This caffeine dose results in a circulating level that is equivalent to 2 cups of coffee in humans. Hypoxic exposure acutely reduced embryonic growth by 30%. Exposure to a single dose of caffeine inhibited cardiac ventricular development by 53% in hypoxia and 37% in room air. Caffeine exposure resulted in inhibition of hypoxia-induced HIF1alpha protein expression in embryos by 40%. When offspring from dams treated with a single dose of caffeine were studied in adulthood, we observed that caffeine treatment alone resulted in a decrease in cardiac function of 38%, as assessed by echocardiography. We also observed a 20% increase in body fat with male mice exposed to caffeine. Caffeine was dissolved in normal saline, so it was used as a control. Room air controls were used to compare to the hypoxic mice. Exposure to a single dose of caffeine during embryogenesis results in both short-term effects on cardiac development and long-term effects on cardiac function.

Few signaling molecules have the potential to influence the developing mammal as the nucleoside adenosine. Adenosine levels increase rapidly with tissue hypoxia and inflammation. Adenosine antagonists include the methlyxanthines caffeine and theophylline. The receptors that transduce adenosine action are the A1, A2a, A2b, and A3 adenosine receptors (ARs). In the postnatal period, A1AR activation may contribute to white matter injury in the preterm infant by altering oligodendrocyte (OL) development. In models of perinatal brain injury, caffeine is neuroprotective against periventricular white matter injury (PWMI) and hypoxic-ischemic encephalopathy (HIE). Supporting the notion that blockade of adenosine action is of benefit in the premature infant, caffeine reduces the incidence of broncho-pulmonary dysplasia and cerebral palsy in clinical studies. In comparison with the adverse effects on the postnatal brain, adenosine acts via A1ARs to play an essential role in protecting the embryo from hypoxia. Embryo protective effects are blocked by caffeine, and caffeine intake during early pregnancy increases the risk of miscarriage and fetal growth retardation. Adenosine and adenosine antagonists play important modulatory roles during mammalian development. The protective and deleterious effects of adenosine depend on the time of exposure and target sites of action.

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 b-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. FASEB J. 30, 3238-3255 (2016). www.fasebj.org