SUMMARY Elevated plasma homocysteine (HCy), which results from folate (folic acid, FA) deficiency, and the mood-stabilizing drug lithium (Li) are both linked to the induction of human congenital heart and neural tube defects. We demonstrated previously that acute administration of Li to pregnant mice on embryonic day (E)6.75 induced cardiac valve defects by potentiating Wnt–β-catenin signaling. We hypothesized that HCy may similarly induce cardiac defects during gastrulation by targeting the Wnt–β-catenin pathway. Because dietary FA supplementation protects from neural tube defects, we sought to determine whether FA also protects the embryonic heart from Li- or HCy-induced birth defects and whether the protection occurs by impacting Wnt signaling. Maternal elevation of HCy or Li on E6.75 induced defective heart and placental function on E15.5, as identified non-invasively using echocardiography. This functional analysis of HCy-exposed mouse hearts revealed defects in tricuspid and semilunar valves, together with altered myocardial thickness. A smaller embryo and placental size was observed in the treated groups. FA supplementation ameliorates the observed developmental errors in the Li- or HCy-exposed mouse embryos and normalized heart function. Molecular analysis of gene expression within the avian cardiogenic crescent determined that Li, HCy or Wnt3A suppress Wnt-modulated Hex (also known as Hhex) and Islet-1 (also known as Isl1) expression, and that FA protects from the gene misexpression that is induced by all three factors. Furthermore, myoinositol with FA synergistically enhances the protective effect. Although the specific molecular epigenetic control mechanisms remain to be defined, it appears that Li or HCy induction and FA protection of cardiac defects involve intimate control of the canonical Wnt pathway at a crucial time preceding, and during, early heart organogenesis.

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Embryonic exposures can increase the risk of congenital cardiac birth defects and adult disease. The present study identifies the predominant pathways modulated by an acute embryonic mouse exposure during gastrulation to lithium or homocysteine that induces cardiac defects. High dose periconceptional folate supplementation normalized development. Microarray bioinformatic analysis of gene expression demonstrated that primarily lipid metabolism is altered after the acute exposures. The lipid-related modulation demonstrated a gender bias with male embryos showing greater number of lipid-related Gene Ontology biological processes altered than in female embryos. RT-PCR analysis demonstrated significant change of the fatty acid oxidation gene Acadm with homocysteine exposure primarily in male embryos than in female. The perturbations resulting from the exposures resulted in growth-restricted placentas with disorganized cellular lipid droplet distribution indicating lipids have a critical role in cardiac-placental abnormal development. High folate supplementation protected normal heart-placental function, gene expression and lipid localization.

Lithium (Li), a mood stabilizing drug, and elevated homocysteine (HCy), a metabolite in the folic acid (FA) cycle, are linked to induction of human congenital heart defects. We determined noninvasively by echocardiography, that exposure of the mouse embryo by an i.p. injection to the dam of Li or HCy during gastrulation on embryonic day E6.75 induces cardiac and valve defects. A single dose of 125 μl Li (25 mM) or HCy (75 μM) on E5.5 to 6.5 results in mouse embryonic lethality; on E6.75 to 7.0, both induce tricuspid and semilunar valve defects (Li: n=131 ; HCy: n=78). The tricuspid valve septal leaflet fails to delaminate, a characteristic of Ebstein’s Anomaly. Use of Li during human pregnancy has been associated with Ebstein’s Anomaly. Li mimics the Wnt/β-catenin (β-cat) signaling pathway by inactivating glycogen synthase kinase-3β. During chick cardiac specification, Li, Wnt 3A, or HCy exposure adversely affects chick cardiogenesis with severity of anomalies based on timing of early exposure. To initiate cardiogenesis, the secreted Wnt antagonist Dickkopf-1 acts extracellularly on the endoderm to upregulate Hex , an inducer of cardiomyogenesis. Exposure of stages 3+/4− chick embryos to Li/Wnt3A/ HCys inhibits Hex and Islet-1 gene expression in the cardiogenic crescent via an intracellular mechanism, thus augmenting inhibitory Wnt/β-cat signaling. FA deficiency leads to elevated HCys levels. We hypothesize that HCys/FA metabolism intersects with Wnt/β-cat signaling and that mechanistically FA supplementation acts by overriding Wnt/β-cat inhibition of gene expression in the embryonic heart fields that leads to cardiac defects. FA, known to protect against HCys-mediated neural tube defects, was tested for protective effects against Li/Wnt3A/HCy during cardiogenesis. With all three experimental exposures, FA addition results in reexpression of the cardiac inducers Hex and Islet-1 at high levels in the chick heart fields. In the mouse, no valve defects were detected in HCys/FA exposed embryos (n=27). Regimen and concentration of FA supplementation necessary to fully rescue Li effects are being tested. In conclusion, folate supplementation potentiates the repression of Wnt/β-cat signaling and protects formation of heart and valve defects. This research has received full or partial funding support from the American Heart Association, AHA Greater Southeast Affiliate (Alabama, Florida, Georgia, Louisiana, Mississippi, Puerto Rico & Tennessee).