Congenital birth defects can arise with embryonic exposure to therapeutic drugs, high levels of normal plasma metabolites, or other environmental factors. Congenital cardiac defects arising from lithium (Li) exposure, a drug used for management of mood disorders, or from elevated plasma homocysteine (HCy) often involve tricuspid, pulmonary or aortic valve defects; a thickened heart wall; and/or defects in the outflow tract. Cardiac abnormalities are accompanied by neural tube defects and craniofacial anomalies that occur through unknown mechanisms. Lithium exposure during gastrulation induces cardiac defects accompanied by potentiation of the Wnt–β-catenin signaling pathway. This suggests that elevated HCy, associated with cardiac defects, may also target the Wnt–β-catenin pathway during neural tube and craniofacial development. It is well known that folic acid (FA) supplementation can reduce the incidence of neural tube defects, but the cause for its preventative action is not known. This work sought to address the ability of FA supplementation to prevent cardiac defects induced by Li, HCy or Wnt3A in the vertebrate embryo.
This study uses Doppler ultrasound to identify functional heart and placental defects in utero on embryonic day (E)15.5 after pregnant mice received a single exposure to HCy or Li on E6.75. Histological analysis shows defects in tricuspid and semilunar valves and altered myocardial thickness in HCy- and Litreated embryos. These embryos, and their placentas, were also smaller than control animals. When FA supplementation was started immediately after fertilization, it prevented these developmental errors. Gene expression studies within the avian heart-forming regions demonstrate that Li, HCy and Wnt3A suppress Wnt-modulated Hex and Isl1 expression, and that FA prevents this effect. Inclusion of myoinositol with FA supplementation potentiates the protection of Li-induced errors.
FA supplementation has been used since 1998 to reduce the occurrence of neural tube defects. The underlying basis of the protection is not known. Our results demonstrate that FA deficiency- and elevated HCy-associated birth defects involve Wnt–β-catenin signaling, and that prophylactic FA supplementation, at levels that are higher than those used currently for neural tube defect prevention, can prevent cardiac defects. Histone and DNA methylation are fundamental processes in the Wnt–β-catenin pathway and gene target regulation. HCy and FA molecules are intermediaries in the S-adenosylmethionine (SAM), the universal methyl donor, synthesis pathway. FA deficiency leads to HCy accumulation and a cellular deficit of SAM, thereby limiting transmethylation reactions in the nucleus. These reactions are involved in epigenetic modifications, meaning that nonlethal Li and HCy effects could extend beyond the period of embryogenesis. Further study on FA effects on the epigenome should facilitate the mechanistic understanding of factors that induce cardiac and neural birth defects so that they may be prevented.