Folate rescues lithium-, homocysteine- and Wnt3A-induced vertebrate cardiac anomalies

Abstract

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-beta-catenin signaling. We hypothesized that HCy may similarly induce cardiac defects during gastrulation by targeting the Wnt-beta-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.

Fig. 1.

Effects of exogenous HCy on early chick heart development after a 24-hour incubation. Immunolocalization of MF20 defines the presence of cardiac tissue (arrows). (A,B) Two extremes of cardiabifida: in A, two small cardiogenic regions are differentiating bilaterally; in B, the two heart fields have moved close to the midline, are almost touching, and are ready to fuse. (C) Light microscopic view of an embryo displaying a severely truncated neural tube with cardiac tissue differentiating cephalad to the neural area (D). (E) Bright-field ventral view of a normal, control, right-looping heart and (F) fluorescence view showing MF20 localization in the heart shown in E. In all panels, the embryonic anterior is at the top. Bars, 300 μm.

Fig. 2.

Hex and Islet-1 (Isl1) gene expression patterns after 8 and 24 hours in control embryos and in Li- or HCy-exposed embryos. (A,B,G,H) In control embryos (Con, top row), both Hex and Isl1 expression are seen at relatively high levels after an 8-hour (A,G) and 24-hour (B,H) incubation. The black arrows indicate in situ gene expression. Hex and Isl1 gene expression are suppressed in chick embryos following Li (C, second row) or HCy (E, bottom row) exposure at HH stages 3+/4–. A recovery in gene expression is noted by 24 hours (D,F), but embryos remain delayed in their development. Modulation of Isl1 is similar: little or no Isl1 gene expression is detectable following 8 hours of exposure to Li (I) or HCy (K). By 24 hours, heart development is delayed and cardiac anomalies arise (J,L). In all panels, the embryonic anterior is at the top. Bars, 300 μm.

Fig. 3.

FA supplementation with embryonic Wnt3A exposure at HH stages 3+/4 increases the percentage of normal heart development. Of the embryos in the control group, 58% displayed normal development in contrast to 18% of embryos in the Wnt3A-exposed group. FA supplementation increased the percentage of Wnt3A-exposed embryos with normal development to the level seen in the control group.

Fig. 4.

FA rescues heart development of Wnt3A-exposed embryos immunostained with MF20 antibody. (A–C) Control embryonic hearts in whole mounts: (A) untreated control (Con) embryo; (B) FA exposure only, 2 μg/ml; and (C) FA exposure only, 10 μg/ml. HH stages 3+/4 embryos exposed to 2 ng/ml (D) or 10 ng/ml (G) of Wnt3A display abnormal heart development. FA supplementation at a concentration of 2 μg/ml rescues embryonic heart development at the 2 ng/ml Wnt3A concentration (E), but only partially rescues heart development in embryos exposed to 10 ng/ml of Wnt3A (H, left-looping heart). FA supplementation at a concentration of 10 μg/ml completely normalized heart development with either a low or high level of Wnt 3A exposure (F,I). (J) Bright-field view of the embryo shown in K, a negative control without primary antibody treatment. In all panels, the embryonic anterior is at the top. Bar, 275 μm.

Fig. 5.

FA rescues Hex expression in Li-, HCy- and Wnt3A-exposed embryos. (A–C) Normal patterns of Hex expression in control embryos (saline added) after 8 hours (A), and after FA-only exposure at 8 hours (B) and at 24 hours (C). (D–F) Gene expression in embryos exposed to only HCy for 8 hours (D), and to HCy with FA supplementation for 8 hours (E) and 24 hours (F). (G–I) Gene expression in embryos exposed to only Li for 8 hours (G), and to Li with FA supplementation for 8 hours (H) and 24 hours (I). (J–L) Gene expression in embryos exposed to only Wnt3A for 8 hours (J), and to Wnt3A with FA supplementation for 8 hours (K) and 24 hours (L). In all panels, the embryonic anterior is at the top. Bars, 300 μm.

Fig. 6.

FA rescues Isl1 expression in Li-, HCy- and Wnt3A-exposed embryos. (A–C) Normal patterns of Isl1 expression in control embryos (saline added to medium) after 8 hrs (A), and after FA-only exposure at 8 hours (B) and at 24 hours (C). (D–F) Gene expression in embryos exposed to only HCy for 8 hours (D), and to HCy with FA supplementation for 8 hours (E) and 24 hours (F) (G–I) Gene expression in embryos exposed to only Li for 8 hours (G), and to Li with FA supplementation for 8 hours (H) and 24 hours (I). (J–L) Gene expression in embryos exposed to only Wnt3A for 8 hours (J), and to Wnt3A with FA supplementation for 8 hours (K) and 24 hours (L). In all panels, the embryonic anterior is at the top. Bars, 300 μm.

Fig. 7.

Doppler velocity waveforms of blood flow on E15.5. Inflow is shown above the zero line and outflow below the line. The outflow is the systolic ejection velocity. (A) A normal pattern of blood flow. (B) Holosystolic AV valve regurgitation (arrow), (C) SL valve regurgitation (arrow), and (D) monophasic inflow pattern with merged E and A waves (arrow). E, early ventricular filling; A, late ventricular filling during atrial contraction; ICT, isovolemic contraction time; IRT, isovolemic relaxation time; ET, ejection time. (E–J) Blood flow velocity waveforms obtained from the descending aorta (E,G,I) and umbilical artery (F,H,J) of a control embryo (E,F), a Li-exposed embryo (G,H), and an HCy-exposed embryo (I,J). The control embryo waveforms show a normal pattern with diastolic flow (E,F), whereas the Li-exposed embryo waveforms show an absence of diastolic flow (G,H). (I,J) Blood flow in the maternal uterine artery (I) and the descending aorta (J) of an HCy-exposed embryo is similiar to control animals. (K–M) Doppler velocity waveforms of blood flow on E15.5 after a single exposure to HCy on E6.75. The waveforms show SL valve regurgitation (K), SL valve stenosis (L), and AV valve regurgitation (M).

Fig. 8.

Pathology of HCy-exposed E15.5 mouse hearts displaying abnormal echo patterns. The panels in the left column depict AV valves (TCV, tricuspid valve; MV, mitral valve); the middle column shows aortic valves (AoV); and the right column shows pulmonary valves (PV). (A–C) A control heart. (D–F) An embryo with AV valve regurgitation. (G–I) An embryo displaying SL and AV valve regurgitation upon echocardiography. (J–L) The echo patterns for this embryo defined pulmonary stenosis. (M–O) Higher magnification of valves in hearts showing valve regurgitation upon echocardiography of an AV valve (M) and SL valves (N,O). RA, right atrium; RV, right ventricle, LA, left atrium; LV, left ventricle; IVS, interventricular septum; Ao, aorta; OFT, outflow tract. In all panels, the embryonic anterior is to the top. Bars, 152 μm.