Concomitant genetic defects potentiate the adverse impact of prenatal alcohol exposure on cardiac outflow tract maturation

Abstract

Background: Prenatal alcohol exposure (PAE) is associated with an increased incidence of congenital heart defects (CHD), in particular outflow tract (OFT) defects. However, the variability in the incidence of CHD following PAE has not been fully explored. We hypothesize that a concomitant, relevant genetic defect would potentiate the adverse effect of PAE and partially explain the variability of PAE-induced CHD incidence.

Methods: The OFT is formed by the second heart field (SHF). Our PAE model consisted of two intraperitoneal injections (3 g/kg, separated by 6 hr) of 30% ethanol on E6.5 during SHF specification. The impact of genetic defects was studied by SHF-specific loss of Delta-like ligand 4 (Dll4), fibroblast growth factor 8 (Fgf8) and Islet1.

Results: Acute PAE alone significantly increased CHD incidence (4% vs. 26%, p = .015) with a particular increase in OFT alignment defects, viz., double outlet right ventricle (0 vs. 9%, p = .02). In embryos with a SHF genetic defect, acute PAE significantly increased CHD incidence (14 vs. 63%, p < .001), including double outlet right ventricle (6 vs. 50%, p < .001) compared to controls. PAE (p = .01) and heterozygous loss of Dll4 (p = .04) were found to independently contribute to CHD incidence, while neither Islet1 nor Fgf8 defects were found to be significant.

Conclusions: Our model recapitulates the increased incidence of OFT alignment defects seen in the clinic due to PAE. The presence of a concomitant SHF genetic mutation increases the incidence of PAE-related OFT defects. An apparent synergistic interaction between PAE and the loss of DLL4-mediated Notch signaling in OFT alignment requires further analysis.

Keywords: Notch; cardiac outflow tract; congenital heart defect; prenatal alcohol.

FIGURE 1

Prenatal alcohol and phosphate-buffered saline (PBS) exposure protocol and blood alcohol content. Acute prenatal alcohol exposure protocol is depicted (a). At embryonic day 6.5 (E6.5) the pregnant dam was administered two intraperitoneal injections of either 3 g/kg PBS or 30% ethanol. The embryos were analyzed for cardiac phenotype on E14.5. Blood alcohol content was found to peak (b) 30 min after each injection. Baseline levels were achieved by 30 min prior to the second injection and 3 hr after the second injection. There was no significant difference in levels between nonpregnant (average peak 0.39 ± 0.04 g/dL after second injection) and pregnant mice (0.34 ± 0.05 g/dL after the second injection, c)

FIGURE 2

The combined deleterious impact of prenatal alcohol exposure (PAE) teratogenicity and second heart field (SHF) genetic defects. Embryos with heterozygous loss of SHF genes were examined for the presence of congenital heart defects (CHD). Comparisons were made across genotypes and between those exposed to phosphate-buffered saline (PBS) or received acute PAE. Wild-type/PAE embryos had a significant (p < .05) increase in CHD frequency (26 vs. 4%). All embryos carrying a SHF mutation with PAE have a higher frequency of CHD than wild-type/PAE mice, but only Dll4 mutations reached statistical significance. Mef2c-cre, Dll4^F/WT and Islet1-cre, Dll4^F/WT/PAE mutants had the most significant increase in overall CHD (p < .001 and p < .005, respectively) and frequency of DORV (67 and 54 vs. 17%). CAT, common arterial trunk; DORV, double outlet right ventricle; Isolated VSD, isolated ventricular septum defect. *p < .05, ***p < .005

FIGURE 3

Phenotypic spectrum of lesions observed with combined Notch mutation and prenatal alcohol exposure (PAE). Typical cardiac anatomy at embryonic day 14.5 (E14.5) is displayed in panels a-a″. The ventricular septum is fully intact (a), the aorta arises from the left ventricle (a′), and the pulmonary artery arises from the right ventricle (a″). PAE and mutations in the second heart field (SHF) result in a variety of cardiac phenotypes. The mildest lesion is an isolated ventricular septal defect (arrow in b) with normally aligned outflow tracts (b′ A indicates aortic valve, b″ P indicates pulmonary valve). Double outlet right ventricle consists of an obligate ventricular septal defect (VSD; arrow in a), observed to be larger than when the VSD was isolated, and malalignment of the outflow tract (OFT). The aorta is situated over the right ventricle (A in c′) as is the pulmonary valve (P in c″). The most severe phenotype observed was persistent truncus arteriosus in a PAE Islet1-cre, Dll4^F/WT embryo. This also consists of an obligate VSD (arrow in d) and is defined by the lack of septation of the OFT into two separate vessels, and instead continues as a single common arterial trunk (A in d′) from which the pulmonary artery arises (P in d″).Images were brightened in ImageJ, which was applied evenly to all parts of each image and equally to all subjects. Scale bar = 400 μm