Epigenetic Regulation of Notch Signaling by Prenatal Alcohol Exposure Impacts Cardiac Outflow Tract Defect Development

Introduction: Prenatal alcohol exposure (PAE) leads to congenital heart defects (CHD) with incomplete penetrance and phenotypic heterogeneity. We have demonstrated a gene-environment interaction in this context wherein acute PAE synergizes with second heart field (SHF) haploinsufficiency of Notch signaling, resulting in highly penetrant outflow tract (OFT) defects. Hypothesis: We hypothesize that acute PAE-induced hyperacetylation at histone marker 3 lysine residue 9 (aH3K9) epigenetically inhibits Notch signaling. This potentiates the impact of a pre-existing genetic defect that results in partial loss of Notch signaling leading to loss of viability of SHF progenitors necessary for OFT elongation and alignment. Methods: We studied the effects of acute PAE in vivo in our murine model (two injections of 3g/kg 30% ethanol on E6.5) on acetylation, SHF viability, OFT length, and Notch intracellular domain (NICD) localization. Complimentarily, we evaluated alcohol exposure in vitro using our established thoracic organ culture model. Acetylation was reversed using pan-histone acetyltransferase inhibitor anacardic acid. Results: At E9.5, acute PAE resulted in a significant increase in H3K9 acetylation in SHF associated with restriction of NICD to the cell membrane. This resulted in a 0.28-fold reduction in Notch signaling, as evidenced by reduced Notch reporter expression, and 0.52-fold reduction in SHF cell proliferation (both p<0.001). Loss of SHF progenitors needed for incorporation into the developing OFT reduced OFT length 26% (p<0.001). In vitro thoracic organ culture analyses recapitulated increased acetylation, NICD restriction to cell membrane and reduced SHF proliferation by 60% (p<0.001). Anacardic acid reversed the increase of aH3K9 and allowed NICD nuclear translocation. Notch signaling and SHF proliferation returned to baseline. Conclusions: Acute PAE induces hyperacetylation in the SHF and inhibits Notch signaling by restricting NICD translocation. Lack of Notch signaling reduces SHF viability and prevents elongation of the developing OFT. This hereto unexplored impact of alcohol on the Notch pathway provides a novel area of investigation regarding gene-environment interaction in alcohol teratogenesis.