Identifying and protecting alcohol-sensitive epigenetic changes in congenital heart disease

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This proposal describes a five-year mentored research and training plan that will facilitate the development of Dr. Stephanie Ford, MD as an independent investigator in the pathogenesis of congenital heart disease. Building upon Dr. Ford’s background as a clinical neonatologist and a basic scientist, she will attain expertise in design of mouse studies, RNA-FISH, and epigenetic mechanisms. She will gain skills through structured mentorship, hands-on laboratory experiences, didactic teaching, and formal classwork at Case Western Reserve University, FAES at the NIH, and Jackson Laboratories. Dr. Michael Jenkins, a pioneer in cardiac optical imaging, and Dr. Cynthia Bearer, an expert in prenatal alcohol exposure models, will provide their expertise and mentorship skills to this project, fostering Dr. Ford’s transition to research independence. An estimated 2.4-4.8% of newborns in the U.S. have fetal alcohol spectrum disorders (FASDs), caused by prenatal alcohol exposure (PAE). PAE induced Congenital Heart Diseases (CHDs) have not been studied as intensively as other FASD outcomes despite their high prevalence rate (40%). The CHDs associated with FASDs, mostly valvuloseptal and outflow tract defects, are life-threatening and impact growth and health. PAE is known to affect methylation and one-carbon metabolism. Normal one-carbon metabolism and its resulting methylation of DNA is crucial for the correct expression of genes. Comparative genomics studies have revealed that there is strong epigenetic conservation across vertebrate species including mice and avians, particularly the hyper-and hypo-methylated DNA sequences of critical genes. We will investigate the PAE-induced changes at times critical to heart development (endocardial cushion and 4 chamber development) in mouse and avian embryonic hearts. All hearts will be imaged with optical coherence tomography to rapidly determine their phenotype. DNA methylation changes will be determined with a combination of methyl-ATAC-seqand bisulfite sequencing. DNA methylation will be compared in both species, as conserved changes in two species are more likely to be relevant to human PAE-induced defects. RNA-FISH will be used to confirm gene expression changes, which will allow us to pinpoint where within the 3D heart, such as the forming valves, gene expression is changing. We will then explore the use of choline and glutathione to prevent the effects of PAE. Choline and glutathione are known to promote methylation in one-carbon metabolism. Choline has been shown in human studies to prevent early neurologic effects of PAE. We have shown in an avian model that glutathione prevents the CHDs and abnormal DNA methylation seen after PAE. We will use both avian and mouse models to determine the effects of alcohol + choline or glutathione on cardiac structure, DNA methylation, and gene expression. We hypothesize that by maintaining normal methylation, and therefore DNA expression, our chosen compounds will prevent the CHDs that result from PAE. Compounds that could prevent PAE-induced CHDs could help thousands of children and their families each year.