The incidence of diabetes is rising in the population; maternal diabetes affects 7% of pregnancies resulting in a 3-5 fold increased risk for fetal cardiac and valve malformations. As a result of elevated maternal blood glucose levels, facilitated maternofetal diffusion through the placenta causes elevated fetal glucose levels leading to chronic hyperglycemic or pulsed hyperglycemic embryonic blood glucose levels. Hyperglycemia modulates the ability of endocardial cells (ECs) to respond to changes in hemodynamic conditions which is critical during development. The objective of this study is to determine the role of hyperglycemia on early embryonic heart development and its effect on heart valve formation. We hypothesize that hyperglycemia in the embryo will result in increased oxidative stress and protein nitration leading to EC dysfunction and alterations of the TGF- and Wnt signaling pathways, which could ultimately lead to cardiac malformations including valve malformation and outflow tract defects. The effect of altered glucose will be established by: determining the degree to which chronic hyperglycemia or pulsed hyperglycemia will alter (1) ECs in the outflow tract of the embryonic heart and (2) the ability of embryonic ECs to transform into mesenchymal cells, a critical step in heart development. Hyperglycemia induced in vivo gene expression alterations will be determined in the outflow tracts of embryonic chick hearts using qPCR, immunohistochemistry, and immunofluorescence (IF). ECs isolated from these outflow tracts will be used to study the in vitro response of the embryonic ECs to hyperglycemia using qPCR, and IF. Protein nitration will be studied by measuring the amount of nitrotryosine in the chick hearts, and the resulting changes in signaling pathways will be determined by measuring gene expression changes in WNT3, BMP10, -Catenin, and E-Cadherin. Preliminary data indicates that pulsed and chronic increases in blood glucose levels lead to developmental delays, decreased cell proliferation and increased mortality. Optical coherence tomography measurements demonstrated a change in the structure of the outflow tracts in response to hyperglycemia, while microarray results indicated a change in the critical developmental cell signaling pathways of TGF- and Wnt. A team of experts in heart development, endothelial cells functions, chick embryos, clinical diabetes, and protein nitration has been assembled to train the applicant. The results of the proposed study will determine the mechanisms by which hyperglycemia in early heart development alters EC activation and will guide future research to prevent the fetal cardiac and valve malformations which result from maternal diabetes. PUBLIC HEALTH RELEVANCE: The worldwide incidence of diabetes is rising worldwide, affecting 7% of pregnancies. Fluctuations in glucose affects cardiac development, specifically maternal diabetes is associated with a 3-5 fold increased risk for cardiac malformations. Research needs to be conducted on the effect of glucose on the development of cardiac malformations.