Program Summary/Abstract Coronary micro- and macrovascular disease is the leading cause of diabetes-associated death. Despite many medications for diabetes, the number of deaths resulting from coronary vascular complications is still high among diabetic patients. Coronary vascular endothelial injury and dysfunction reduce coronary blood flow by inhibiting vasodilatation and decreasing capillary density in the heart. Sustained coronary flow reduction not only increases the risk to exaggerate cardiac damage after ischemia/reperfusion, but also leads to cardiac dysfunction. Our long-term goal is to define the mechanisms of diabetes-associated coronary endothelial dysfunction and ultimately develop novel therapies for coronary microvascular disease in diabetes. Posttranscriptional regulation of gene expression is a critical step in mRNA biogenesis. The RNA-binding protein HuR serves as a key regulator of mRNA stability and translation via binding to specific elements within mRNAs. The objective of this study is to examine if HuR contributes to the development of coronary endothelial dysfunction in diabetes by regulating hexokinase 2 (HK2), a modifier of mitochondrial [Ca2+], and connexin 40 (Cx40), a key component of gap junction (GJ) in endothelial cells (ECs). We will use a) inducible and genetically modified diabetic mice (type 1 and type 2 diabetic mice) for in vivo and ex vivo experiments, b) mouse coronary ECs (MCECs) freshly-isolated from control and diabetic mice and human coronary ECs (HCEC) from control subjects and diabetic patients for molecular biological and electrophysiological experiments, and c) small coronary arteries (CAs, 3rd or 4th order) for isometric tension experiments. Our preliminary data indicate that i) HuR deletion in ECs lowered coronary flow velocity reserve and cardiac contractility, increased EC apoptosis, and decreased capillary density in the heart, ii) protein levels of HuR, HK2 and Cx40 were decreased in diabetic HCECs and MCECs compared to the controls, iii) HuR binds to Cx40 and HK2 mRNAs and increases their protein expression, iv) overexpression of HuR, HK2 or Cx40 in diabetic ECs restored endothelial function to the level close to control ECs. Based on these data, we hypothesize that downregulation of HuR leads to coronary endothelial dysfunction and subsequently results in coronary microvascular disease in diabetes via downregulation of HK2 and Cx40 in ECs. Three Specific Aims are proposed to test the hypothesis: 1) To explore the role of HuR in the development of endothelial dysfunction in diabetic mice, 2) To characterize the HuR-mediated posttranscriptional modification of HK2 and to examine whether HK2 contributes to coronary endothelial dysfunction in diabetes via mitochondrial Ca2+ overload, and 3) To examine whether HuR regulates Cx40 expression levels and if overexpression of HuR restores GJ intercellular communication through increasing Cx40 levels, enhances vascular relaxation, and improves cardiac function in diabetes. Completion of this study will provide important insights into developing new and effective therapeutic interventions for coronary microvascular disease in diabetes.