Summary: Microvascular rarefaction with reduced coronary flow reserve (CFR) has been shown to contribute to diastolic dysfunction. Although a significant coronary microvascular rarefaction occurs in type II diabetes/obesity, surprisingly, little is known about capillary rarefaction in diastolic dysfunction. In the last grant cycle, we have showed that obesity/diabetes disrupts angiopoietins/Tie-2 system in favor Ang-2, thus leading to microvascular phenotypic alterations and capillary rarefaction with a reduction of CFR. The microvascular phenotypic alterations/rarefaction and reduced CFR may render cardiomyocyte vulnerable to hypoxia, thus leading to a diastolic dysfunction in diabetes/obesity. However, the molecular mechanisms that lead to coronary microvascular phenotypic changes and capillary rarefaction in diabetes/obesity is not clearly defined. This renewal proposal will specific investigate Sirtuin 3 (Sirt3), a metabolic sensor, on EC glycolytic metabolism and capillary rarefaction. Aim 1: To define the mechanism(s) by which ablation of endothelial Sirt3 leads to coronary microvascular dysfunction with a focus on impairment of endothelial glycolytic metabolism. First, we will determine whether improvement of glycolysis increases angiogenesis and mitochondrial function, and reduces ROS formation by overexpression of HIF-2? in Sirt3 deficient EC in vitro. Further, we will determine whether endothelial specific overexpression of HIF-2? upregulates PFKFB3 expression, increases capillary density, restores CFR, and improves diastolic function in Sirt3ECKO mice and Sirt3ECKO- DIO mice. Aim 2: To test the hypothesis that disruption of endothelial Sirt3-PFKFB3 signaling pathway promotes microvascular rarefaction in diabetes. In vitro, we will determine whether overexpression of PFKFB3 rescues impaired glycolysis, thus leading to increase in the expression of angiogenic growth factors, reducing ROS formation and inflammation in Sirt3KO-EC. We also determine whether specific overexpression of PFKFB3 in EC reduces ROS formation, attenuates EC inflammation and prevents capillary rarefaction, and restores CFR and diastolic function in Sirt3ECKO-DIO mice and diabetic db/db mice. Aim 3: To test the hypothesis that ablation of endothelial Sirt3 promotes capillary rarefaction by a mechanism involving upregulation of Ang-2 and loss of pericytes in diabetes. We will determine whether blockade of Ang-2 rescues impaired EC/pericyte coverage and attenuates coronary microvascular rarefaction in Sirt3ECKO and Sirt3ECKO-DIO mice. Using Notch3KO/db/db mouse as a model of loss of pericytes, we will further determine whether inhibition of Ang-2 attenuates loss of cardiac pericytes and coronary microvascular rarefaction, improves CFR and diastolic function in diabetes.