Project Abstract Telomerase a ribo-nucleoprotein that counteracts telomere shortening has recently been shown by our investigative team to have a non-canonical role in attenuating formation of mitochondrial reactive oxygen species (mtROS) in coronary arterioles from subjects with coronary artery disease (CAD). We demonstrated that activation of TERT can reverse the mechanism of flow-induced endothelium-dependent dilation from H2O2- to NO, restoring the phenotype to one observed in subjects without CAD. In this proposal, we aim to investigate the role of mitochondrial specific effects of telomerase activity and whether the dominant negative splice variant β del TERT is critical in this phenotypic change in dilator mechanism. Our central hypothesis is that mitochondrial DNA damage is one of the underlying causes that leads to increase in ROS production. mtROS is known to promote development of arteriolosclerosis and endothelial dysfunction predisposing individuals to vascular complications. NO has a well-known inhibitory effect on mtROS generation and has also been demonstrated to increase telomerase. Whether nuclear or mitochondrial telomerase activity contributes to cardiovascular protection is not defined. We developed novel inhibitors of nuclear (nucTERT) or mitochondrial (mitoTERTi) telomerase activity to differentiate the roles of nuclear and mitochondrial telomerase in mediating vascular protective phenotypes. We will identify the role of mitochondrial telomerase in this change of mechanism from health (NO mediation) to disease (H2O2 mediation) in mouse and human resistance vessels. We hypothesize that mitochondrial telomerase plays a protective role by preventing mtDNA damage in normal conditions, while expression of β del TERT in disease suppresses this protective effect and elevates vascular cellular oxidative stress, and induces the conversion from NO to H2O2 as the mediator of FMD. This will be tested by addressing two specific aims. First, we will determine whether mitochondrial localization of TERT is necessary and sufficient to maintain NO rather than mtH2O2 as the mediator of flow-induced dilation in the human microcirculation. Second, we will investigate whether the mechanism by which CAD elicits a switch from NO to H2O2 as the mediator of FMD and impairs mitochondrial function involves accumulation of β-del TERT. We will use existing pharmacological and genetic tools that will lead to strategies for restoration of microvascular function in disease. This novel hypothesis has important translational potential, identifying new therapeutic targets for moderating the pathological changes associated with microvascular disease.