PROJECT SUMMARY The goal of this project is to gain a better understanding of mechanisms that underlie metabolic coupling between myocardial oxygen demand and oxygen supply via changes in coronary blood flow to the heart. Redox-sensitive voltage-gated potassium channels (i.e., Kv1.x) in coronary vascular smooth muscle (CVSM) are known to be essential to the enhancement of myocardial blood flow (MBF) in response to increased cardiac workload. How Kv1 channel activity is enhanced in CVSM in response to metabolic signals from an active myocardium is unknown. Here, we propose an essential role for metabolic regulation of Kv1 channel activity by the tetrameric assembly of cytosolic auxiliary Kvβ subunits, which are members of the aldo-keto reductase (AKR) superfamily and bind oxidized and reduced pyridine nucleotides (e.g., NAD(H)) with high affinity. Our preliminary data are consistent with the global hypothesis that interactions between NAD(H) and Kvβ1 and Kvβ2 impart precise control over the coupling between regional MBF with cardiac oxygen demand. In Aim 1, we will delineate the role of coronary Kvβ subunits in the regulation of the metabolic hyperemia response. To do this, we will use non-invasive myocardial contrast echocardiography (MCE) to measure MBF as a function of cardiac workload in anesthetized WT and Kvβ-null animals upon administration of norepinephrine (NE). The cell-specific contribution of Kvβ1 and Kvβ2 in CVSM to metabolic hyperemia will be examined in double transgenic animals with inducible smooth muscle-specific expression of Kvβ in null background animals. In vitro electrophysiology and myography will test the relative roles for Kvβ in altering the voltage-dependence of Kv1 activation and inactivation and metabolic vasodilation, respectively. In Aim 2, we will elucidate the mechanism of Kvβ-mediated metabolic coupling. We will measure, using electrophysiology, the relative functional contribution of Kvβ subunits to regulation of Kv1 activity upon specific manipulations in cellular metabolism that alter the redox ratio of NADH:NAD+ in CVSM. We will quantify changes in NADH/NAD+ redox in CVSM using genetically-encoded fluorescent biosensors and MALDI-MS imaging, and determine the role of NAD(H) turnover by Kvβ catalysis in regulation of coronary vasodilation and enhancement of MBF. In Aim 3, we will clarify the role of Kvβ in cardiovascular adaptation to exercise conditioning. To do this, we will subject WT and genetically-modified animals to a forced treadmill running exercise protocol before measuring adaptations in MBF, coronary vasodilatory capacity, and Kv activation and inactivation properties. This aim will also address the overall dependence of physiological myocardial adaptations and enhancement of exercise capacity on coronary Kvβ-dependent changes in MBF.