PROJECT SUMMARY/ABSTRACT Nitric oxide (NO) regulates blood pressure by binding reduced heme iron (Fe2+) in soluble guanylyl cyclase (sGC) activating production of cyclic guanosine 3', 5'-monophosphate (cGMP) and relaxing vascular smooth muscle cells (SMC). In cardiovascular diseases, increased oxidative stress can cause oxidation (Fe3+) or loss (apo-) of the sGC heme impairing NO binding and causing vasoconstriction. A new class of therapeutic compounds, termed sGC activators have been developed to target the oxidized heme sGC and, more robustly, heme-deficient apo-sGC to restore cGMP production and induce vasodilation independent of NO or disease. However, these sGC activators have failed Phase 2 clinical trials due in part to patients developing sustained, systemic hypotension. Surprisingly, little is known about the impact of sGC activator use under physiological conditions, representing a major gap in our knowledge of sGC function. Our preliminary vascular function studies have uncovered that resistance arteries were log-orders more sensitive to sGC activator than conduit arteries in non- stressed conditions. This suggests, for the first time, that resistance arteries may contain a previously unrecognized physiological pool of oxidized heme and/or apo-sGC that can be activated independent of NO or disease. We have previously shown in cultured SMC that cytochrome b5 reductase 3 (CYB5R3) regulates sGC redox state by reducing oxidized heme sGC back to its NO-sensitive reduced state (Fe3+->Fe2+). Our preliminary studies in transgenic CYB5R3 overexpression (CYB5R3 OE) mice show that resistance, but not conduit, arteries had a diminished response to sGC activator induced vasodilation and blood pressure lowering. We also found pre-treatment of resistance arteries with reactive oxygen species scavengers or anti-oxidant compounds reduced resistance vessel vasodilation to sGC activator. Thus we hypothesize in physiological conditions, resistance arteries have a pool of oxidized heme or apo-sGC and CYB5R3 and endogenous O2- and H2O2 can modulate sensitivity to sGC activator treatment potentially through governing sGC redox state. This grant will focus on non-stressed resistance arteries and 1) explore basal sGC redox state and if it is modulated by CYB5R3, and 2) determine if CYB5R3, O2-, and/or H2O2 confer their sensitivity to sGC activator therapy. For Aim 1, we will assess vasodilation and cGMP production in CYB5R3 OE, SMC-specific CYB5R3 knockout, and littermate control mice in response to sodium nitroprusside (reduced heme sGC), sGC activator BAY 58-2667 (oxidized heme and apo- sGC), and protoporphyrin IX (apo-sGC). In Aim 2, we will use a combination of pharmacological compounds and mouse models to modulate levels of CYB5R3, and non-mitochondrial and mitochondrial sources of O2- and H2O2 to assess if this can impact resistance artery sensitivity to sGC activator induced vasodilation. Completion of these aims will provide greater insight into physi...