The cGMP-dependent protein kinase 1 alpha (PKG1α) opposes pathological left ventricular (LV) hypertrophy and remodeling via roles in the cardiac myocyte (CM) and regulates blood pressure by promoting vascular smooth muscle cell (VSMC) relaxation and vasodilation. Drugs which activate PKG1, including nitrates, sacubitril/valsartan, vericiguat, and others have improved mortality in heart failure with reduced LV ejection fraction (HFrEF) and thus represent a central advance in HFrEF treatment. However, these therapies remain limited by incomplete efficacy in HFrEF. Furthermore, hypotension from PKG1-induced vasodilation has severely limited the practical use of these agents. The overarching hypothesis of this program is that identifying downstream PKG1α anti-remodeling substrates in the LV can reveal novel therapeutic candidates to overcome these critical limitations of current PKG1-activating drugs. We have identified Mixed Lineage Kinase 3 (MLK3) as a novel PKG1α-interacting protein and anti-remodeling molecule. We propose to explore the following exciting findings which identify MLK3 as a translationally relevant molecule in HFrEF. 1) PKG1α-MLK3 interaction declines in the failing LV, and MLK3 is required for PKG1α-mediated therapeutic effects of sildenafil on LV function after pressure overload, thus identifying disruption of myocardial MLK3 regulation by PKG as promoting LV remodeling and decreasing the efficacy of PKG1-activating drugs in HF. 2) MLK3 kinase function opposes pathological CM and LV dysfunction and remodeling but does not affect blood pressure in vivo. 3) MLK3 deletion promotes hypertension in vivo, but MLK3 regulation of blood pressure occurs through MLK3 kinase independent mechanisms and independently of signaling by PKG1α. We propose to test a two-part novel model in which 1) PKG1α activation of MLK3 promotes LV compensation to pressure overload through MLK3 kinase-dependent mechanisms in the CM; and 2) MLK3 opposes hypertension through kinase-independent effects on vascular stiffness through a role in the VSMC. SA1 will use novel MLK3 cell-specific deletion models developed in our lab to determine the CM and SMC-specific roles of MLK3 in basal regulation of LV function and blood pressure and in the chronic LV response to pressure overload or myocardial infarction. SA2 will determine the kinase dependent versus kinase independent effects of MLK3 on LV function and blood pressure. SA3 will determine the translational relevance of MLK3 to HF treatment by testing the requirement of MLK3 for LV therapeutic effects versus blood pressure effects of currently available PKG1-activating drugs. These studies will define novel mechanisms through which MLK3 regulates blood pressure and through which MLK3 blood pressure- independent functions mediate the therapeutic effect of current PKG1-activating drugs. These studies have the potential to identify MLK3 kinase activation as a novel therapeutic strategy to promote PKG1 therapeutic effects...