PROJECT SUMMARY Cardiac fibrosis is a common adverse myocardial remodeling event that worsens heart failure caused by chronic ventricular pressure overload and other cardiac pathologies such as heart failure with preserved ejection fraction. Due to our incomplete understanding of the pathogenesis of cardiac fibrosis, no specific antifibrotic therapy exists, and affected patients are usually treated by heart transplantation, a therapy that has many limitations. Motivated by this clinical problem, our research has recently uncovered new insights into the regulation of cardiac collagen content and fibrosis, and, in this application, we propose to extend this work to gain further understanding into cellular and molecular mechanisms. In our recent work, we discovered that global deficiency of SLIT3, a secreted glycoprotein which binds to the ROBO receptors, leads to a marked decrease in cardiac collagen content, improved diastolic function in adult mice, and decreased cardiac fibrosis and improved survival after left ventricle (LV) pressure overload induced by transverse aortic constriction (TAC). We also found that SLIT3 stimulates cardiac fibroblast function, intracellular signaling, and collagen production in vitro. Collectively, these results support a developmental link between SLIT3 and collagen deposition during both normal cardiac homeostasis and pathologic stress. However, as these studies relied on SLIT3 global knockout mice where SLIT3 knockout potentially affected developmental programs, the more specific, postnatal functions of SLIT3 - as well as the details of the cellular and molecular signaling - remain undefined in the adult state. New preliminary data using SLIT3 floxed mice and inducible Cre promoters, we find that SLIT3, generated by either cardiac pericytes or fibroblasts, regulates collagen expression in vitro and in vivo in the postnatal state, most likely, by signaling through ROBO1. Based on these results, our overarching hypothesis is that cardiac pericyte and fibroblast-mediated SLIT3 regulate fibroblast activation and function via ROBO1 during conditions of homeostasis and pressure overload. As such, we propose the following aims: (1) Characterize the role of SLIT3 in regulating adult cardiac fibroblast function in vivo. (2) Determine the details of how SLIT3/ROBO1 regulates adult cardiac fibroblast activation and function in vivo and in vitro. (3) Characterize the role of cardiac pericyte and fibroblast-mediated SLIT3 on the ROBO1-dependent response of cardiac fibroblasts during LV pressure overload in vivo. This research will reveal new cellular and molecular concepts of cardiac fibroblast regulation and will further advance our understanding of the pathogenesis of cardiac fibrosis. The results of this research may provide the rationale for targeting SLIT3-ROBO1 to mitigate pressure overload-induced cardiac fibrosis, thereby benefiting patients with heart failure and other forms of cardiac disease.