PROJECT SUMMARY/ABSTRACTHypertension stimulates cardiac fibroblast (CF) expansion activation and excess extracellular matrix (ECM)production. Although there are no approved treatments for cardiac fibrosis angiotensin converting enzymeinhibition (ACEi) limits CF activation and ECM accumulation. Recent findings from the laboratory of the PIdemonstrate that resident CFs once considered functionally homogeneous consist of physiologically distinctpopulations that differentiate to diverse phenotypes in response to pressure overload. The premise for thisapplication is based on these findings in which hypertensive rats were transiently treated with an ACEi prior tosingle cell RNA sequencing on resident CFs. Pre-treatment with ACEi shifts CF subpopulations to generatehomeostatic CFs with a reduced capacity for fibrosis. This effect persists after treatment is stopped indicatingmemory is retained. The proposed studies will reveal the mechanisms by which CF subpopulations shift todetermine how to reprogram CFs to display a homeostatic less fibrogenic phenotype. Following ACEihomeostatic CFs comprise the largest subpopulation of resident CFs and are the least fibrogenic. Trajectoryanalysis revealed a gateway CF subpopulation that is the immediate precursor to activated CFs and thisgateway cluster was the most depleted by ACEi. Gateway CFs were defined by high expression of Spp1encoding for the protein osteopontin which induces several pro-fibrotic genes and represents a critical targetcandidate to maintain the activated CF pool. ACEi altered expression of epigenetic genes indicating changesin chromatin structure may drive the persistent shift from gateway to homeostatic CF subpopulations. Thesecompelling preliminary results led to the central hypothesis: transient reduction in angiotensin II signaling altersCF memory to protect against left ventricle (LV) fibrosis by fibroblast subpopulation-specific reprogramming ofchromatin structure to shift an osteopontin-producing gateway subpopulation toward a homeostaticsubpopulation with low fibrogenic capacity. To test the hypothesis the following specific aims are proposed:Aim 1) elucidate the degree to which reduction in angiotensin II signaling mediates the persistent shift inresident CF physiology that protects from future fibrosis; Aim 2) determine the impact of chromatin structuralmodification on shifting the gateway cluster toward the homeostatic cluster; and Aim 3) ascertain the degree towhich reduction in osteopontin mediates the shift to a less fibrogenic phenotype. In this application theresearch team uses a multidisciplinary approach employing in vivo and in vitro methodologies to test thehypothesis. Successful completion of these experiments will determine whether reduction in angiotensin IIsignaling mediates the expansion of a subset of homeostatic CFs that renders the LV resistant to fibrosis. It isexpected that the key drivers regulating the shift from a gateway to a homeostatic subpopulation of CFs will beidentified. Impact: These anticipated findings will have a positive impact in developing CF-targeted therapies forthe treatment and prevention of fibrotic remodeling that underlies heart disease.