Targeting Resident Cardiac Fibroblast Subpopulations for Protection Against Fibrosis

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PROJECT SUMMARY/ABSTRACT Hypertension stimulates cardiac fibroblast (CF) expansion, activation, and excess extracellular matrix (ECM) production. Although there are no approved treatments for cardiac fibrosis, angiotensin converting enzyme inhibition (ACEi) limits CF activation and ECM accumulation. Recent findings from the laboratory of the PI demonstrate that resident CFs, once considered functionally homogeneous, consist of physiologically distinct populations that differentiate to diverse phenotypes in response to pressure overload. The premise for this application is based on these findings in which hypertensive rats were transiently treated with an ACEi prior to single cell RNA sequencing on resident CFs. Pre-treatment with ACEi shifts CF subpopulations to generate homeostatic CFs with a reduced capacity for fibrosis. This effect persists after treatment is stopped, indicating memory is retained. The proposed studies will reveal the mechanisms by which CF subpopulations shift to determine how to reprogram CFs to display a homeostatic, less fibrogenic phenotype. Following ACEi, homeostatic CFs comprise the largest subpopulation of resident CFs and are the least fibrogenic. Trajectory analysis revealed a gateway CF subpopulation that is the immediate precursor to activated CFs, and this gateway cluster was the most depleted by ACEi. Gateway CFs were defined by high expression of Spp1, encoding for the protein osteopontin, which induces several pro-fibrotic genes and represents a critical target candidate to maintain the activated CF pool. ACEi altered expression of epigenetic genes, indicating changes in chromatin structure may drive the persistent shift from gateway to homeostatic CF subpopulations. These compelling preliminary results led to the central hypothesis: transient reduction in angiotensin II signaling alters CF memory to protect against left ventricle (LV) fibrosis by fibroblast subpopulation-specific reprogramming of chromatin structure to shift an osteopontin-producing gateway subpopulation toward a homeostatic subpopulation 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 in resident CF physiology that protects from future fibrosis; Aim 2) determine the impact of chromatin structural modification on shifting the gateway cluster toward the homeostatic cluster; and Aim 3) ascertain the degree to which reduction in osteopontin mediates the shift to a less fibrogenic phenotype. In this application, the research team uses a multidisciplinary approach employing in vivo and in vitro methodologies to test the hypothesis. Successful completion of these experiments will determine whether reduction in angiotensin II signaling mediates the expansion of a subset of homeostatic CFs that renders the LV resistant to fibrosis. It is expected that the key drivers regulating the shift from a gateway to a homeostatic subpopulation of CFs will be identified. Impact: These anticipated findings will have a positive impact in developing CF-targeted therapies for the treatment and prevention of fibrotic remodeling that underlies heart disease.