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Single Cell RNA Sequencing Reveals a Shift in Cardiac Fibroblast Sub‐Populations in Hypertensive Rats After Transient ACE Inhibition in vivo

Cardioprotection afforded by angiotensin converting enzyme inhibitors (ACEI) may be related in part to fibroblast‐specific changes. We have demonstrated that beneficial effects of ACEI treatment in adult spontaneously hypertensive rats (SHR) persist even after cessation of treatment. Specifically, transient ACEI treatment protects SHR hearts from future injury‐induced cardiac inflammation, fibrosis, and dysfunction. Further, isolated cardiac fibroblasts (CF) from rats transiently treated with an ACEI prior to injury display decreased chemokine production, proliferation rate, and collagen gene expression; thereby linking fibroblast phenotype and ACEI‐induced cardioprotection in vivo. We thus hypothesize that transient ACEI treatment produces a persistent change in hypertensive CF populations characterized by an anti‐fibrotic and anti‐inflammatory transcriptome. In the present study we used single cell RNA‐Seq to determine whether transient ACEI treatment produced persistent changes in CF sub‐populations. Adult male SHRs were treated for 2 weeks with an ACEI, enalapril (30mg/kg/day, p.o.), or water followed by a 2‐week washout period (n=7/group). CFs were isolated from the left ventricle and cultured for 3 days and collected (P0) or passaged to P1. P0 CFs were subjected to single cell RNA‐Seq using Chromium Single Cell 3′ V3 kit (10X Genomics). Media was collected from P1 CFs to evaluate secreted levels of monocyte chemoattractant protein 1 (MCP1) and osteopontin (OPN; a marker of activated CF) by ELISA. Single cell RNA‐Seq analysis revealed 9 distinct clusters of CFs. Transient ACEI treatment produced significant changes within and across clusters. Notably, ACEI treatment significantly increases the percentage of cells in Cluster 0, which is characterized by significantly reduced expression levels of genes involved in CF activation, extracellular matrix (ECM) production, and inflammation. Furthermore, within a cluster of cells expressing genes most related to activated CF (Cluster 1), cells from ACEI‐treated rats have significantly reduced expression of ECM genes (Col1a1, Col3a1, Fn1) vs. control rats suggesting a reduced degree of activation. Lastly, the percentage of cells in Cluster 6 is significantly reduced by ACEI treatment. Projecting cells onto pseudotime analysis arranges Cluster 6 as a potential precursor to Cluster 1. Since treatment significantly reduces the number of CFs in Cluster 6, prior ACEI treatment impacts the proportion of CFs that may be directed toward Cluster 1 (activated CF). CFs from rats previously treated with an ACEI displayed significantly reduced secretion of MCP1 (24%) and OPN (53%) in culture media. In conclusion, transient ACEI treatment produces a significant shift in CF populations, characterized by a less fibrogenic and inflammatory CF phenotype. This may mediate our previously demonstrated reduced inflammatory and fibrotic response to an injurious stimulus. Future investigation into the mechanisms by which ACEI treatment produces this persistent change in CF physiology may reveal novel targets for anti‐fibrotic therapy.