Abstract 15359: Multi-Omics Integration Reveals Mechanosensitive Regulatory Pathways in Cardiac Fibroblasts

Abstract

Introduction: Regionalized myocardial fibrosis exists in the inferobasal myocardium of patients undergoing surgical repair for Mitral Valve Prolapse, and supports a process in which cells within mechanically stressed myocardial regions alter their phenotype in order to increase tissue stiffness, eventually causing establishment of a fibrotic scar. How cells behave in different fibromechanical environments is not currently well understood. Hypothesis: Integrating RNA Seq and ATAC Seq datasets will identify mechanosensitive regulatory pathways in cardiac fibroblasts. Methods: Primary human cardiac fibroblasts (HCFs) were cultured on stiff (TC Plastic, ~2GPa), as well as soft (2kPa) substrates coated with type 1 collagen. Following 48 hours in serum-free conditions, HCFs were lysed and subjected to immunofluorescence (IF), Western blotting (WB), RNA Seq, and ATAC Seq. Differential analysis of RNA Seq, and ATAC Seq data was performed, followed by an integration analysis which linked differentially expressed genes to chromatin that opened/closed within 10 kB. Finally, motif enrichment analysis was performed using the online bioinformatics package Homer. Results: IF and WB revealed that HCFs cultured on a stiff substrate expressed markers of fibroblast activation (alpha SMA, P<0.0001), as well as increased amounts of Connexin 43 (P<0.0001). Additionally, there were higher numbers of Ki-67 positive cells (7.2% vs 2.1% P=0.001). RNA Seq revealed differential changes in 4324 genes (FDR adjusted P<0.05), several of which were subsequently validated using western blots. ATAC Seq identified 5855 chromatin regions which opened/closed in response to different mechanical environments in fibroblasts (adjusted P<0.05). Integrating RNA Seq and ATAC Seq datasets revealed 803 Chromatin changes that fell within 10kb of differentially expressed genes, with a predicted enrichment of TEAD binding sites in differentially opened chromatin (P=1.0*10-40). Conclusions: The mechanical environment plays a major role in cellular phenotype and directly regulates chromatin organization and expression of pro-fibrotic genes, with a possible role for TEAD as a driver of fibroblast activation.