Abstract 402: Exclusion of Major Cell Types in the Murine Heart Enriches for Cardiac Pericytes

Abstract

Cardiac fibrosis is one of the multitude of cardiac pathologies that occur in the event of acute myocardial injury and heart failure. Formation of scar tissue in the myocardium results in disrupted cardiomyocyte gap junction networks and reduced cardiac contractility. Cardiac pericytes are a heterogeneous population of mural cells that have been attributed to having a pro-fibrotic role in injury. However, due to the lack of specific markers for this cell, there has been difficulty in properly characterizing this population. We aimed to conduct single cell RNA-sequencing on this population to discover novel markers for cardiac pericytes and identify pro-fibrotic subpopulations that promote perivascular fibrosis. To avoid using current markers for cardiac pericytes, which lack specificity, we utilized an exclusion-based approach to enrich for the cardiac pericyte population in an unbiased manner. Using a combination of transgenic mice (αMHC GFP/+ ;Col1a1 GFP/+ ) and fluorescence-activated cell sorting (FACS), we excluded all major cardiac cell types, including cardiomyocytes, fibroblasts, endothelial cells, hematopoietic cells, smooth muscle cells, and cardiac neurons. We used Drop-sequencing technology to capture single cells and conducted RNA-sequencing. Cardiomyocytes (isolated by Langendorff), fibroblasts (FACS sorted Col1a1 + cells), endothelial cells (FACS sorted CD31 + cells), and smooth muscle cells (isolated from murine aortas) were also subjected to single-cell RNA-sequencing for comparison of gene expression profiles. Cells collected by the exclusion-based approach exhibited high expression of known pericyte markers and low expression of markers for other cell types. The t-SNE plots of all the populations revealed that the majority of cardiac pericytes shared similar gene expression profiles with smooth muscle cells while some cells clustered closer with fibroblasts. The use of an unbiased exclusion-based approach, coupled with single cell RNA-sequencing, allows for the identification of unique subpopulations of cardiac pericytes. Further investigation as to the functional roles of these cells will uncover potential therapeutic targets for treating cardiac fibrosis.