Abstract 251: miRNA Mapping of Cardiac Endothelial and Fibroblast Cells during Hypertrophy Progression

Abstract

MicroRNAs (MiRNA/miRs) are known key players in cardiovascular disorders. Here we created and analyzed a global miRNA map in isolated cardiac endothelial and fibroblast cells during hypertrophy progression. Hypertrophy was induced in male C57BL/6 mice by trans-aortic constriction (TAC). Hypertrophic phenotyping was performed at 3 days (3d), 2 weeks (2w) or 4 weeks (4w) post- SHAM/TAC operation, using Millar system and echocardiography. Thereafter, pure single cell fractions from hearts were recovered by retrograde collagenase-II perfusion, followed by pre-plating of fibroblasts and magnetic sorting of endothelial cells. Transcriptomic analysis was performed on RNA isolated from different heart cell fractions at 3d, 2w or 4w post-SHAM/TAC. Additional 6w and 13w SHAM/TAC groups were included for validation of selected miRNAs. Cytokine secretome was performed using multiplex assay after transfection of a miR library to primary cardiac fibroblasts. Unsupervised hierarchical clustering revealed specific miRNA profile of each cardiac cell fraction. Principle component analysis projected strong effect of individual cellular compartments. Based on miRnome screening, miR-709, miR-30e-5p, miR-146a, miR-34a and miR-204, miR-1187 were validated by RTPCR to confirm spatial and temporal regulation (n=3-5, p<0.05) of these miRNAs in non-cardiomyocyte fractions. Corresponding time-dependent downregulation of miR-146a targets, Pten and Timp-2, was also observed. Cytokine secretome analysis upon miR-precursor library transfection in cardiac fibroblasts confirmed an increase in FGF, LIF-1, MCP-1, MIP-1 secretion (1.5-5 folds) by miR-146a and miR-34a. Gradual activation of the TGF beta pathway in endothelial cells may initiate endothelial to mesenchymal transition, whereas cytokine secretion from fibroblasts may affect the cellular hemostasis. Our study represents a global miRNome of cardiac endothelial and fibroblast compartments during progressive hypertrophy to better understand the time dependent molecular changes in non-cardiomyocyte compartment during pressure-overload induced cardiac remodeling. The collective influence of miRNA deregulation may be linked to different pathways responsible for cell-proliferation, inflammation and fibrosis with the progression of hypertrophy.