Abstract 426: Effects of Non-syndromic Mitral Valve Prolapse on Valvular Interstitial Cell Extracellular Vesicle-associated Non-coding RNA in a Spontaneous Canine Disease Model

Abstract

Non-syndromic mitral valve prolapse (MVP), or Barlow’s Disease, is a valvular heart disease with a prevalence of 2-3% in the human adult population. Consequences of MVP include mitral regurgitation that without surgical treatment can progress to congestive heart failure, arrhythmias and sudden death. Since MVP is a degenerative disease that worsens with age, surgical risk is of concern with an older population, necessitating the development of alternative strategies for early detection and effective medical intervention. Similar to humans, canine MVP is age-related with a lifetime prevalence close to 90%, providing a large population for study. Canine MVP represents a relevant spontaneous large animal model to dissect the mechanistic underpinnings of disease pathogenesis and evaluate novel treatment. We hypothesize that extracellular vesicles (EVs) and their associated non-coding RNA (ncRNA) may play a role in this disease by enabling propagation of the fibroblast-to-myofibroblast phenotypic switch in mitral valvular interstitial cells (VICs). VICs were isolated from 12 normal and 14 diseased canine mitral valves and cultured in defined chemical media for 48 hours. EVs were isolated from conditioned media using size exclusion chromatography. Total RNA was isolated from EVs with the Qiagen miReasy Plasma/Serum kit, and from VIC cells with the mirVana miRNA Isolation Kit. Sequencing libraries were created using QIAseq miRNA Library Kit, and RNAseq was performed with Illumina HiSeq 2500 sequencer with single read 100 base format. Reads were mapped to canine ncRNA databases (miRbase, RFam). VICs from diseased valves had higher expression of αSMA than cells from normal valves (p = 0.002) based on RT-qPCR. Sequence analysis showed decreased content of tRNA (p = 0.023) and miRNA precursor (p = 0.036) in EVs from diseased VICs, in addition to decreased expression of let-7f. Cells from diseased valves had lower Y-RNA content (p = 0.007). miR-8859a and miR-451 were preferentially packaged into EVs from both normal and diseased VICs. Preferential packaging of miR-155 was seen in normal VIC EVs and miR-191 in diseased VIC EVs. Functional roles of these miRs will need to be confirmed to determine their therapeutic potential for treatment of MVP.