Abstract 16657: MicroRNA-184 is Epigenetically Silenced by CpG Methylation and Contributes to the Pathogenesis of Arrhythmogenic Cardiomyopathy Phenotype

Abstract

Introduction: A phenotypic characteristic of arrhythmogenic cardiomyopathy (AC) is a gradual replacement of cardiac myocytes by fibro-adipocytes, which leads to cardiac arrhythmias, dysfunction and sudden death. Hypothesis: Differentially expressed miRNAs contribute to the pathogenesis of AC Results: Control and plakophilin-2 (PKP2)-suppressed HL-1 myocytes (HL-1Pkp2-shRNA) were screened for the expression of 750 miRNA using Taqman low-density microfluidic panels. Thirty-one miRNAs were up- and 28 were down-regulated in the HL-1Pkp2-shRNA myocytes. MiR-184 was the most down-regulated miRNA (~10-fold). It was also down-regulated in the heart of mouse models of AC. MiR-184 was developmentally regulated in the mouse heart as its levels were the highest in cardiac myocytes islated from the newborn mice, the lowest in adult myocytes and intermediate in myocytes isolated from 3-week old mice. Ingenuity pathway analysis of paired miR-184 and mRNA sequencing data identified cell proliferation, differentiation and death as the major affected functions. Knock down of miR-184 by shRNA reduced cellular proliferation/viability, increased apoptosis, and enhanced adipogenesis. Levels of over a dozen regulators of lipid synthesis were increased along with fat droplets. Over-expression of miR-184 had reciprocal effects. Bisulfite sequencing identified differential hypermethylation of the CpG sites at the upstream region of miR-184. Treatment with 5-aza-2’-deoxycytidine, a demethylation agent, partially rescued suppressed miR-184 levels. However, activation or suppression of the Hippo and the canonical Wnt signaling pathways, implicated in the pathogenesis of AC, did not affect miR-184 levels. Likewise, miR-184 over-expression or suppression did not affect the Hippo and Wnt signaling. Conclusions: miR-184 is developmentally regulated with the highest levels of expression in the newborn myocytes. It levels are suppressed in the AC models, partially because of hypermethylation of the CpG sites at its genomic regions. MiR-184 by regulating cellular proliferation and differentiation contributes to the pathogenesis of AC.