Microarray analysis of miRNAs in atrial tissue from chronic AF patients

OBJECTIVES: This study aims to define a microRNA signature in chronic AF (CAF) and to unravel key target genes contributing to the atrial remodelling. METHODS: Total RNA from atrial biopsies of 2 sinus rhythm (SR) and 4 CAF patients was analyzed by microRNA(miR)-array. Expression of most deregulated miRs and their actions on specific targets were confirmed by qRT-PCR in human and ovine samples and validated in HL-1 cells and myocytes isolated from CAF patients. After bioinformatics prediction of targets and signaling, and through lentiviral miR-transduction or mimics-miR transfection of HL-1 cells, effects on main predicted regulatory pathways were analyzed. RESULTS: 38 microRNAs showed significant changes in expression in CAF atrial samples (>2-fold) and among them, miR-208a and miR-208b were confirmed to be significantly upregulated (>2 and >5-fold, respectively). Bioinformatics analysis predicted 70 pairs of CAF-altered mRNAs/miR-208a/b interactions. We confirmed that miR-208a and b directly targeted muscle cardiac gene program and canonical Wnt-signaling receptors and seemed to repress indirectly CX43. L-type Ca(2+) channel genes (CACNA1C and CACNB2) and sarcoplasmic reticulum-calcium pump SERCA2 were repressed by miR-208b at transcriptional, posttranscriptional and functional levels. CONCLUSION:In this work we report for the first time the role of miR-208b upregulation in the control of calcium balance in CAF. To determine the miRNA repertoires of human AF atrial tissue, we extracted total RNA from 4 AF patients, 2 SR patients and 2 control (no AF) patients, after disruption and homogeneization of small pieces of frozen atrial appendage biopses from AF and SR patients. Total RNA, including miRNAs, was purified by Qiagen miRNeasy Mini Kit - catalog number 217004, following manufacturer instructions.Isolated RNA was accurately quantified using Bioanalyzer