MiR-182 regulates IL-6 in the cardiac context: implications for human atrial electrical remodelling

Background The progression of cardiac electrical remodelling and the onset of proarrhythmic events are multifactorial processes, with many factors contributing to the development of atrial fibrillation (AF). Recently, proinflammatory mediators have emerged as new key players: in particular, independent preclinical evidence has identified interleukin-6 (IL-6) and miR-182 as causative factors of arrhythmogenesis in animal models. MiR-182 regulates a wide range of pathways, including the expression of inflammatory mediators; however, in the cardiac context, the potential relationship between these two factors remains unknown. Methods Human induced pluripotent stem cells (hiPSCs) were differentiated into cardiomyocytes (CMs) to study the role of miR-182-overexpression (OE) on IL-6 expression/secretion by RT-PCR/Elisa assays. Functional consequences were assessed by measuring spontaneous electrical activity by using MULTIPLE-High-Throughput and Intracell systems, with/without autonomic stimulation. Hearts from the Tg(myl7:GAL4,EGFP) x Tg(Sce.4xUAS:miR-182,cry:EGFP) zebrafish line [ Tg(myl7 > miR-182) ] were used to measure the expression of dre-il6 . Human IL-6 protein (5.4pg/nL) was microinjected in the pericardial region of 2dpf Tg(myl7:EGFP) wt -like zebrafish embryos and heart rate was recorded. Expression analyses were performed on human left atrial samples of 49 patients (11 controls, CT; 18 left atrial dilation (LA-D); 20 chronic AF). Results MiR-182-OE in hiPS-CMs significantly incremented IL-6 expression and secretion, and was associated with a reduced and irregular spontaneous beating rate, as well as enhanced response to acetylcholine. Accordingly, MiR-182-OE downregulated the expression of HCN4, encoding for the pacemaker f-current , and dysregulated genes associated with atrial pathology. On the contrary, 24-hour incubation with IL-6 (50ng/mL) did not change miR-182-5p expression levels in CT hiPS-CMs. The IL-6 receptor antagonist tocilizumab (TOC, 10µg/mL) partially rescued HCN4 expression in miR-182-OE hiPS-CMs. Zebrafish heart samples from Tg(myl7 > miR-182) exhibited increased il6 expression levels. Pericardial injection of human IL-6 in wt zebrafish embryos decreased heart rate. Finally, miR-182-5p was found to be overexpressed in human biopsies from patients with LA-D, with the highest expression levels observed in patients with permanent AF; remarkably, miR-182 levels positively correlated with IL-6 expression. Conclusion The results support the hypothesis of a causative link between miR-182-OE and IL-6 production in the cardiac context. This molecular axis may represent a prognostic factor predisposing to arrhythmogenesis. Overall, our findings reveal novel pathophysiological mechanisms and suggest novel pharmacological targets within the complex AF setting.