# 95 High resolution multidimensional proteomics detects candidate arrhythmia biomarkers {#article-title-2} Purpose: Although biomarkers can predict prognosis, studies are limited to small panels, chosen a priori. Mass spectroscopy proteomic techniques powerfully and simultaneously demonstrate differential candidate proteins in an unbiased fashion but must overcome issues of resolving power caused by high abundant proteins. This study utilised a novel whole proteome technique to identify candidate biomarkers associated with arrhythmia. Methods: Consecutive device clinic patients were recruited. Baseline sera were later pooled according to death or arrhythmia occurrence. Samples underwent stable isotope labelling, fractionation and immunodepletion, followed by tandem mass spectroscopy. A separate validation set was tested using ELISA. Results: 243 patients (54% male, age 71±6) were included in the analysis. During follow up of 40 months, there were 10 cardiovascular deaths. 29 experienced VT>182bpm or VF, whilst 48 never experienced VT/VF (controls). Of 634 proteins identified, 94 were differentially expressed in those who died or experienced VT>182 or VF compared to controls. 9 proteins were selected for ELISA validation (table 1). Tropomyosin and VDAC1 were differentially expressed in both discovery and validation sets. Conclusion: This study provides proof of principle that proteomic techniques can identify candidate proteins for use as biomarkers of arrhythmia risk. Further investigation is needed to select proteins with potential for clinical application before testing in a prospective setting.[⇓][1] # 96 Regulation of the small conductance calcium-activated potassium channel 3 by microRNA-499 - potential role in atrial fibrillation {#article-title-3} Purpose: MicroRNAs have emerged as important regulators of gene expression, including those involving electrical remodeling in atrial fibrillation (AF). Recently, KCNN3, the gene that encodes the small conductance calcium-activated potassium channel 3 (SK3), was found to be strongly associated with AF. This study sought to evaluate the changes in atrial myocardial microRNAs in patients with permanent AF and to determine the role of microRNA on the regulation of cardiac SK3 expression. Methods: Atrial tissue obtained during cardiac surgery from patients (4 sinus rhythm and 4 permanent AF) was analyzed by microRNA arrays. Potential targets of microRNAs were predicted by software programs. The effects of specific microRNAs on target gene expression were evaluated in HL-1 cells from a continuously proliferating mouse hyperplastic atrial cardiomyocyte cell line. Interactions between microRNAs and targets were further evaluated by luciferase reporter assay using a vector expressing luciferase with the 3' UTR of SK3 mRNA subcloned into it. Expression of luciferase activity in cells transfected with the vector in the presence of specific microRNAs and microRNA negative controls were determined. The interaction between SK3 mRNA and specific microRNAs were also evaluated by Argonaute pull-down assay. Results: Twenty one microRNAs showed significant, greater than two-fold changes in AF. miR-499 was upregulated by 2.33 fold (p<0.01) in AF atria, whereas SK3 protein expression was down-regulated by 46% (n=3, p<0.05). Transfection of miR-499 mimic in HL-1 cells resulted in the downregulation of SK3 protein expression (reduction by 52%, n=3, p<0.05), while that of miR-499 inhibitor upregulated SK3 expression (54% increase, n=3, p<0.05). Binding of miR-499 to the 3' UTR of SK3 was confirmed by luciferase reporter assay. Exposure of miR-499 mimic to HL-1 cells transfected with luciferase reporter vector containing the SK3 3' UTR reduced luciferase activity by 53% (n=4, p<0.001) compared to cells exposed to microRNA negative control. Immunoprecipitates pull-downed by antibodies against Argonaute 1 and Argonaute 2 showed enhanced presence of SK3 mRNA in the microRNA-induced silencing complexes (RISC) after transfection with miR-499 mimic. Conclusion: Atrial microRNA expression profiles were different in patients with permanent AF compared to those with sinus rhythm. miRNA-499 binds to the 3' UTR of SK3 mRNA and effectively enriches SK3 mRNA in RISCs. Atrial miRNA-499 is significantly upregulated in AF, leading to SK3 downregulation and possibly contributing to the electrical remodeling in AF. [1]: #T1