Na v 1.5, the pore-forming ␣ subunit of the cardiac voltagegated Na ؉ channel complex, is required for the initiation and propagation of the cardiac action potential. Mutations in Na v 1.5 cause cardiac arrhythmias and sudden death. The cardiac Na ؉ channel functions as a protein complex; however, its complete components remain to be fully elucidated. A yeast two-hybrid screen identified a new candidate Na v 1.5-interacting protein, ␣B-crystallin. GST pull-down, co-immunoprecipitation, and immunostaining analyses validated the interaction between Na v 1.5 and ␣B-crystallin. Whole-cell patch clamping showed that overexpression of ␣B-crystallin significantly increased peak sodium current (I Na ) density, and the underlying molecular mechanism is the increased cell surface expression level of Na v 1.5 via reduced internalization of cell surface Na v 1.5 and ubiquitination of Na v 1.5. Knock-out of ␣B-crystallin expression significantly decreased the cell surface expression level of Na v 1.5. Co-immunoprecipitation analysis showed that ␣B-crystallin interacted with Nedd4-2; however, a catalytically inactive Nedd4-2-C801S mutant impaired the interaction and abolished the up-regulation of I Na by ␣B-crystallin. Na v 1.5 mutation V1980A at the interaction site for Nedd4-2 eliminated the effect of ␣B-crystallin on reduction of Na v 1.5 ubiquitination and increases of I Na density. Two disease-causing mutations in ␣B-crystallin, R109H and R151X (nonsense mutation), eliminated the effect of ␣B-crystallin on I Na . This study identifies ␣B-crystallin as a new binding partner for Na v 1.5. ␣B-Crystallin interacts with Na v 1.5 and increases I Na by modulating the expression level and internalization of cell surface Na v 1.5 and ubiquitination of Na v 1.5, which requires the protein-protein interactions between ␣B-crystallin and Na v 1.5 and between ␣B-crystallin and functionally active Nedd4-2.Na v 1.5 is the pore-forming ␣ subunit of the major cardiac voltage-gated Na ϩ channel complex. It generates the sodium current (I Na ) 4 that plays an essential role in the initiation and propagation of the cardiac action potential (1-3). Mutations in the SCN5A gene (encoding Na v 1.5) cause several inherited arrhythmias, including atrial fibrillation, Brugada syndrome, long QT syndrome, progressive cardiac conduction defect disease, sick sinus syndrome, and dilated cardiomyopathy (4). Na v 1.5 exists in vivo in a multiprotein complex, which interacts with the actin cytoskeleton and the extracellular matrix to provide an important functional link between channel complexes, cardiac structure, and electrical functioning (5, 6). Several proteins have been reported to bind to Na v 1.5 (5-7). We have previously reported a small protein, MOG1, with a function in nucleocytoplasmic protein transport that interacts directly with Na v 1.5, promotes trafficking of Na v 1.5 to the cell surface, and increases peak I Na density (4, 6). Specifically, MOG1 facilitates export of Na v 1.5 from the endoplasmic reticulum as well as targeting of Na v 1.5...

Edited by Ronald C. Wek Coronary artery disease (CAD) is the leading cause of death worldwide. Long noncoding RNAs (lncRNAs) are a class of noncoding transcripts of > 200 nucleotides and are increasingly recognized as playing functional roles in physiology and disease. ANRIL is an lncRNA gene mapped to the chromosome 9p21 genetic locus for CAD identified by the first series of genomewide association studies (GWAS). However, ANRIL's role in CAD and the underlying molecular mechanism are unknown. Here, we show that the major ANRIL transcript in endothelial cells (ECs) is DQ485454 with a much higher expression level in ECs than in THP-1 monocytes. Of note, DQ485454 expression was down-regulated in CAD coronary arteries compared with non-CAD arteries. DQ485454 overexpression significantly reduced monocyte adhesion to ECs, transendothelial monocyte migration (TEM), and EC migration, which are critical cellular processes involved in CAD initiation, whereas siRNA-mediated ANRIL knockdown (KD) had the opposite effect. Microarray and follow-up quantitative RT-PCR analyses revealed that the ANRIL KD down-regulated expression of AHNAK2, CLIP1, CXCL11, ENC1, EZR, LYVE1, WASL, and TNFSF10 genes and up-regulated TMEM100 and TMEM106B genes. Mechanistic studies disclosed that overexpression of CLIP1, EZR, and LYVE1 reversed the effects of ANRIL KD on monocyte adhesion to ECs, TEM, and EC migration. These findings indicate that ANRIL regulates EC functions directly related to CAD, supporting the hypothesis that ANRIL is involved in CAD pathogenesis at the 9p21 genetic locus and identifying a molecular mechanism underlying lncRNA-mediated regulation of EC function and CAD development. Coronary artery disease (CAD) 4 is the most common cardiovascular disease and the leading cause of death worldwide (1). It occurs as a chronic inflammatory response to endothelial injuries in coronary arteries. Then the coronary arteries develop an atherosclerotic plaque inside the intima (referred to as atherosclerosis), leading to hardening and narrowing of the arteries and blocking of blood flow to the heart. Typical CAD complications include stable angina, unstable angina, myocardial infarction (MI), arrhythmias, heart failure, and sudden death (2). For the development of atherosclerosis and CAD, monocyte adhesion to the endothelium and transendothelial migration of monocytes (TEM) into the intima have been established as two of the most important cellular processes by multiple studies (1, 2). An increased serum concentration of oxidized low-density lipoproteins (ox-LDLs) or other inflammatory molecules such as TNF␣ recruits monocytes to the region where ROS-induced endothelial dysfunction occurs (1-3). This leads to monocyte adhesion to the endothelium, followed by transmigration of monocytes across the endothelium into the intima area (1-3). The monocytes are then transformed into lipid-engorged macrophages and foam cells, leading to the formation of plaques (1-3).

Edited by Mike ShipstonNa v 1.5 is the ␣-subunit of the cardiac sodium channel complex. Abnormal expression of Na v 1.5 on the cell surface because of mutations that disrupt Na v 1.5 trafficking causes Brugada syndrome (BrS), sick sinus syndrome (SSS), cardiac conduction disease, dilated cardiomyopathy, and sudden infant death syndrome. We and others previously reported that Ran-binding protein MOG1 (MOG1), a small protein that interacts with Na v 1.5, promotes Na v 1.5 intracellular trafficking to plasma membranes and that a substitution in MOG1, E83D, causes BrS. However, the molecular basis for the MOG1/Nav1.5 interaction and how the E83D substitution causes BrS remains unknown. Here, we assessed the effects of defined MOG1 deletions and alanine-scanning substitutions on MOG1's interaction with Na v 1.5. Large deletion analysis mapped the MOG1 domain required for the interaction with Na v 1.5 to the region spanning amino acids 146 -174, and a refined deletion analysis further narrowed this domain to amino acids 146 -155. Site-directed mutagenesis further revealed that Asp-148, Arg-150, and Ser-151 cluster in a peptide loop essential for binding to Na v 1.5. GST pulldown and electrophysiological analyses disclosed that the substitutions E83D, D148Q, R150Q, and S151Q disrupt MOG1's interaction with Na v 1.5 and significantly reduce its trafficking to the cell surface. Examination of MOG1's 3D structure revealed that Glu-83 and the loop containing Asp-148, Arg-150, and Ser-151 are spatially proximal, suggesting that these residues form a critical binding site for Na v 1.5. In conclusion, our findings identify the structural elements in MOG1 that are crucial for its interaction with Na v 1.5 and improve our understanding of how the E83D substitution causes BrS. 3 The abbreviations used are: BrS, Brugada syndrome; SSS, sick sinus syndrome; aa, amino acid; GST-Na v 1.5-LII, GST-Na v 1.5-loop II.