BS-515-03 ATRIAL FIBRILLATION ASSOCIATED COMMON INTRONIC RISK VARIANTS IN SYNE2 LEAD TO LOWER EXPRESSION OF NESPRIN-2A1, INCREASED NUCLEAR STIFFNESS AND EARLY AFTER DEPOLARIZATIONS IN CARDIOMYOCYTES

Abstract

Atrial fibrillation (AF) genome-wide association studies (GWAS) identified significant associations with rs1152591 and linked variant rs1152595 in the SYNE2 gene, encoding the nesprin-2 protein that connects the nuclear membrane with the cytoskeleton. To determine the effects of the AF-associated SNPs on SYNE2 expression and investigate the mechanisms for their association with AF. RNA sequencing was performed on 234 AF human left atrial appendage (LAA) tissues. Human induced pluripotent stem cell-derived cardiomyocytes (iCMs) were used to study the cellular effects of SYNE2 knockdown (KD) or GFP-SYNE2α1 overexpression. Reporter gene vectors was used to study the regulatory roles of the SNPs. Nuclear size and stiffness were measured by immunofluorescent microscopy and atomic force microscopy. Calcium transient assay was performed using epifluorescence microscopy and Fura-2 AM. RNA sequencing of LAA tissues indicated that rs1152591 and rs1152595 were significantly associated with the expression of short SYNE2α1 isoform, without affecting the expression of the full-length SYNE2 mRNA. Risk vs. reference alleles of rs1152591 and rs1152595 had decreased promoter or enhancer activity. SYNE2 siRNA KD or nesprin-2α1 overexpression in human iCMs resulted in ∼12.5% larger nuclear area compared to controls (p<0.001). SYNE2 KD or nesprin-2α1 overexpression led to 57.5% or 33.2% decreases, respectively, in nuclear stiffness compared to controls (p< 0.0001). Nesprin-2α1 overexpression rescued the effects of SYNE2 siRNA KD on calcium transient, significantly decreasing the intracellular calcium concentration (p<0.001) and the incidence of early afterdepolarizations (EADs) (p<0.001). AF-associated SNPs rs1152591 and rs1152595 downregulate the expression of SYNE2α1 in the human LAA. SYNE2α1 has a dominant-negative effect on the nucleus, decreasing nuclear-cytoskeletal connectivity and nuclear stiffness that may protect iCMs from repetitive motion stress, and also has a gain of function activity on the calcium cycling, decreasing EADs that may protect against atrial arrhythmia.