Atrial fibrillation associated common risk variants in SYNE2 lead to lower expression of nesprin-2α1 and increased nuclear stiffness

Rationale: Atrial fibrillation (AF) genome-wide association studies (GWAS) identified significant associations for rs1152591 and linked variants in the SYNE2 gene encoding the nesprin-2 protein that connects the nuclear membrane with the cytoskeleton. Objective: Determine the effects of the AF-associated rs1152591 and rs1152595, two linked intronic single nucleotide polymorphisms (SNPs), on SYNE2 expression and investigate the mechanisms for their association with AF. Methods and Results: RNA sequencing of human left atrial appendage (LAA) tissues indicated that rs1152591 and rs1152595 were significantly associated with the expressions of SYNE2α1, a short mRNA isoform, without an effect on the expression of the full-length SYNE2 mRNA. SYNE2α1 mRNA uses an alternative transcription start site and encodes an N-terminal deleted 62 kDa nesprin-2α1 isoform, which can act as a dominant-negative on nuclear-cytoskeleton connectivity. Western blot and qPCR assays confirmed that AF risk alleles of both SNPs were associated with lower expression of nesprin-2 in human LAA tissues. Reporter gene transfections demonstrated that the risk vs. reference alleles of rs1152591 and rs1152595 had decreased enhancer activity. SYNE2 siRNA knockdown (KD) or nesprin-2 overexpression studies in human stem cell-derived induced cardiomyocytes (iCMs) resulted in ~12.5 % increases in the nuclear area compared to controls (p<0.001). Atomic force microscopy demonstrated that SYNE2 KD or nesprin-2α1 overexpression led to 57.5% or 33.2% decreases, respectively, in nuclear stiffness compared to controls (p< 0.0001). Conclusions: AF-associated SNPs rs1152591 and rs1152595 downregulate the expression of SYNE2α1, increasing nuclear-cytoskeletal connectivity and nuclear stiffness. The resulting increase in mechanical stress may play a role in the development of AF.