Project 1 Summary: Atrial fibrillation (AF) increases risk of heart failure, stroke and death, and its incidence increases with age and obesity. Human genome wide association studies (GWAS) have identified ~140 genetic loci associated with AF susceptibility, but it still takes dedicated functional and molecular studies to identify the causal gene, the causal genetic variant, and the mechanisms for AF association. We are very excited to pursue two AF risk loci on chromosomes 10q22 (SYNPO2L and MYOZ1 genes) and 14q23 (SYNE2 gene). We are poised to discover the mechanism by which these two loci increase AF susceptibility, and identify gene centric repurposable drugs, which may be targeted to individuals carrying these risk alleles. We chose both loci for study as the GWAS single nucleotide polymorphism (SNP) was associated with expression of a nearby gene; thus, the causal SNP, which can be in linkage disequilibrium with the GWAS SNP, may work by regulating gene expression. These associations with gene expression were for specific transcript isoforms of SYNPO2L and SYNE2 due to alternative transcription start sites. In Aim 1, we will study the complex AF locus on chromosome 10q22, where the AF risk allele is associated with decreased expression of MYOZ1, but increased expression of the shorter isoforms of SYNPO2L. Both of these genes encode Z disk proteins, which may directly affect contractility and secondarily alter Ca2+ handling that may impact cellular electrophysiology. We will study how changes in the expression of MYOZ1 and the SYPO2L isoforms alter contractility and electrophysiology in human stem cells differentiated into atrial-like cardiomyocytes (a-iCMs) and engineered heat tissue (EHT). In Aim 2, we will build on our preliminary human iCM studies, where we found that the chromosome 14q23 AF risk allele is associated with less expression of a SYNE2 short isoform. SYNE2 encodes a protein that connects the nucleus to the cytoplasm, but the short isoform does not bind to the cytoskeleton, and acts as a dominant negative to disrupt the nuclear-cytoplasm connection. RNAseq from SYNE2 short isoform overexpression in a-iCMs showed a large effect on Ca2+ handling proteins. We looked at Ca2+ handling and action potentials; knockdown of all SYNE2 isoforms led to increased early afterdepolarizations, which was rescued by over expression of the short isoform. Short isoform over expression also decreased peak Ca2+ content. We discovered that the SYNE2 short isoform also binds to the sarcoplasmic reticulum. In this aim we are poised to determine if over expression of the SYNE2 short isoform can protect against AF in a mouse model of spontaneous AF. In collaboration with SC3 and P3, Aim 3 will use the RNAseq data from Aims 1 and 2 to identify “gene effect modules”, for which we will identify module altering repurposalbe drugs, which will be tested for beneficial effects in a-iCMs and EHTs. Successful completion of our aims will make significant contrib...