Project 1 - Genes to Function: Causal Genes and their Roles in Cardiomyocyte and Atrial Physiology

项目1总结： 房颤(房颤)增加心力衰竭、中风和死亡的风险，其发病率随着年龄和年龄的增加而增加。 肥胖。人类基因组全关联研究(GWAS)已经确定了大约140个与 房颤易感性，但仍需要专门的功能和分子研究来确定致病基因，即 因果遗传变异，以及房颤关联的机制。我们很高兴能追踪到两个房颤风险基因 染色体10q22(SYNPO2L和MYOZ1基因)和14q23(SYNE2基因)。我们将会发现 这两个基因座增加房颤易感性和识别以基因为中心的可再利用的机制 药物，可能针对携带这些风险等位基因的个人。我们选择了这两个基因座作为研究对象 单核苷酸多态(SNP)与邻近基因的表达相关；因此， 因果SNP可能与GWASSNP处于连锁不平衡状态，可能通过调控基因发挥作用 表情。这些与基因表达的关联是SYNPO2L和SYNPO2L的特定转录异构体和 SYNE2是由于选择性转录起始点所致。在目标1中，我们将研究复杂的房颤基因座 染色体10q22，其中房颤风险等位基因与MYOZ1表达降低相关，但 SYNPO2L较短的异构体表达增加。这两个基因都编码Z盘蛋白，它 可能直接影响细胞的收缩能力，继而改变钙离子的处理，从而影响细胞电生理。 我们将研究MYOZ1和SYPO2L亚型表达的变化如何改变收缩性和 人干细胞分化为心房样心肌细胞的电生理学研究 热组织(EHT)。在目标2中，我们将建立在我们初步的人类ICM研究的基础上，在那里我们发现 染色体14q23AF风险等位基因与SYNE2短亚型表达减少有关。SYNE2 编码一种连接细胞核和细胞质的蛋白质，但短的异构体不与 细胞骨架，并作为显性负性干扰核质连接。RNASeq来自 α-ICM中SYNE2短异构体的过表达对钙离子调节蛋白有较大影响。我们看了一下 钙离子处理和动作电位；所有SYNE2亚型的敲除导致早期增加 后去极化，这是通过过度表达短的异构体而挽救的。短异构体过度表达 也降低了峰值钙含量。我们发现SYNE2短的异构体也与 肌浆网。在这一目标中，我们准备确定SYNE2短亚型是否过度表达 能在自发性房颤小鼠模型中预防房颤。在与SC3和P3的合作中，Aim 3将使用 来自AIMS 1和AIMS 2的RNAseq数据用来识别“基因效应模块”，对于这些模块，我们将识别模块改变 重新调整用途的药物，将在a-ICM和EHTS中进行有益效果测试。成功完成 我们的目标将为房颤功能基因组学和治疗学做出重大贡献。

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 contributions to AF functional genomics and therapeutics.