Molecular determinants of the cardiac pacemaker automaticity

描述（由申请人提供）：窦房结（SA结或SAN）是一个精细调谐的结构，它启动和设置心跳的节奏。最近对胚胎发育的研究发现，T-box （Tbx）转录因子是窦房结发育的关键决定因素。特别是Tbx18，已被证明在开发过程中对于SA节点的规范是不可或缺的。然而，对于指定窦房结形态发生的tbx驱动的基因调控途径，以及这些途径如何导致起搏器细胞的自动性，我们知之甚少。我们试图验证Tbx18的重新表达足以将出生后心肌细胞重编程为起搏器细胞的一般假设。我们建议揭示tbx18支配的基因调控途径，从而产生从头开始的自动性。同时，我们将描述赋予正常静止心室肌细胞自动性的电生理通路的变化，并将起搏的重编程机制与天然SA结肌细胞中的起搏机制进行比较，后者是真正起搏细胞的金标准。了解自动性基因调控途径的主要障碍是缺乏研究SA节点转录调控途径特异性靶点的系统。这是因为胚胎发育过程中时间和空间的快速变化使得研究转录调控的具体目标变得困难。相反，我们提出的对出生后心肌细胞的研究提供了一个相对缓慢变化（新生儿）或稳态（成人）的电生理环境。AIMs 2和3旨在深入了解单细胞、双细胞起搏单元、2D单层和3D结构中tbx18重新编程的自动性。我们的细胞培养系统可以很容易地应用于其他转录因子或疾病介导的细胞电生理研究。这项研究即将带来三项科学创新。第一，AIMs 1和2的数据将提供对自动性的分子决定因素的见解，因为静止的肌细胞在Tbx18重新表达后开始自发和自主地跳动。第二，AIM 3的结果将为SAN生理学中的源-汇不匹配现象提供重要见解。第三，在拟议研究的结论中，生物起搏器的候选物可以被确定为电子起搏器设备的替代品。此外，全基因组关联研究（GWAS）已经确定并将T-box转录因子基因与先天性心脏缺陷和传导系统异常联系起来。tbx18诱导通路的失调可能导致传导系统形态发生异常，并可能导致心律失常。从AIMs 1、2和3中获得的知识将为这些心律失常的临床表现提供第一个因果解释。

DESCRIPTION (provided by applicant): The sinoatrial node (SA node or SAN) is a finely-tuned structure that initiates and sets the rhythm of the heartbeat. Recent insights into embryonic development have pinpointed T-box (Tbx) transcription factors as key determinants of SA node development. Tbx18, in particular, has been shown to be indispensable for the specification of the SA node during development. However, little is known about Tbx-driven gene regulatory pathways which specify morphogenesis of the SA node, and how these pathways lead to automaticity in pacemaker cells. We seek to test the general hypothesis that re-expression of Tbx18 suffices to reprogram postnatal cardiomyocytes to pacemaker cells. We propose to reveal Tbx18-dictated gene regulatory pathways that give rise to de novo automaticity. In parallel, we will characterize the changes in electrophysiological pathways which confer automaticity on normally-quiescent ventricular myocytes, and compare the reprogrammed mechanisms of pacing to those which are operative in native SA nodal myocytes, as the gold standard for genuine pacemaker cells. The main impediment to understanding the gene regulatory pathways to automaticity is a lack of a system to study specific targets of SA nodal transcriptional regulatory pathways. This is because the rapid temporal and spatial changes during embryonic development make it difficult to study specific targets of transcriptional regulation. In contrast, our proposed studies in postnatal cardiomyocytes offer a milieu for relatively slow-changing (neonatal) or steady-state (adult) electrophysiology. AIMs 2 and 3 are designed to gain insights into the Tbx18-reprogrammed automaticity in single-cell, two-cell pacing unit, 2D monolayers, and 3D structures. Our cell culture systems could readily be applied for other transcription factor- or disease-mediated studies of cellular electrophysiology. Three scientific innovations are imminent from this study. One, data from AIMs 1 and 2 will provide insights into molecular determinants of automaticity as quiescent myocytes begin to beat spontaneously and autonomously upon Tbx18 re-expression. Two, outcomes of AIM 3 will provide important insights into the source-sink mismatch phenomenon in SAN physiology. Three, at the conclusion of the proposed studies, a candidate for a biological pacemaker could be identified as an alternative to electronic pacemaker devices. Furthermore, Genome wide association studies (GWAS) have identified and linked T-box transcription factor genes with congenital heart defects and conduction system abnormalities. Dysregulation of Tbx18-guided pathways may cause improper morphogenesis of conduction system and may lead to arrhythmias. Knowledge gained from AIMs 1, 2, and 3 will provide the first cause-effect explanations for clinical manifestations of these arrhythmias. PUBLIC HEALTH RELEVANCE: Abnormally slow or fast heart rhythms, known as cardiac arrhythmias, affect many in North America and the number of people affected by this disease is increasing steadily with our aging populace. A key to treating these pathologic conditions is a fundamental understanding of the cardiac rhythm generation. We propose a detailed mechanistic study to investigate the initiation and propagation of a heartbeat, which will lead to better understanding and treatment of cardiac arrhythmias.