Molecular determinants of the cardiac pacemaker automaticity

描述（由申请人提供）：窦房结（SA结或SAN）是一种微调结构，可启动和设定心跳节律。最近对胚胎发育的认识已经确定了T-box（Tbx）转录因子是SA结发育的关键决定因素。特别是，TBX 18已被证明是开发期间SA节点规范不可或缺的。然而，很少有人知道Tbx驱动的基因调控途径，指定SA结的形态发生，以及这些途径如何导致起搏细胞的自律性。我们试图测试Tbx 18的重新表达足以将出生后的心肌细胞重新编程为起搏细胞的一般假设。我们建议揭示Tbx 18决定的基因调控途径，引起从头自律性。与此同时，我们将表征电生理通路的变化，赋予正常静止的心室肌细胞的自律性，并比较重新编程的起搏机制，这些机制是在本地SA结肌细胞，真正的起搏细胞的金标准。了解基因调控途径的自动性的主要障碍是缺乏一个系统来研究特定的目标SA节点转录调控途径。这是因为胚胎发育过程中快速的时间和空间变化使得研究转录调控的特定靶点变得困难。相比之下，我们提出的研究在出生后心肌细胞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。AIM 2和3旨在深入了解单细胞、双细胞起搏单元、2D单层和3D结构中的Tbx 18重编程自动性。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学研究。这项研究有三个科学创新。第一，AIM 1和2的数据将提供对自动性分子决定因素的深入了解，因为静止的肌细胞在Tbx 18重新表达后开始自发和自主跳动。第二，AIM 3的结果将为SAN生理学中的源-汇失配现象提供重要的见解。第三，在拟议的研究结束时，可以确定生物起搏器的候选者作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病和传导系统异常联系起来。Tbx 18引导的通路调节失调可能会导致传导系统形态发生不当，并可能导致心律失常。从AIM 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。 公共卫生相关性：不正常的缓慢或快速的心脏节律，称为心律失常，影响许多在北美和受这种疾病影响的人数正在稳步增加，随着我们的人口老龄化。治疗这些病理状况的关键是对心律产生的基本理解。我们提出了一个详细的机制研究，调查的启动和传播的心跳，这将导致更好地理解和治疗心律失常。

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.