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。最近对胚胎发育的深入研究已确定 T-box (Tbx) 转录因子是 SA 节点发育的关键决定因素。特别是，Tbx18 已被证明对于开发过程中 SA 节点的规范是不可或缺的。然而，人们对 Tbx 驱动的基因调控途径知之甚少，这些途径指定了 SA 节点的形态发生，以及这些途径如何导致起搏细胞的自动化。我们试图检验 Tbx18 的重新表达足以将出生后心肌细胞重新编程为起搏细胞的一般假设。我们建议揭示 Tbx18 决定的基因调控途径，从而产生从头自动化。与此同时，我们将描述电生理通路的变化，这些变化赋予正常静止的心室肌细胞自主性，并将起搏的重新编程机制与天然 SA 结肌细胞中起作用的机制进行比较，作为真正起搏细胞的黄金标准。了解自动化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定靶标的系统。这是因为胚胎发育过程中快速的时空变化使得研究转录调控的特定靶点变得困难。相比之下，我们提出的出生后心肌细胞研究为相对缓慢变化（新生儿）或稳态（成人）电生理学提供了环境。 AIM 2 和 3 旨在深入了解单细胞、两细胞起搏单元、2D 单层和 3D 结构中 Tbx18 重编程的自动化。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。这项研究即将带来三项科学创新。第一，来自 AIM 1 和 2 的数据将提供对自动化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地、自主地跳动。第二，AIM 3 的结果将为 SAN 生理学中的源-宿不匹配现象提供重要的见解。第三，在拟议研究的结论中，生物起搏器的候选者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) 已鉴定出 T-box 转录因子基因与先天性心脏缺陷和传导系统异常之间的关系。 Tbx18 引导通路的失调可能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。

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.