Genetic Analyst of Early Conduction System Development

描述(由申请人提供)：了解专门的室性心脏传导系统的发展对于开发心律失常的生物治疗方法和掌握心脏组织再生至关重要。心律失常和猝死是几种伴有异常小梁或致密化不全的心肌病的特征。小梁状心肌的发育和维持是心脏器官发生的关键过程，并通过ERBB2/neuregin(NRG)信号通路与成熟的心脏传导模式的正常发育有关。已知ERBB2/NRG通路在传导系统的发育中发挥作用。然而，人们既不了解骨小梁的过程，也不了解它在为发展中的传导系统奠定基础方面所起的作用。这项研究的长期目标是描述专门化传导系统的早期结构和功能，并确定小梁形成与传导系统发育的关系。这个项目的具体目标有两个。首先，我们将建立斑马鱼特殊的心脏传导系统的分子标记。我们将通过分析这些标记物的表达来检验这样的假设，即传导系统的第一个细胞位于小梁形成心肌层，而小梁形成突变体缺乏传导系统细胞。其次，我们将检验这一假设，即传导系统的发育需要小梁形成过程，而不仅仅是NRG/ERBB2途径。斑马鱼是这些实验的有用的模式生物，因为它的遗传易驯化，半透明的胚胎发生，以及与人类电生理表型和疾病的相似性。为了鉴定传导细胞，我们将在整体和切片斑马鱼心脏上使用原位杂交和候选标记的抗体染色。为了评估野生型、突变型和转基因心脏的传导系统发育，我们将使用光学标测和胚胎心电监测。我们将评估ERBB2突变体以及注入心脏特异显性负向结构的野生型胚胎的传导表型，这些负向结构旨在阻断参与小梁形成的其他途径。研究发育中的心脏传导系统的结构和功能及其与小梁形成的关系将加深我们对先天性和获得性心肌病心律失常的理解，并将提供有助于通过操纵传导细胞来治疗室性心律失常和猝死的工具和知识。 与公共健康相关：这些实验建议调查心脏的电线系统(传导系统)是如何发育的，以及在发育过程中心肌的增厚(小梁)是否需要传导系统的正常发育。这项工作将有助于我们进一步了解遗传性心肌缺陷和获得性心脏病中的心律失常和猝死。

DESCRIPTION (provided by applicant): Understanding development of the specialized ventricular cardiac conduction system is critical to generating biologic treatments for arrhythmia and mastering cardiac tissue regeneration. Arrhythmia and sudden death characterize several cardiomyopathies with aberrant trabeculation or noncompaction. The development and maintenance of trabeculated ventricular myocardium is a key process in heart organogenesis and appears linked to the normal development of a mature ventricular cardiac conduction pattern by way of erbb2/neuregulin (nrg) signaling. The erbb2/nrg pathway is already known to play a part in conduction system development. However, neither the process of trabeculation nor its role in setting the stage for the developing conduction system is understood. The long-term goals of this research are to describe the early structure and function of the specialized conduction system, and to determine how trabeculation relates to conduction system development. The specific aims of this project are twofold. First, we will establish molecular markers of the specialized ventricular cardiac conduction system in zebrafish. We will test the hypotheses that the location of the first cells of the conduction system are located in the trabeculating myocardial layer, and that trabeculation mutants lack conduction system cells as assessed by analyzing the expression of these markers. Second, we will test the hypothesis that the process of trabeculation, and not just the nrg/erbb2 pathway, is required for conduction system development. The zebrafish is a useful model organism for these experiments due to its genetic tractability, translucent embryogenesis, and similarity to human electrophysiological phenotypes and disease. To identify conduction cells, we will use in situ hybridization and antibody staining of candidate markers on whole-mount and sectioned zebrafish hearts. To assess conduction system development in wild-type, mutant, and genetically altered hearts, we will use optical mapping and embryonic ECG monitoring. We will assess conduction phenotypes in erbb2 mutants, as well as in wild-type embryos injected with heart-specific dominant negative constructs designed to block other pathways involved in trabeculation. Studying the structure and function of the developing cardiac conduction system and its relationship to trabeculation will further our understanding of arrhythmia in both congenital and acquired cardiomyopathies and will provide tools and knowledge that can help treat ventricular arrhythmia and sudden death by manipulating conduction cells. PUBLIC HEALTH RELEVANCE: The experiments proposed investigate how the electrical wiring system (conduction system) of the heart develops, and whether thickening of the heart muscle during development (trabeculation) is required for the conduction system to develop properly. This work will help further our understanding of arrhythmias and sudden death in inherited heart muscle defects and acquired heart diseases.