Irx3 as a critical transcriptional regulator in the maturation and specification of the ventricular conduction system

心室传导系统(VCS)是由一种特殊形式的心肌细胞(心肌细胞)组成的生物电线网络，它使快速的电传播能够触发同步的心室收缩。我们已经证明，IRX3是易洛魁(Iroquis，IRX)转录因子家族的成员之一，在VCS中特异表达，而IRX3的缺失导致电传导缓慢，提示IRX3在VCS功能中起着至关重要的作用。IRX3KO小鼠心脏的这种表型可归因于缝隙连接Cx40(Gja5)和Cx43(Gja1)基因的异常表达以及出生后成熟缺陷导致的VCS结构发育不良。然而，IRX3介导的VCS规范和成熟的分子机制(即下游靶基因)在很大程度上仍不清楚。*有趣的是，我们用新生小鼠心肌细胞进行的初步研究表明，IRX3的过表达通过诱导VCS的分子特征而增加自发搏动频率，例如超极化激活的环核苷酸门控通道基因(HCn2和HCN4)以及浦肯野纤维标记Contactin 2(Cntn2)。这些数据表明，IRX3过表达可以将收缩心肌细胞转化为VCS样细胞。此外，我们还发现IRX3对Tbx3激活VCS特异性基因具有协同作用，而Tbx3在这一过程中的作用依赖于IRX3。这些新颖的观察结果表明，IRX3和TBX3的转录编程能力将为剖析和理解VCS规范中的分子机制提供一个有前途的工具。*我们建议进一步阐明IRX3在VCS中的分子机制如下：(1)为了深入了解VCS细胞的指定和分化，并建立IRX3介导的VCS编程的机制基础，我们将构建IRX3在心脏中的基因调控网络，重点是与TBX3的相互作用；(2)我们将验证IRX3和TBX3的靶基因，并表征IRX3-TBX3编程的VCS样细胞的电生理特性；以及(3)我们将利用心脏过表达或缺失IRX3的小鼠来研究IRX3介导的VCS编程和规范的体内效应。特别是，我们将通过在不同的时间点(例如胚胎、新生儿和成人)诱导IRX3的过度表达来测试IRX3的VCS规范能力是否依赖于发育阶段。*本提案中概述的研究将揭示与IRX3在VCS的建立和规范中的调节作用相关的新的机制细节。这也将是建立我的研究项目的基础，该项目旨在调查心脏发育和功能的遗传调节。

The ventricular conduction system (VCS) is a network of biological electric wires comprised of a specialized form of cardiac muscle cells (cardiomyocytes), which enables the rapid electric propagation to trigger a synchronous ventricular contraction. We have demonstrated that a member of Iroquois (Irx) transcription factor family, Irx3 is specifically expressed in the VCS, while loss of Irx3 led to slow electrical conduction, suggesting an essential role in the VCS function. This phenotype in Irx3KO mouse heart can be attributed to abnormal expression of gap junction Cx40 (Gja5) and Cx43 (Gja1) genes as well as VCS structural hypoplasia caused by a postnatal maturation defect. Nevertheless, molecular mechanisms underlying Irx3-mediated VCS specification and maturation (i.e. downstream target genes) remain largely unknown. ******Interestingly, our preliminary study using neonatal mouse ventricular myocytes revealed that Irx3 overexpression increased the spontaneous beating rate with induction of molecular signatures of the VCS, such as hyperpolarization-activated cyclic nucleotide-gated channel genes (Hcn2 and Hcn4) as well as Purkinje fiber marker Contactin2 (Cntn2). These data suggest Irx3 overexpression can transform contractile myocytes into VCS-like cells. Moreover, we found that Irx3 exerts synergistic effects on the activation of VCS-specific genes with Tbx3, while Tbx3 function in this process is dependent on Irx3. These novel observations suggest that transcriptional programing capacity of Irx3 in cooperation with Tbx3 will provide a promising tool to dissect and understand their molecular mechanisms in the VCS specification. ******Here we propose to further delineate the molecular mechanisms of Irx3 in the VCS as follows: (1) To gain molecular insights into the specification and differentiation of VCS cells, and to establish a mechanistic basis of Irx3-medated VCS programming, we will construct a gene regulatory network of Irx3 in the heart with a particular emphasis on interactions with Tbx3; (2) we will validate Irx3 and Tbx3 target genes and characterize electrophysiological properties of Irx3-Tbx3 programmed VCS-like cells; and (3) We will investigate the in vivo effect of Irx3-mediated VCS programming and specification by utilizing mice overexpressing or lacking Irx3 in the heart. Particularly, we will test whether VCS specification capacity of Irx3 is dependent on the developmental stages by inducing Irx3 overexpression in different time points (e.g. embryo, neonatal and adult). ******The studies outlined in this proposal will unveil novel mechanistic detail related to the regulatory action of Irx3 in the establishment and specification of the VCS. It will also be fundamental to build my research program which is aimed to investigate the genetic regulation of heart development and function.