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，它使快速的电传播触发同步心室收缩。我们已经证明Iroquois （Irx）转录因子家族的成员Irx3在VCS中特异性表达，而Irx3的缺失导致电传导缓慢，这表明在VCS功能中起重要作用。Irx3KO小鼠心脏中的这种表型可归因于间隙连接Cx40 （Gja5）和Cx43 （Gja1）基因的异常表达以及由出生后成熟缺陷引起的VCS结构发育不全。然而，irx3介导的VCS规范和成熟（即下游靶基因）的分子机制在很大程度上仍然未知。******有趣的是，我们对新生小鼠心室肌细胞的初步研究显示，Irx3过表达通过诱导VCS的分子特征，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物conttin2 (Cntn2)，增加了自发跳动率。这些数据表明，Irx3过表达可以将收缩性肌细胞转化为vcs样细胞。此外，我们发现Irx3与Tbx3对vcs特异性基因的激活具有协同作用，而Tbx3在这一过程中的功能依赖于Irx3。这些新颖的观察结果表明，Irx3与Tbx3合作的转录编程能力将为在VCS规范中解剖和理解它们的分子机制提供一个有前途的工具。******在此，我们建议进一步阐述Irx3在VCS中的分子机制：(1)为深入了解VCS细胞的规范和分化，并建立Irx3介导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，重点研究其与Tbx3的相互作用；(2)验证Irx3和Tbx3靶基因，表征Irx3-Tbx3编程vcs样细胞的电生理特性；(3)我们将利用心脏中过表达或缺乏Irx3的小鼠，研究Irx3介导的VCS编程和规范在体内的影响。特别是，我们将通过诱导不同时间点（如胚胎、新生儿和成体）的Irx3过表达来测试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的建立和规范中的监管作用相关的新机制细节。这也将是建立我的研究计划的基础，该计划旨在研究心脏发育和功能的遗传调控。

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.