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

心室传导系统（Ventricular Conduction System，简称VCS）是由特定形式的心肌细胞（cardiomyocytes）组成的生物电线网络，它能够使快速的电传播触发同步的心室收缩。我们已经证明，Iroquois（Irx）转录因子家族的成员，Irx 3特异性地表达在细胞中，而Irx 3的缺失导致缓慢的电传导，这表明在细胞功能中起着至关重要的作用。Irx 3 KO小鼠心脏中的这种表型可归因于间隙连接Cx40（Gja 5）和Cx43（Gja 1）基因的异常表达以及由出生后成熟缺陷引起的心脏结构发育不全。尽管如此，Irx 3介导的转录因子特异化和成熟（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，我们使用新生小鼠心室肌细胞的初步研究显示，Irx 3过表达增加了自发搏动率，诱导了心肌细胞的分子特征，如超极化激活的环核苷酸门控通道基因（Hcn 2和Hcn 4）以及浦肯野纤维标记物接触蛋白2（Cntn 2）。这些数据表明Irx 3过表达可以将收缩性心肌细胞转化为VCS样细胞。此外，我们发现Irx 3与Tbx 3对VCS特异性基因的激活具有协同作用，而Tbx 3在此过程中的功能依赖于Irx 3。这些新的观察结果表明，Irx 3与Tbx 3合作的转录编程能力将提供一个有前途的工具来剖析和理解它们在转录调控中的分子机制。** 在此，我们拟进一步阐明Irx 3在心肌细胞中的分子机制，具体如下：（1）为了从分子水平深入了解心肌细胞的特化和分化，并建立Irx 3介导的心肌细胞程序化的机制基础，我们将构建Irx 3在心脏中的基因调控网络，特别强调Irx 3与Tbx 3的相互作用;（2）我们将验证Irx 3和Tbx 3靶基因，并表征Irx 3-Tbx 3编程的VCS样细胞的电生理学特性;以及（3）我们将通过利用心脏中过表达或缺乏Irx 3的小鼠来研究Irx 3介导的VCS编程和特化的体内作用。特别地，我们将通过在不同时间点（例如胚胎、新生儿和成人）诱导Irx 3过表达来测试Irx 3的特异性表达能力是否依赖于发育阶段。** 本提案中概述的研究将揭示与Irx 3在建立和规范生物学行为中的调节作用相关的新机制细节。这也将是建立我的研究计划，旨在调查心脏发育和功能的遗传调控的基础。

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.