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

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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.