Functional Analysis of the Cardiac Transcription Factor Nkx2.5

本项目的长期目标是通过对心脏转录因子Nkx 2.5（Csx）的研究，进一步了解心脏发育的分子机制，以及先天性心脏病，特别是法洛四联症（TOF）的发病机制。NKX2.5是迄今为止在人类中报告的唯一基因，其突变占非综合征型散发性TOF患者的显著部分（4-5%）。Nkx2.5是最早在心前中胚层中表达的基因之一。Nkx2.5的纯合缺失导致早期胚胎死亡，原因是心脏流出道阻塞、心室功能障碍和心室形成停滞。Nkx2.5在整个发育到成年阶段的心脏中的持续表达表明Nkx2.5蛋白的靶基因也可能在心脏中表达。 对出生后心脏功能的维持很重要。事实上，我们的初步结果表明，Nkx2.5杂合小鼠对肌病应激表现出独特的遗传反应，并且更容易发生心律失常和心力衰竭。我们的初步结果还表明，Nkx2.5在不同的发育阶段和心脏的不同区域具有不同的靶基因。此外，已知Nkx2.5与其他重要的心脏转录因子，如Tbx 5，GATA 4和SRF直接相互作用。这些结果表明，Nkx2.5基因功能的进一步研究可能会产生许多重要的先天性心脏病，特别是在人类TOF的发病机制的新见解。因此，我们将在本提案中提出以下具体目标：具体目标1：确定Nkx2.5杂合突变小鼠心力衰竭易感性增加的机制。具体目的2：通过在Nkx2.5中相同位置的靶向突变来创建TOF小鼠模型 在患者中发现，并且，如果必要的话，与具有圆锥动脉干表型的其他突变小鼠杂交。具体目标3：对TOF患者的RV活检进行转录谱分析，以比较小鼠模型和人类中的基因表达模式。具体目标4：确定Nkx2.5在流出道发育中的靶点，其改变可导致圆锥动脉干异常。具体目标五：利用蛋白质组学方法鉴定Nkx2.5相关蛋白，研究蛋白质间相互作用如何为Nkx2.5蛋白调控靶基因提供特异性。

The long term goal of this project is to further the understanding of the molecular mechanism of cardiac development end the pathogenesis of congenital heart disease, particularly that of the tetralogy of Fallot (TOF), through the study of the cardiac transcription factor Nkx2.5 (Csx). NKX2.5 is the only gene, so far, reported in humans whose mutations accounts for a significant portion (4-5%) of patients with non-syndromic sporadic TOF. Nkx2.5 is among the first genes to be expressed in the precardiac mesoderm. Homozygous deletion of Nkx2.5 causes early embryonic lethality due to cardiac outflow tract obstruction, ventricular dysfunction and the arrest of chamber formation. The continued expression of the Nkx2.5 in the heart throughout development into the adult stage suggests that target genes for Nkx2.5 protein may also be important to the maintenance of cardiac function postnatally. Indeed our preliminary results indicate that Nkx2.5 heterozygous mice display a distinct genetic response to myopathic stress and are much more susceptible to arrhythmias and heart failure. Our preliminary results also suggest that Nkx2.5 have different target genes at different stages of development and different regions of the heart. In addition, Nkx2.5 is known to interact directly with other important cardiac transcription factors, such as Tbx5, GATA4 and SRF. These results suggest that further studies of the Nkx2.5 gene function are likely to yield many important new insights into the pathogenesis of congenital heart disease, particularly that of TOF in humans. Accordingly, we will address the following Specific Aims in this proposal: Specific Aim 1: To identify the mechanism of an increased susceptibility to heart failure in Nkx2.5 heterozygous mutant mice. Specific Aim 2: To create mouse models of TOF by targeted mutations in Nkx2.5 in the identical positions found in patients, and, if necessary, crossing with other mutant mice that have a conotruncal phenotype. Specific Aim 3: To perform transcriptional profiling of RV biopsies of patients with TOF to compare the pattern of gene expression in the mouse model and humans. Specific Aim 4: To identify targets of Nkx2.5 in the developing outflow truct, whose alterations can cause conotruncal abnormalities. Specific Aim 5: To identify Nkx 2.5 associated proteins by proteomics approach and study how protein - protein interactions provide specificity to target gene regulation by Nkx2.5 protein.