Institute of Child Health, University College London, UK.
Circ Res. 2012 Jul 6;111(2):e19-31. doi: 10.1161/CIRCRESAHA.111.260695. Epub 2012 May 29.
Nkx2.5 is one of the most widely studied cardiac-specific transcription factors, conserved from flies to man, with multiple essential roles in both the developing and adult heart. Specific dominant mutations in NKX2.5 have been identified in adult congenital heart disease patients presenting with conduction system anomalies and recent genome-wide association studies implicate the NKX2.5 locus, as causative for lethal arrhythmias ("sudden cardiac death") that occur at a frequency in the population of 1 in 1000 per annum worldwide. Haploinsufficiency for Nkx2.5 in the mouse phenocopies human conduction disease pathology yet the phenotypes, described in both mouse and man, are highly pleiotropic, implicit of unknown modifiers and/or factors acting in epistasis with Nkx2.5/NKX2.5.
To identify bone fide upstream genetic modifier(s) of Nkx2.5/NKX2.5 function and to determine epistatic effects relevant to the manifestation of NKX2.5-dependent adult congenital heart disease.
A study of cardiac function in prospero-related homeobox protein 1 (Prox1) heterozygous mice, using pressure-volume loop and micromannometry, revealed rescue of hemodynamic parameters in Nkx2.5(Cre/+); Prox1(loxP/+) animals versus Nkx2.5(Cre/+) controls. Anatomic studies, on a Cx40(EGFP) background, revealed Cre-mediated knock-down of Prox1 restored the anatomy of the atrioventricular node and His-Purkinje network both of which were severely hypoplastic in Nkx2.5(Cre/+) littermates. Steady state surface electrocardiography recordings and high-speed multiphoton imaging, to assess Ca(2+) handling, revealed atrioventricular conduction and excitation-contraction were also normalized by Prox1 haploinsufficiency, as was expression of conduction genes thought to act downstream of Nkx2.5. Chromatin immunoprecipitation on adult hearts, in combination with both gain and loss-of-function reporter assays in vitro, revealed that Prox1 recruits the corepressor HDAC3 to directly repress Nkx2.5 via a proximal upstream enhancer as a mechanism for regulating Nkx2.5 function in adult cardiac conduction.
Here we identify Prox1 as a direct upstream modifier of Nkx2.5 in the maintenance of the adult conduction system and rescue of Nkx2.5 conduction disease phenotypes. This study is the first example of rescue of Nkx2.5 function and establishes a model for ensuring electrophysiological function within the adult heart alongside insight into a novel Prox1-HDAC3-Nkx2.5 signaling pathway for therapeutic targeting in conduction disease.
Nkx2.5 是研究最多的心脏特异性转录因子之一，从苍蝇到人类都有保守性，在心脏发育和成年心脏中都有多种重要作用。在患有传导系统异常的成年先天性心脏病患者中发现了 NKX2.5 中的特定显性突变，最近的全基因组关联研究表明 NKX2.5 基因座是导致全球每年每 1000 人中发生 1 次致死性心律失常（“心源性猝死”）的原因。在小鼠中 Nkx2.5 的单倍不足可模拟人类传导疾病病理学，但在小鼠和人类中描述的表型高度多效性，暗示了未知的修饰因子和/或与 Nkx2.5/NKX2.5 起上位作用的因子。
鉴定 Nkx2.5/NKX2.5 功能的真实上游遗传修饰因子，并确定与 NKX2.5 依赖性成年先天性心脏病表现相关的上位效应。
使用压力-容积环和微压测量法对 Prospero 相关同源框蛋白 1 (Prox1) 杂合子小鼠的心脏功能进行了研究，结果显示 Nkx2.5(Cre/+)；Prox1(loxP/+) 动物的血液动力学参数得到了挽救，而 Nkx2.5(Cre/+)对照动物则没有。在 Cx40(EGFP)背景下进行的解剖学研究显示，Cre 介导的 Prox1 敲低恢复了房室结和 His-Purkinje 网络的解剖结构，而 Nkx2.5(Cre/+)同窝仔鼠的这些结构严重发育不良。通过体表心电图记录和高速多光子成像来评估 Ca(2+)处理，结果显示房室传导和兴奋-收缩也通过 Prox1 单倍不足得到了正常化，而 Nkx2.5 下游的传导基因的表达也得到了正常化。体外的染色质免疫沉淀结合功能获得和丧失报告基因实验显示，Prox1 通过一个近端上游增强子募集核心抑制因子 HDAC3 来直接抑制 Nkx2.5，作为调节成年心脏传导中 Nkx2.5 功能的机制。
在这里，我们将 Prox1 鉴定为维持成年传导系统和挽救 Nkx2.5 传导疾病表型的 Nkx2.5 的直接上游修饰因子。本研究首次证明了 Nkx2.5 功能的挽救，并建立了一个在成年心脏中确保电生理功能的模型，同时为传导疾病的治疗靶点提供了一个新的 Prox1-HDAC3-Nkx2.5 信号通路的见解。
