We screened the complete exome sequence database of the Pediatrics Cardiac Genomics Consortium and identified a cohort with a de novo CHD4 (chromodomain helicase DNA-binding protein 4) proband, CHD4M202I, with congenital heart defects. We engineered a humanized mouse model of CHD4M202I (mouse CHD4M195I). Histological analysis, immunohistochemistry, flow cytometry, transmission electron microscopy, and echocardiography were used to analyze cardiac anatomy and function. Ex vivo culture, immunopurification coupled with mass spectrometry, transcriptional profiling, and chromatin immunoprecipitation were performed to deduce the mechanism of CHD4M195I-mediated ventricular wall defects.
CHD4M195I/M195I mice developed biventricular hypertrabeculation and noncompaction and died at birth. Proliferation of cardiomyocytes was significantly increased in CHD4M195I hearts, and the excessive trabeculation was associated with accumulation of ECM (extracellular matrix) proteins and a reduction of ADAMTS1 (ADAM metallopeptidase with thrombospondin type 1 motif 1), an ECM protease. We rescued the hyperproliferation and hypertrabeculation defects in CHD4M195I hearts by administration of ADAMTS1. Mechanistically, the CHD4M195I protein showed augmented affinity to endocardial BRG1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4). This enhanced affinity resulted in the failure of derepression of Adamts1 transcription such that ADAMTS1-mediated trabeculation termination was impaired.
Our study reveals how a single mutation in the chromatin remodeler CHD4, in mice or humans, modulates ventricular chamber maturation and that cardiac defects associated with the missense mutation CHD4M195I can be attenuated by the administration of ADAMTS1.
方法:我们筛选了儿科学心脏基因组学联盟的完整外显子组序列数据库,并确定了一个具有从头CHD4(色域解旋酶DNA结合蛋白4)先证者的队列,CHD4M202I,先天性心脏缺陷.我们设计了CHD4M202I的人源化小鼠模型(小鼠CHD4M195I)。组织学分析,免疫组织化学,流式细胞术,透射电子显微镜,采用超声心动图对心脏解剖结构和功能进行分析。离体培养,免疫纯化与质谱联用,转录分析,并进行染色质免疫沉淀以推断CHD4M195I介导的心室壁缺损的机制。
结果:CHD4M195I/M195I小鼠出现双室高纤维化和心肌致密化,出生时死亡。CHD4M195I心脏的心肌细胞增殖显著增加,过度的小梁形成与ECM(细胞外基质)蛋白的积累和ADAMTS1(具有血小板反应蛋白1型基序1的ADAM金属肽酶)的减少有关,ECM蛋白酶。我们通过施用ADAMTS1挽救了CHD4M195I心脏的过度增殖和过度分化缺陷。机械上,CHD4M195I蛋白对心内膜BRG1(SWI/SNF相关,矩阵关联,肌动蛋白依赖性染色质调节因子,亚科A,成员4)。这种增强的亲和力导致Adamts1转录的去抑制失败,从而损害了ADAMTS1介导的小梁形成终止。
结论:我们的研究揭示了染色质重塑剂CHD4中的单个突变是如何在小鼠或人类中,调节心室成熟,并且与错义突变CHD4M195I相关的心脏缺陷可以通过施用ADAMTS1来减轻。