Kim Yuri, Kim Seong Won, Saul David, Neyazi Meraj, Schmid Manuel, Wakimoto Hiroko, Slaven Neil, Lee Joshua H, Layton Olivia, Wasson Lauren K, Letendre Justin H, Xiao Feng, Ewoldt Jourdan K, Gkatzis Konstantinos, Sommer Peter, Gobert Bénédicte, Wiest-Daesslé Nicolas, McAfee Quentin, Singhal Nandita, Lun Mingyue, Gorham Joshua M, Arany Zolt, Sharma Arun, Toepfer Christopher N, Oudit Gavin Y, Pu William T, Dickel Diane E, Pennacchio Len A, Visel Axel, Chen Christopher S, Seidman J G, Seidman Christine E
Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
Division of Cardiovascular Medicine, Brigham and Women's Hospital, Boston, Massachusetts, USA.
J Clin Invest. 2024 Dec 17;135(4):e183353. doi: 10.1172/JCI183353.
Heterozygous truncating variants in the sarcomere protein titin (TTN) are the most common genetic cause of heart failure. To understand mechanisms that regulate abundant cardiomyocyte (CM) TTN expression, we characterized highly conserved intron 1 sequences that exhibited dynamic changes in chromatin accessibility during differentiation of human CMs from induced pluripotent stem cells (hiPSC-CMs). Homozygous deletion of these sequences in mice caused embryonic lethality, whereas heterozygous mice showed an allele-specific reduction in Ttn expression. A 296 bp fragment of this element, denoted E1, was sufficient to drive expression of a reporter gene in hiPSC-CMs. Deletion of E1 downregulated TTN expression, impaired sarcomerogenesis, and decreased contractility in hiPSC-CMs. Site-directed mutagenesis of predicted binding sites of NK2 homeobox 5 (NKX2-5) and myocyte enhancer factor 2 (MEF2) within E1 abolished its transcriptional activity. In embryonic mice expressing E1 reporter gene constructs, we validated in vivo cardiac-specific activity of E1 and the requirement for NKX2-5- and MEF2-binding sequences. Moreover, isogenic hiPSC-CMs containing a rare E1 variant in the predicted MEF2-binding motif that was identified in a patient with unexplained dilated cardiomyopathy (DCM) showed reduced TTN expression. Together, these discoveries define an essential, functional enhancer that regulates TTN expression. Manipulation of this element may advance therapeutic strategies to treat DCM caused by TTN haploinsufficiency.
肌节蛋白肌联蛋白（TTN）中的杂合截短变异是心力衰竭最常见的遗传原因。为了解调节心肌细胞（CM）中TTN高表达的机制，我们对高度保守的内含子1序列进行了表征，这些序列在人诱导多能干细胞分化为心肌细胞（hiPSC-CM）的过程中，染色质可及性呈现动态变化。小鼠中这些序列的纯合缺失导致胚胎致死，而杂合小鼠则表现出Ttn表达的等位基因特异性降低。该元件的一个296 bp片段（称为E1）足以驱动hiPSC-CM中报告基因的表达。E1的缺失下调了TTN的表达，损害了肌节生成，并降低了hiPSC-CM的收缩性。对E1内预测的NK2同源盒5（NKX2-5）和肌细胞增强因子2（MEF2）结合位点进行定点诱变，消除了其转录活性。在表达E1报告基因构建体的胚胎小鼠中，我们验证了E1在体内的心脏特异性活性以及对NKX2-5和MEF2结合序列的需求。此外，在一名不明原因扩张型心肌病（DCM）患者中鉴定出的预测MEF2结合基序中含有罕见E1变异的同基因hiPSC-CM显示TTN表达降低。总之，这些发现定义了一个调节TTN表达的重要功能增强子。对该元件的操控可能会推进治疗由TTN单倍体不足引起的DCM的治疗策略。
