Reading Frame Repair of TTN Truncation Variants Restores Titin Quantity and Functions.
Reading Frame Repair of TTN Truncation Variants Restores Titin Quantity and Functions.
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TTN截短变异体的阅读框修复恢复了肌联蛋白的数量和功能。
DOI：
10.1161/circulationaha.120.049997
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发表时间：
2022-01-18
期刊：
影响因子：
37.8
通讯作者：
Hinson JT
中科院分区：
文献类型：
--
作者：
Romano R;Ghahremani S;Zimmerman T;Legere N;Thakar K;Ladha FA;Pettinato AM;Hinson JT
肌动蛋白截断变异(TTNtvs)是扩张型心肌病(DCM)最常见的遗传危险因素，扩张型心肌病是一种发病率和死亡率都很高的疾病。TTNtvs的致病性与结构定位有关，因为重叠肌球蛋白重链结合区的A带变异比I带变异更具致病性，其机制尚不完全清楚。阐明A-带变异对扩张型心肌病高致病性的原因，可以为扩张型心肌病的发病机制、TTN功能和治疗靶点提供新的见解。我们利用诱导多能干细胞和CRISPR技术构建了在A带和I带结构域内携带DCM相关TTNtv的人心肌细胞模型。我们分别通过TTN蛋白凝胶电泳法和免疫荧光法对正常TTN异构体和变异型特异性截短肽的表达水平和心肌细胞定位进行了研究。通过引入近端I带TTNtv，利用CRISPR去除A带变异特异性截短肽，我们研究了单个心肌细胞的遗传机制和三维仿生心脏微组织功能分析。最后，我们设计了全长TTN蛋白报告实验，并利用下一代测序分析开发了用于体细胞基因组编辑TTNtvs的CRISPR治疗。A波段TTNtv可剂量依赖性地损伤心肌微组织的扭体力，降低全长TTN水平，并产生丰富的TTN截短肽。TTN截短肽整合到新生的肌原纤维样结构中，并损害肌原纤维的形成。使用近端I-波段TTNtv的CRISPR消融TTN截短肽，部分恢复了心脏微组织的扭动力缺陷。心肌细胞基因组编辑使用SpCas9和TTNtv特异的引导RNA恢复了TTN蛋白读框，在心脏微组织分析中增加了全长TTN蛋白水平，减少了TTN截短肽，并增加了肌节功能。A波段TTNtv对肌节功能的削弱程度大于I波段TTNtv，与估计的DCM致病性成比例。虽然两个TTNtv都导致全长TTN单倍体功能不全，但只有A带TTNtv产生TTN截短肽，从而损害肌纤维形成和肌节功能。CRISPR介导的A-带TTNtv阅读框修复恢复了功能缺陷，可作为一种“一劳永逸”的基因组编辑策略，以靶向约30%的DCM相关TTNtv。
Titin truncation variants (TTNtvs) are the most common inheritable risk factor for dilated cardiomyopathy (DCM), a disease with high morbidity and mortality. The pathogenicity of TTNtvs has been associated with structural localization as A-band variants overlapping myosin heavy chain-binding domains are more pathogenic than I-band variants by incompletely understood mechanisms. Demonstrating why A-band variants are highly pathogenic for DCM could reveal new insights into DCM pathogenesis, TTN functions and therapeutic targets. We constructed human cardiomyocyte models harboring DCM-associated TTNtvs within A-band and I-band structural domains using induced pluripotent stem cell and CRISPR technologies. We characterized normal TTN isoforms and variant-specific truncation peptides by their expression levels and cardiomyocyte localization using TTN protein gel electrophoresis and immunofluorescence, respectively. Using CRISPR to ablate A-band variant-specific truncation peptides through introduction of a proximal I-band TTNtv, we studied genetic mechanisms in single cardiomyocyte and 3-dimensional, biomimetic cardiac microtissue functional assays. Finally, we engineered a full-length TTN protein reporter assay and utilized next-generation sequencing assays to develop a CRISPR therapeutic for somatic cell genome editing TTNtvs. An A-band TTNtv dose-dependently impaired cardiac microtissue twitch force, reduced full-length TTN levels, and produced abundant TTN truncation peptides. TTN truncation peptides integrated into nascent myofibril-like structures and impaired myofibrillogenesis. CRISPR-ablation of TTN truncation peptides using a proximal I-band TTNtv partially restored cardiac microtissue twitch force deficits. Cardiomyocyte genome-editing using SpCas9 and a TTNtv-specific guide RNA restored TTN protein reading frame, which increased full length TTN protein levels, reduced TTN truncation peptides, and increased sarcomere function in cardiac microtissue assays. An A-band TTNtv diminished sarcomere function greater than an I-band TTNtv in proportion to estimated DCM pathogenicity. While both TTNtvs resulted in full-length TTN haploinsufficiency, only the A-band TTNtv produced TTN truncation peptides that impaired myofibrillogenesis and sarcomere function. CRISPR-mediated reading frame repair of the A-band TTNtv restored functional deficits, and could be adapted as a “one-and-done” genome editing strategy to target ~30% of DCM-associated TTNtvs.