Establishing and reversing the functional consequences of Titin truncation mutations

项目概要/摘要 心肌病发生在大约 1:200 的个体中，通常是由基因变异遗传引起的，这些变异 编码调节肌节（心脏细胞产生力的细胞器）的蛋白质。由于不完整 目前，医生对变异致病性和心肌病发病机制的了解还有限 他们为心肌病患者提供诊断、预后和治疗选择的能力。变种 TTN 基因编码肌节蛋白肌联蛋白，是最常见的遗传性病变 扩张型心肌病（DCM），其特征是心室扩张、收缩功能降低、 猝死和进行性心力衰竭的风险。 DCM 中最常见的 TTN 变异类型是 预计会缩短 TTN 蛋白长度并减少 TTN 蛋白数量的截断突变。 值得注意的是，位于远端 TTN 结构域的截短变异比那些本地化的截短变异更具致病性。 局限于近端结构域，但这种关系的机制基础尚不确定。它仍然存在 不完全了解 TTN 截断变异通常如何导致 DCM，而我们的缺乏使情况变得更加复杂 了解 TTN 变异致病性的“长度依赖性”。这些知识差距限制了疾病 DCM 患者的预测、生物标志物识别和治疗开发。的中心目标是 我们的研究旨在确定 TTN 变体对 TTN 长度和剂量的破坏如何导致 DCM，并利用这一点 为 TTN 变异携带者开发 DCM 疗法的知识。我们假设健康的心脏 收缩功能和结构取决于TTN长度和剂量的调节，并且不同 TTN 截短的致病性可以通过相关的不同结构和功能后果来解释 具有特定的截断位点。在目标 1 中，我们将确定 TTN 截断的功能后果 通过利用由人类心肌细胞组成的 3 维心脏组织模型来跨结构域 与诱导多能干细胞不同，诱导多能干细胞中通过 CRISPR 介导引入了变异体 基因组编辑。我们将询问这些模型的组织机械表型（例如被动张力 和 Frank-Starling 行为）、TTN 蛋白质长度和水平（使用专门方法）、蛋白质稳态应激 途径反应（使用免疫印迹），以及机械转导信号和选择性剪接（使用 分别是表达分析和转录组学）。在目标 2 中，我们将使用以下方法恢复 TTN 蛋白水平： 最近开发的 CRISPR 激活方法应用于 DCM 工程心脏组织模型 总体评估 TTN 亚型的功能以及作为 DCM 概念验证治疗的功能。通过这些 目标是，我们将对 DCM 相关 TTN 截短变异的病理生理学获得重要的新见解， 揭示特征来解释 DCM 患者中发现的可变致病性，并开发一种治疗方法 直接定位 TTN。我们预计这一新知识将提高医生诊断、预测、 并治疗因 TTN 变异而导致的扩张型心肌病 (DCM) 患者。

PROJECT SUMMARY/ABSTRACT Cardiomyopathies occur in ~1:200 individuals and are commonly caused by inheritance of variants in genes that encode proteins that regulate the sarcomere, the force-producing organelle of heart cells. Due to an incomplete understanding of variant pathogenicity and cardiomyopathy pathogenesis, physicians are currently limited in their ability to provide diagnoses, prognoses, and therapeutic options for cardiomyopathy patients. Variants in the TTN gene, which encodes the sarcomere protein titin, are the most frequently identified genetic lesion in dilated cardiomyopathy (DCM), which is characterized by heart chamber dilation, reduced contractile function, risk of sudden death, and progressive heart failure. The most frequent type of TTN variant identified in DCM is a truncation mutation that would be predicted to shorten TTN protein length and to reduce TTN protein quantities. Significantly, truncation variants localized to distal TTN structural domains are more pathogenic than those localized to proximal structural domains, but the mechanistic basis for this relationship is uncertain. It remains incompletely understood how TTN truncation variants cause DCM generally, which is compounded by our lack of understanding of the ‘length dependence’ of TTN variant pathogenicity. These knowledge gaps limit disease prognostication, biomarker identification, and therapeutic development for DCM patients. The central goal of our study is to define how disruptions in TTN length and dosage by TTN variants cause DCM, and exploit this knowledge to develop DCM therapeutics for TTN variant carriers. We hypothesize that healthy cardiac contractile function and structure depends on the regulation of TTN length and dosage, and that varying pathogenicity of TTN truncation can be explained by distinct structural and functional consequences associated with the specific site of truncation. In Aim 1, we will determine the functional consequences of TTN truncations across structural domains by harnessing 3-dimensional heart tissue models composed of human cardiomyocytes differentiated from induced pluripotent stem cells in which variants have been introduced by CRISPR-mediated genome editing. We will interrogate these models for tissue mechanical phenotypes (such as passive tension and Frank-Starling behavior), TTN protein length and levels (using specialized methods), proteostasis stress pathway responses (using immunoblotting), and mechanotransduction signaling and alternative splicing (using expression analysis and transcriptomics, respectively). In Aim 2, we will restore TTN protein levels using the recently developed method of CRISPR activation applied to DCM engineered heart tissue models for both evaluating the function of TTN isoforms generally and as a DCM proof-of-concept therapeutic. Through these Aims, we will gain critical new insights into the pathophysiology of DCM-associated TTN truncation variants, uncover features to explain the variable pathogenicity identified in DCM patients, and develop a therapeutic to target TTN directly. We anticipate this new knowledge will improve physicians’ capacity to diagnose, prognose, and treat patients with DCM due to TTN variants.