Establishing and reversing the functional consequences of Titin truncation mutations

项目摘要/摘要 心肌病发生在大约1：200人中，通常是由以下基因变异引起的 编码调节肌节的蛋白质，肌节是心脏细胞的产生力量的细胞器。由于一个不完整的 对变异致病机制和心肌病发病机制的了解，目前医生仅限于 他们为心肌病患者提供诊断、预后和治疗选择的能力。中的变体 TTN基因编码肌节蛋白titin，是人类最常见的遗传损伤 扩张型心肌病(DCM)，其特征是心腔扩张，收缩功能降低， 有猝死和进行性心力衰竭的风险。在DCM中发现的最常见的TTN变异类型是 一种截断突变，预计会缩短TTN蛋白长度，减少TTN蛋白数量。 值得注意的是，定位于TTN远端结构域的截断变体比那些 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截断变量是如何导致DCM的，这一点由于我们缺乏 了解TTN变异体致病的“长度依赖性”。这些知识差距限制了疾病 扩张型心肌病患者的预后、生物标记物识别和治疗进展。的中心目标是 我们的研究是定义TTN变种对TTN长度和剂量的干扰如何导致DCM，并利用这一点 对TTN变异型携带者开发扩张型心肌病疗法的知识。我们假设健康的心脏 收缩功能和结构依赖于TTN的长度和剂量的调节，而这种变化 TTN截断的致病性可以用不同的结构和功能后果来解释 带有截断的特定位置。在目标1中，我们将确定TTN截断的功能后果 利用由人心肌细胞组成的三维心脏组织模型跨越结构域 CRISPR介导的变异体诱导多能干细胞分化 基因组编辑。我们将询问这些模型的组织机械表型(如被动张力 和Frank-Starling行为)、TTN蛋白长度和水平(使用特殊方法)、蛋白稳定应激 途径反应(使用免疫印迹)、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 新开发的CRISPR激活方法在DCM工程心脏组织模型中的应用 评估TTN异构体的一般功能和作为DCM概念验证治疗的作用。通过这些 目的，我们将获得对DCM相关TTN截断变体的病理生理学的关键的新见解， 揭示在扩张型心肌病患者中发现的不同致病性的特征，并开发一种治疗方法 直接瞄准TTN。我们预计这一新知识将提高医生的诊断、预后、 以及治疗由于TTN变异而导致的扩张型心肌病患者。

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.