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Establishing and reversing the functional consequences of Titin truncation mutations

建立并逆转肌联蛋白截断突变的功能后果

基本信息

批准号：
10640157
负责人：
STUART G CAMPBELL
金额：
$ 52.91万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10640157
关键词：
3-Dimensional Adrenergic Agents Agar Gel Electrophoresis Alternative Splicing Base Pairing Behavior Biological Assay Biology Biomimetics CRISPR-mediated transcriptional activation CRISPR/Cas technology Cardiac Cardiac Myocytes Cardiomyopathies Cells Clustered Regularly Interspaced Short Palindromic Repeats Communities Computing Methodologies DNA Data Dependence Development Diagnosis Dilated Cardiomyopathy Disease Distal Dose Extracellular Matrix Fibroblasts Functional disorder Future Genes Genetic Goals Guide RNA Heart Heart failure Heterozygote Human Individual Kinetics Knowledge Learning Length Lesion Link Location Measures Mechanics Mediating Methods Modeling Molecular Mus Mutation Myocardial Contraction Myocardium Nonmuscle Myosin Type IIA Organelles Pathogenesis Pathogenicity Pathologic Pathway interactions Patients Phenotype Physicians Physiological Prognosis Protein Isoforms Proteins Publications RNA Binding Reagent Regulation Regulatory Element Research Resources Risk Sarcomeres Series Signal Transduction Site Stress Structure Sturnus vulgaris Sudden Death Technology Therapeutic Tissue Engineering Tissue Model Tissue-Specific Gene Expression Transcription Coactivator Variant Western Blotting Work adverse outcome biological adaptation to stress biomarker development biomarker identification cardiac tissue engineering cohort confocal imaging connectin disease prognosis dosage genome editing heart cell heart dimension/size heart function high risk human model improved induced pluripotent stem cell induced pluripotent stem cell derived cardiomyocytes inherited cardiomyopathy insight mechanotransduction mutation carrier prevent promoter proteostasis response scaffold sudden cardiac death therapeutic development therapeutic target transcriptomics

项目摘要

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.

项目总结/文摘