Dysregulated mechanosignaling in dilated cardiomyopathy caused by defective Filamin C

Filamin C 缺陷引起的扩张型心肌病的机械信号失调

基本信息

批准号：
10877387
负责人：
JOSEPH D. POWERS
金额：
$ 24.9万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10877387
关键词：
Address Atomic Force Microscopy Attenuated Biological Markers Biophysics Cardiac Cardiac Myocytes Cardiomyopathies Cells Clinical Cytoskeletal Proteins Cytoskeleton Defect Development Diagnosis Diastole Dilated Cardiomyopathy Equilibrium Extracellular Matrix Fibroblasts Fluorescence Resonance Energy Transfer Frameshift Mutation Functional disorder Gene Expression Gene Expression Profile Gene Expression Regulation Gene Targeting Genes Genetic Geometry Goals Heart Heart Diseases Heart failure Heterozygote Homologous Gene Human Human Pathology Hypertrophy Image In Vitro Investigation Knock-out Knockout Mice Length Life Link Maintenance Measurement Mechanics Mediating Mentors Microfilaments Modeling Molecular Mus Mutation Myocardium Nonsense Mutation Pathogenicity Pathologic Pathway interactions Patients Phase Phenotype Play Proteins Reporting Research Role Sarcomeres Signal Pathway Stimulus Stress Structural defect Structure Subcellular structure Systole Techniques Testing Tissues Translating Variant Ventricular Ventricular Remodeling X ray diffraction analysis biomechanical test biophysical techniques design extracellular filamin functional loss heart cell in vitro Model induced pluripotent stem cell derived cardiomyocytes loss of function mutation mechanical force mechanical load mechanical signal mechanical stimulus mouse model multi-scale modeling novel novel therapeutics post-doctoral training prevent response sensor tool transcriptomics transmission process ventricular hypertrophy

项目摘要

PROJECT SUMMARY / ABSTRACT A common and deadly form of familial heart disease is dilated cardiomyopathy (DCM), which is typically characterized by adverse cellular and ventricular remodeling and systolic dysfunction. DCM is often associated with loss-of-function mutations in genes encoding sarcomeric or cytoskeletal proteins. Mechanotransmission and mechanosignaling in cardiomyocytes (CMs) rely on these protein networks, particularly in the costamere, which provides a direct mechanical link between the extracellular matrix (ECM) and the Z-disk of the sarcomere. The costamere may therefore regulate both ‘inside-out’ mechanotransmission (transmitting sarcomere-born forces out to the ECM) and ‘outside-in’ mechanosignaling (transmitting/transducing extracellular mechanical signals into the CM)—the dysfunction of either of which may be central to DCM progression. My overall hypothesis is that the costamere and cortical cytoskeleton of cardiomyocytes provide key mechanosensitive protein networks that regulate mechanical signalling pathways initiated by intracellular and extracellular forces, and that specific defects in these structures inhibits their ability to transmit and transduce mechanical forces, causing contractile dysfunction and pathological cell remodelling. Supporting this, the costamere protein Filamin C (FLNC) has recently been implicated in a variety of human cardiomyopathies, including DCM. During my F32 postdoctoral training, I used a new mouse model that exploits cardiac-specific and inducible homozygous FLNC deletion to trigger rapid DCM development. I found that a loss of FLNC causes significant reductions in the tissue- and cell- level contractility, as well as significant CM remodeling accompanied by a reduction in cortical cytoskeleton stiffness. However, whether FLNC mutations in humans with DCM cause similar defects in cortex structure and mechanics, systolic mechanotransmission, and mechanosensitive gene regulation requires further investigation. Thus, the goal of my proposed research is to integrate quantitative subcellular-level structural and biomechanical measurements with quantitative measurements of intracellular stress distributions and hypertrophic gene expression patterns in response to intra- and extra-cellular mechanical perturbations using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing a patient- specific FLNC-truncating mutation. To accomplish this, I will: (1) combine X-ray diffraction imaging, atomic force microscopy, and multiscale computational modeling to test the hypothesis that a loss of FLNC disrupts ‘inside-out’ mechanotransmission of sarcomeric forces by dysregulating myofilament lattice geometry via altered cortical cytoskeleton mechanics in murine CMs, (2) apply these biophysical methods and hypotheses to a new human DCM model made from gene-edited hiPSC-CMs expressing a patient-specific FLNC-truncating variant, and (3) combine FRET-based molecular tension sensor imaging, in-vitro extracellular mechanical loading techniques, and quantitative transcriptomics to test the hypothesis that truncated FLNC dysregulates ‘outside- in’ mechanosignaling in hiPSC-CMs and promotes DCM remodeling.

项目概要/摘要扩张型心肌病 (DCM) 是一种常见且致命的家族性心脏病，通常是以不利的细胞和心室重塑为特征的DCM通常与收缩功能障碍有关。编码肌节或细胞骨架蛋白的基因发生功能丧失突变，以及心肌细胞 (CM) 中的机械信号传导依赖于这些蛋白质网络，特别是在肋节中，提供细胞外基质 (ECM) 和肌节 Z 盘之间的直接机械连接。因此，肋节可能调节“由内而外”的机械传递（传递肌节产生的力）到 ECM）和“由外向内”机械信号传递（传输/转换细胞外机械信号到 CM）——其中任何一个的功能障碍可能是 DCM 进展的核心。心肌细胞的肋节和皮质细胞骨架提供了关键的机械敏感蛋白网络调节由细胞内和细胞外力启动的机械信号传导途径，以及特定的这些结构中的缺陷抑制了它们传递和转换机械力的能力，导致收缩肋纤维蛋白 C (FLNC) 可以支持这一点。最近在我的 F32 博士后期间发现与多种人类心肌病有关，包括 DCM。在训练中，我使用了一种新的小鼠模型，该模型利用心脏特异性和可诱导的纯合 FLNC 缺失来我发现 FLNC 的缺失会导致组织和细胞的显着减少。水平收缩性，以及显着的 CM 重塑，并伴有皮质细胞骨架的减少然而，患有 DCM 的人的 FLNC 突变是否会导致皮质结构和僵硬的类似缺陷。力学、收缩性机械传递和机械敏感基因调控需要进一步研究。因此，我提出的研究的目标是整合定量的亚细胞水平结构和生物力学测量与细胞内应力分布的定量测量响应细胞内和细胞外机械扰动的肥大基因表达模式使用人类诱导多能干细胞衍生的心肌细胞（hiPSC-CM）表达患者- 为了实现这一目标，我将：（1）结合X射线衍射成像、原子分析。力显微镜和多尺度计算模型来检验 FLNC 丢失会破坏这一假设通过改变肌丝晶格几何形状失调来实现肌节力的“由内而外”机械传递小鼠 CM 中的皮质细胞骨架力学，(2) 将这些生物物理方法和假设应用于新的由表达患者特异性 FLNC 截短变体的基因编辑 hiPSC-CM 制成的人类 DCM 模型， (3)结合基于FRET的分子张力传感器成像、体外细胞外机械加载技术和定量转录组学来检验截短的 FLNC 失调“外部- hiPSC-CM 中的机械信号传导并促进 DCM 重塑。