Dysregulated mechanosignaling in dilated cardiomyopathy caused by defective Filamin C

项目摘要/摘要 扩张型心肌病(DCM)是家族性心脏病的一种常见且致命的形式，典型的 以不利的细胞和心室重塑和收缩功能障碍为特征。DCM通常与 编码肌瘤或细胞骨架蛋白的基因发生功能丧失突变。机械传动和 心肌细胞(CMS)中的机械信号依赖于这些蛋白质网络，特别是在胞浆中， 在细胞外基质(ECM)和肌节的Z盘之间提供直接的机械连接。这个 因此，胞壁可能调节由内而外的机械传递(传递肌节所产生的力量 Out to the ECM)和‘Outside-In’机械信号(传输/转导细胞外机械信号 进入CM)-其中任何一个的功能障碍可能是DCM进展的中心。我的总体假设是 心肌细胞的胞间和皮质细胞骨架提供了关键的机械敏感蛋白质网络 调节由细胞内力和细胞外力启动的机械信号通路，以及特定的 这些结构中的缺陷抑制了它们传递和传递机械力的能力，导致收缩 功能障碍和病理性细胞重塑。支持这一点的是，胞间蛋白质丝状C(Flnc)具有 最近被认为与多种人类心肌病有关，包括扩张型心肌病。在我的F32博士后期间 在训练中，我使用了一种新的小鼠模型，它利用心脏特异的和可诱导的纯合子Flc缺失来 引发DCM的快速发展。我发现FLNC的丢失会导致组织和细胞的显著减少- 水平收缩能力，以及伴随皮质细胞骨架减少的显著CM重塑 僵硬。然而，在患有扩张型心肌病的人类中，FlnC突变是否会导致类似的皮质结构和 力学、收缩力学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将量化的亚细胞级别的结构和 生物力学测量和细胞内应力分布的定量测量 肥大基因表达模式对细胞内外机械扰动的响应 利用人诱导多能干细胞来源的心肌细胞(hiPSC-CMS)表达患者- 特定的Flnc-截断突变。要做到这一点，我将：(1)结合X射线衍射成像，原子 力显微镜和多尺度计算模型来测试损失的FLNC破坏的假设 通过改变肌丝晶格结构来实现肌肌力的“由内而外”的机械传递 小鼠CMS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 人DCM模型由表达患者特异性FlnC截断变异体的基因编辑的hiPSC-CMS制成， (3)将基于FRET的分子张力传感器成像、体外细胞外机械载荷 技术和定量转录学，以检验截断FLNC功能失调的假说。 In‘在HiPSC-CMS中的机械信号转导，促进DCM重塑。

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.