Dysregulated mechanosignaling in dilated cardiomyopathy caused by defective Filamin C

项目总结/摘要 家族性心脏病的一种常见且致命的形式是扩张型心肌病（DCM），其典型表现为： 其特征在于不利的细胞和心室重塑以及收缩功能障碍。DCM通常与 在编码肌节或细胞骨架蛋白的基因中存在功能缺失突变。机械传动和 心肌细胞（CM）中的机械信号依赖于这些蛋白质网络，特别是在肋节中， 在细胞外基质（ECM）和肌节的Z盘之间提供直接的机械连接。的 因此，肋节可以调节“由内而外”的机械传递（传递肌节产生的力 输出到ECM）和“由外向内”机械信号传导（传递/转导细胞外机械信号 进入CM）-其中任一者的功能障碍可能是DCM进展的中心。我的总体假设是 心肌细胞的肋节和皮层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引发的机械信号传导途径， 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，costamere蛋白细丝蛋白C（FLNC）具有 最近被认为与包括DCM在内的多种人类心肌病有关。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，该模型利用心脏特异性和可诱导的纯合FLNC缺失， 引发扩张型心肌病的快速发展我发现FLNC的缺失会导致组织和细胞的显著减少 水平的收缩力，以及伴随皮质细胞骨架减少的显著CM重塑 刚度然而，患有DCM的人的FLNC突变是否会导致皮质结构的类似缺陷， 力学、收缩机械传递和机械敏感性基因调控需要进一步研究。 因此，我提出的研究目标是整合定量亚细胞水平的结构和 生物力学测量，定量测量细胞内应力分布， 细胞内和细胞外机械扰动对肥大基因表达的影响 使用表达患者的人诱导多能干细胞衍生的心肌细胞（hiPSC-CM）， 特异性FLNC截短突变。为了实现这一点，我将：（1）结合联合收割机X射线衍射成像，原子 力显微镜和多尺度计算模型来检验FLNC的损失破坏了 通过改变肌丝晶格几何结构失调的肌节力的“由内而外”机械传递 小鼠CM的皮质细胞骨架力学，（2）将这些生物物理学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的hiPSC-CM制成的人DCM模型， （3）结合基于联合收割机的FRET分子张力传感器成像、体外细胞外机械负荷 技术和定量转录组学来检验截短的FLNC失调的假设， 在hiPSC-CM中的机械信号传导并促进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.