Biomechanical characterization of striated muscle cells from R155C VCP knock-in and W2710X filamin C knock-in mice: a novel approach to understand the pathogenesis of myofibrillar myopathies

肌原纤维肌病(MFMs)是一组散发性和遗传性骨骼肌和心肌疾病，可导致严重的身体残疾和过早死亡。MFMS是由编码结蛋白、细丝蛋白C、粘附素、VCP、FHL1、ZASP、myotilin、α-B晶体蛋白和BAG3的基因突变引起的。首先，我们对原代人成肌细胞进行的生物力学研究结果表明，与对照细胞相比，携带结蛋白和粘附素突变的原代人成肌细胞具有更高的机械易损性，从而增加了硬度，降低了机械应力耐受性。我们假设，突变细胞的较高硬度导致生理拉伸和剪切变形时较高的细胞内应力，这反过来又会触发肌肉纤维退化。在本项目中，我们将使用从两个MFM小鼠模型中获得的永生化成肌细胞来验证这一假设。通过DFG研究联盟FOR1228，我们获得了两个敲入小鼠模型(R155C VCP和W2710X微丝C)，它们含有最常见的人类致病VCP和微丝C突变。使用牵引力显微镜、磁钳微观流变学和细胞拉伸器以及高分辨率(时间和空间)共聚焦显微镜，我们将解决两个关键问题：(I)这些突变对培养的成肌细胞和来源于骨骼肌组织的肌管的生物力学功能有什么影响；(Ii)导致这些细胞机械应激耐受性改变的分子过程是什么。该项目将提供对VCP和细丝素C相关肌病发病机制的生物力学方面的第一次洞察。

Myofibrillar myopathies (MFMs) are a group of sporadic and hereditary skeletal and cardiac muscle diseases that lead to severe physical disability and premature death. MFMs are caused by mutations in genes encoding desmin, filamin C, plectin, VCP, FHL1, ZASP, myotilin, alpha-B-crystallin, and BAG3. First results from our biomechanical studies on primary human myoblasts carrying desmin -and plectin mutations showed an increased stiffness and reduced mechanical stress tolerance in the form of higher mechanical vulnerability compared to control cells. We hypothesize that the higher stiffness of mutant cells leads to higher intracellular stress at physiologic stretch and shear deformations, which in turn triggers muscle fiber degeneration. In the present project, we will test this hypothesis using immortalized myoblast cells obtained from two MFM mouse models. Through the DFG research consortium FOR1228, we have access to two knock-in mouse models (R155C VCP, and W2710X filamin C), which harbor the most frequent human pathogenic VCP and filamin C mutations. Using traction force microscopy, magnetic tweezer microrheology, and a cell stretcher together with high resolution (temporal and spatial) confocal microscopy, we will address two key questions: (i) what is the influence of these mutations on the biomechanical function of cultured myoblasts and myotubes derived from skeletal muscle tissue, and (ii) what are the molecular processes that lead to altered mechanical stress tolerance in these cells. This project will provide the first insight into the biomechanical aspects of the pathogenesis of VCP- and filamin C-related myopathies.