Familial Hypertrophic Cardiomyopathy-related myosin mutations. Functional effects studied by single molecule approaches

β-心肌肌球蛋白重链(β-MHC)点突变是人类家族性肥厚型心肌病(FHC)最常见的原因。虽然在肌球蛋白头部结构域和其他肌瘤蛋白中发现了大量不同的突变，但对常见FHC表型的致病途径尚不清楚。对于肌球蛋白头部区域的FHC突变，我们假设它们诱导了运动活动的变化，这是FHC表型发展的触发因素。因此，可以想象，选择性地纠正突变肌球蛋白头部功能行为的主要变化可能会阻止明显的FHC表型的发展。我们提出的研究的主要目标是全面了解肌球蛋白头部因点突变而改变的通信通路和功能域。该项目旨在通过研究FHC突变对运动特性的特定影响，如ATPase动力学、行程大小、力量产生和肌动蛋白细丝的运动，来表征由FHC突变引起的β-MHC头部区域的功能变化。例如，我们小组以前的工作报告说，受影响细胞对某些突变的钙敏感性降低，而转换域中的其他FHC突变由于头部僵硬增加而增加了收缩力量。这说明有必要区分FHC突变对肌球蛋白功能的广泛不同影响。大多数FHC患者同时产生突变型和野生型β-MHC。使用单分子技术，我们可以逐个分子地探测样品，从而即使在混合样品中也能分别研究野生型和突变型分子。我们通过全内反射荧光显微镜(TIRFM)和零模波导(ZMWG)研究了Cy3-EDA-ATP驻留和等待时间对ATPase动力学的影响。我们使用β-MHC单个头域的光学陷阱来直接测量难以从纤维或细胞中得出的参数，如力产生、行程大小和弹性变形阻力(硬度)。为了克服从患者/捐赠者活检中获得有限的天然蛋白的限制，我们在人类心肌细胞中产生了带有FHC突变的β-MHC头部结构域。建立这种新的表达系统的主要优点是肌球蛋白重链与其内源性辅助蛋白(必需的和调节的轻链)的正确组成，这对于精确比较天然的β-MHC和重组的对应物是至关重要的。此外，还将在心肌细胞中产生合理设计的突变蛋白，以进一步阐明通讯途径及其对肌球蛋白马达中化学-机械耦合的重要性。

Point mutations in the beta-cardiac myosin heavy chain (beta-MHC) are the most frequent cause of Familial Hypertrophic Cardiomyopathy (FHC) in humans. While a large number of different mutations were identified in the myosin head domain and other sarcomeric proteins, the pathogenic path to a common FHC-phenotype is not understood. For FHC mutations in the myosin head domain we hypothesize that they induce changes in motor activity that act as the trigger for the development of the FHC-phenotype. Thus, it is conceivable that selective rectification of the primary change in the functional behaviour of mutant myosin heads may prevent the development of a noticeable FHC-phenotype. The main goal of our proposed research is to gain comprehensive understanding of functional domains and altered communication pathways in the myosin head, as a consequence of point mutations. The project aims to characterize functional changes in the beta-MHC head domain, induced by FHC-mutations, by investigating specific effects of FHC mutations on motor properties, such as ATPase kinetics, stroke size, force production, and movement of actin filaments. Previous work from our group reported e.g., reduced calcium sensitivity of affected cells for some mutations while other FHC-mutations in the converter domain increased contractile forces due to increased head stiffness. This illustrates the need to distinguish the widely varying effects of FHC mutations on myosin function. Most FHC-patients produce both mutant and wild-type beta-MHC. Using single molecule techniques, we can probe samples, molecule by molecule, and thus study wildtype and mutant molecules separately even in mixed samples. We investigate ATPase kinetics via Cy3-EDA-ATP dwell- and waiting-times by total internal reflection fluorescence microscopy (TIRFM) and zero mode waveguide (ZMWG) based assay. We use optical trapping of beta-MHC individual head domains to directly measure parameters that are difficult to derive from fibres or cells, such as force generation, stroke size, and resistance to elastic deformation (stiffness). To overcome restrictions due to the limited availability of native protein from patient/donor biopsies, we produce the beta-MHC head domain with FHC-mutations in human cardiomyocytes. The major advantage of establishing this new expression system is the correct composition of myosin heavy chain with its endogenous accessory proteins (essential and regulatory light chains), which is vital for precise comparison of the native beta-MHC function with the recombinant counterpart. Additionally, rationally designed mutant proteins will be produced in cardiomyocytes to further elucidate communication pathways and their importance for chemo-mechanical coupling in the myosin motor.