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

β -心肌肌球蛋白重链（β - mhc）的点突变是人类家族性肥厚性心肌病（FHC）的最常见原因。虽然在肌凝蛋白头部结构域和其他肉瘤蛋白中发现了大量不同的突变，但常见的fhc表型的致病途径尚不清楚。对于肌凝蛋白头部结构域的FHC突变，我们假设它们诱导运动活动的变化，作为FHC表型发展的触发因素。因此，可以想象的是，选择性纠正突变型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凝蛋白头部的功能域和由于点突变而改变的通信途径。该项目旨在通过研究FHC突变对运动特性的特定影响，如atp酶动力学、中风大小、力量产生和肌动蛋白丝的运动，来表征由FHC突变诱导的β - mhc头部结构域的功能变化。我们小组之前的研究报告，例如，一些突变降低了受影响细胞的钙敏感性，而转换结构域的其他fhc突变由于头部刚度增加而增加了收缩力。这说明需要区分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。利用单分子技术，我们可以一个分子一个分子地探测样品，从而在混合样品中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）检测，通过Cy3-EDA-ATP停留和等待时间来研究atp酶动力学。我们使用β - mhc个体头部区域的光学捕获来直接测量难以从纤维或细胞中获得的参数，例如力产生，笔划大小和抗弹性变形（刚度）。为了克服患者/供体活检中天然蛋白有限的限制，我们在人心肌细胞中产生了具有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及其内源性辅助蛋白（必需和调节轻链）的正确组成，这对于精确比较天然β - mhc与重组对应物的功能至关重要。此外，合理设计的突变蛋白将在心肌细胞中产生，以进一步阐明通信途径及其在肌球蛋白运动中化学-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.