Effect of Cardiomopathy Mutations on Myosin and Actin

β-心肌肌球蛋白重链和α-心肌肌动蛋白的点突变导致家族性肥厚型心肌病（FHC）或扩张型心肌病（DCM）。要达到一个明确的机制，这两种疾病的主要原因，我们建议进行广泛的动力学，机械和结构分析突变的心肌肌球蛋白和肌动蛋白。 目的#1将通过比较R403 Q，检查异构体骨架对功能的影响 通过在转基因小鼠中过表达获得的α-或β-鼠心肌肌球蛋白重链（MHC）同种型中的突变。突变的MHC将在N-末端被HIS标记，以便于通过金属螯合物亲和色谱法分离。突变肌球蛋白将通过稳态和瞬时动力学进行酶学表征，并通过测量速度和平均力进行机械表征。野生型和突变型肌球蛋白亚型之间的结构差异将通过基于计算机的拟合晶体结构到通过电子冷冻显微镜获得的肌动球蛋白复合物的3D重建进行研究。类似的分析将扩展到导致FHC（G741 R，R453 C）和DCM（S532和F764）的点突变。为了更好地理解人肌球蛋白点突变的功能后果，目标#2将分析从表达人β-心肌肌球蛋白（R403 Q）基因的转基因兔中分离的肌球蛋白。与此同时，将探讨几种新的策略，在体外表达的人β-心肌肌球蛋白，包括使用果蝇S2表达系统，并添加分子伴侣，以增加产量的横纹肌肌球蛋白亚型。目的#3将试图描述导致FHC或DCM的肌动蛋白点突变的影响。在杆状病毒/昆虫细胞系统中表达的α-心脏肌动蛋白使我们能够研究正确骨架中突变的影响，而不是目前使用的酵母肌动蛋白系统。肌动蛋白内在丝状结构的改变，以及肌动蛋白与肌球蛋白的相互作用，将通过许多与肌球蛋白突变相同的方法进行评估。该提案的总体目标是阐明FHC和DCM中涉及的突变如何影响肌球蛋白和肌动蛋白的机械性能，并确定在原发突变的效应和最终疾病表型之间是否存在任何相关性。

Point mutations in both the beta-cardiac myosin heavy chain and in alpha-cardiac actin lead to either familial hypertrophic cardiomyopathy (FHC) or dilated cardiomyopathy (DCM). To arrive at a definitive mechanism for the primary cause of these two diseases, we propose to undertake an extensive kinetic, mechanical, and structural analysis of mutated cardiac myosins and actins. Aim #1 will examine the effect of isoform backbone on function by comparing the R403Q mutation in an alpha- or beta-murine cardiac myosin heavy chain (MHC) isoform obtained by overexpression in transgenic mice. Mutated MHCs will be HIS-tagged at the N-terminus to facilitate isolation by metal chelate affinity chromatography. The mutant myosins will be characterized enzymatically by steady-state and transient kinetics, and mechanically by measurements of velocity and average force. Structural differences between wildtype and mutant myosin isoforms will be investigated by computer-based fitting of crystal structures into 3D-reconstructions of actomyosin complexes obtained by electron cryomicroscopy. Similar analyses will be extended to point mutations leading to FHC (G741R, R453C) and DCM (S532 and F764). For a better understanding of the functional consequences of a point mutation in human myosin, Aim #2 will analyze myosin isolated from transgenic rabbits that express a human beta-cardiac myosin (R403Q) gene. In parallel, several new strategies for expression of human beta-cardiac myosin in vitro will be explored, including use of the Drosophila S2 expression system, and the addition of chaperones to increase the yield of striated muscle myosin isoforms. Aim #3 will seek to characterize the effect of point mutations in actin that lead to FHC or DCM. alpha-cardiac actin expressed in the baculovirus/insect cell system allows us to investigate the effect of mutations in the correct backbone, rather than in the currently used yeast actin system. Alterations to actin's intrinsic filamentous structure, and to its interactions with myosin, will be assessed by many of he same approaches as described for the myosin mutations.The overall goal of the proposal is to elucidate how mutations implicated in FHC and in DCM affect the mechanical performance of myosin and actin, and to determine if any correlation can be made between the effect of the primary mutation and the ultimate disease phenotype.