Wang Qun, Moncman Carole L, Winkelmann Donald A
Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
J Cell Sci. 2003 Oct 15;116(Pt 20):4227-38. doi: 10.1242/jcs.00709. Epub 2003 Sep 2.
We have investigated the functional impact on cardiac myofibril organization and myosin motor activity of point mutations associated with familial hypertrophic cardiomyopathies (FHC). Embryonic chicken cardiomyocytes were transfected with vectors encoding green fluorescent protein (GFP) fused to a striated muscle myosin heavy chain (GFP-myosin). Within 24 hours of transfection, the GFP-myosin is found co-assembled with the endogenous myosin in striated myofibrils. The wild-type GFP-myosin had no effect on the organization of the contractile cytoskeleton of the cardiomyocytes. However, expression of myosin with the R403Q FHC mutation resulted in a small but significant decrease in myofibril organization, and the R453C and G584R mutations caused a more dramatic increase in myofibril disarray. The embryonic cardiomyocytes beat spontaneously in culture and this was not affected by expression of the wild-type or mutant GFP-myosin. For the biochemical analysis of myosin motor activity, replication defective adenovirus was used to express the wild-type and mutant GFP-myosin in C2C12 myotubes. The R403Q mutation enhanced actin filament velocity but had no effect on the myosin duty ratio. The R453C and G584R mutations impaired actin filament movement and both increased the duty ratio. The effects of these mutations on myosin motor activity correlate with changes in myofibril organization of live cardiomyocytes. Thus, mutations associated with hypertrophic cardiomyopathies that alter myosin motor activity can also impair myofibril organization.
我们研究了与家族性肥厚型心肌病（FHC）相关的点突变对心肌肌原纤维组织和肌球蛋白运动活性的功能影响。将编码与横纹肌肌球蛋白重链融合的绿色荧光蛋白（GFP）的载体转染到胚胎鸡心肌细胞中。转染后24小时内，发现GFP-肌球蛋白与内源性肌球蛋白在横纹肌原纤维中共组装。野生型GFP-肌球蛋白对心肌细胞收缩细胞骨架的组织没有影响。然而，具有R403Q FHC突变的肌球蛋白表达导致肌原纤维组织略有但显著减少，而R453C和G584R突变导致肌原纤维紊乱更显著增加。胚胎心肌细胞在培养物中自发搏动，这不受野生型或突变型GFP-肌球蛋白表达的影响。为了对肌球蛋白运动活性进行生化分析，使用复制缺陷型腺病毒在C2C12肌管中表达野生型和突变型GFP-肌球蛋白。R403Q突变提高了肌动蛋白丝速度，但对肌球蛋白工作比没有影响。R453C和G584R突变损害了肌动蛋白丝的运动，并都增加了工作比。这些突变对肌球蛋白运动活性的影响与活心肌细胞肌原纤维组织的变化相关。因此，与肥厚型心肌病相关的改变肌球蛋白运动活性的突变也会损害肌原纤维组织。
