Narshi Aruna, Denz Christopher R, Nakatsugawa Masako, Zajdel Robert W, Dube Syamalima, Poiesz Bernard J, Dube Dipak K
Department of Medicine, SUNY Upstate Medical University, Syracuse, NY 13210, USA.
Cardiovasc Toxicol. 2005;5(1):1-8. doi: 10.1385/ct:5:1:001.
Although the role of tropomyosin is well-defined in striated muscle, the precise mechanism of how tropomyosin functions is still unclear. It has been shown that extension of either N- or C-terminal ends of sarcomeric tropomyosin do not affect cardiac myofibrillogenesis, but it is not known whether simultaneous extension of both ends affects the process. For studying structural/functional relationships of sarcomeric tropomyosin, we have chosen the Ambystoma mexicanum because cardiac mutant hearts are deficient in sarcomeric tropomyosin. In this study, we have made an expression construct, pEGFP.TPM4alpha.E-L-FLAG, that, on transfection into normal and mutant axolotl hearts in organ culture, expresses GFP.TPM4alpha.E-L-FLAG fusion protein in which both the N- and C-termini of TPM4alpha are being extended. TPM4alpha is one of the three tropomyosins expressed in normal axolotl hearts. Both confocal and electron microscopic analyses show that this modified sarcomeric tropomyosin can form organized myofibrils in axolotl hearts.
尽管原肌球蛋白在横纹肌中的作用已得到明确界定，但其发挥功能的精确机制仍不清楚。研究表明，肌节原肌球蛋白的N端或C端的延伸均不影响心肌纤维生成，但尚不清楚两端同时延伸是否会影响这一过程。为了研究肌节原肌球蛋白的结构/功能关系，我们选择了墨西哥钝口螈，因为其心脏突变体缺乏肌节原肌球蛋白。在本研究中，我们构建了一个表达载体pEGFP.TPM4alpha.E-L-FLAG，将其转染到器官培养的正常和突变蝾螈心脏中后，可表达GFP.TPM4alpha.E-L-FLAG融合蛋白，其中TPM4alpha的N端和C端均得到了延伸。TPM4alpha是正常蝾螈心脏中表达的三种原肌球蛋白之一。共聚焦显微镜和电子显微镜分析均表明，这种修饰后的肌节原肌球蛋白能够在蝾螈心脏中形成有组织的肌原纤维。
