Rhee D, Sanger J M, Sanger J W
Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia 19104-6058.
Cell Motil Cytoskeleton. 1994;28(1):1-24. doi: 10.1002/cm.970280102.
When cardiac muscle cells are isolated from embryonic chicks and grown in culture they attach to the substrate as spherical cells with disrupted myofibrils, and over several days in culture, they spread and extend lamellae. Based on antibody localizations of various cytoskeletal proteins within the spreading cardiomyocyte, three types of myofibrils have been identified: 1) fully formed mature myofibrils that are centrally positioned in the cell, 2) premyofibrils that are closest to the cell periphery, and 3) nascent myofibrils located between the premyofibrils and the mature myofibrils. Muscle-specific myosin is localized in the A-bands in the mature, contractile myofibrils, and along the nascent myofibrils in a continuous pattern, but it is absent from the premyofibrils. Antibodies to non-muscle isoforms of myosin IIB react with the premyofibrils at the cell periphery and with the nascent myofibrils, revealing short bands of myosin between closely spaced bands of alpha-actinin. In the areas where the nascent myofibrils border on the mature myofibrils, the bands of non-muscle myosin II reach lengths matching the lengths of the mature A-bands. With the exception of a small transition zone consisting of one myofibril, or sometimes several sarcomeres, bordering the nascent myofibrils, there is no reaction of these non-muscle myosin IIB antibodies with the mature myofibrils in spreading myocytes. C-protein is found only in the mature myofibrils, and its presence there may prevent co-polymerization of non-muscle and muscle myosins. Antibodies directed against the non-muscle myosin isoforms, IIA, do not stain the cardiomyocytes. In contrast to the cardiomyocytes, the fibroblasts in these cultures stain with antibodies to both non-muscle myosin IIA and IIB. The premyofibrils near the leading edge of the lamellae show no reaction with antibodies to either titin or zeugmatin, whereas the nascent myofibrils and mature myofibrils do. The spacings of the banded alpha-actinin staining range from 0.3 to 1.4 microns in the pre- and nascent myofibrils and reach full spacings (1.8-2.5 microns) in the mature myofibrils. Based on these observations, we propose a premyofibril model in which non-muscle myosin IIB, titin, and zeugmatin play key roles in myofibrillogenesis. This model proposes that pre- and nascent myofibrils are composed of minisarcomeres that increase in length, presumably by the concurrent elongation of actin filaments, the loss of the non-muscle myosin II filaments, the fusion of dense bodies or Z-bodies to form wide Z-bands, and the capture and alignment of muscle myosin II filaments to form the full spacings of mature myofibrils.
从胚胎小鸡中分离出心肌细胞并在培养中生长时，它们会以肌原纤维被破坏的球形细胞形式附着于底物上，并且在培养的数天时间里，它们会铺展并伸出片状伪足。基于对铺展心肌细胞内各种细胞骨架蛋白的抗体定位，已鉴定出三种类型的肌原纤维：1）完全形成的成熟肌原纤维，位于细胞中央；2）最靠近细胞周边的前肌原纤维；3）位于前肌原纤维和成熟肌原纤维之间的新生肌原纤维。肌肉特异性肌球蛋白定位于成熟的收缩性肌原纤维的A带中，并以连续模式沿着新生肌原纤维分布，但在前肌原纤维中不存在。针对肌球蛋白IIB非肌肉同工型的抗体与细胞周边的前肌原纤维以及新生肌原纤维发生反应，在紧密排列的α - 辅肌动蛋白带之间显示出肌球蛋白的短带。在新生肌原纤维与成熟肌原纤维接壤的区域，非肌肉肌球蛋白II的带达到与成熟A带长度匹配的长度。除了由一条肌原纤维或有时几条肌节组成的与新生肌原纤维接壤的小过渡区外，这些非肌肉肌球蛋白IIB抗体在铺展的心肌细胞中与成熟肌原纤维无反应。C蛋白仅在成熟肌原纤维中发现，其在那里的存在可能会阻止非肌肉和肌肉肌球蛋白的共聚。针对非肌肉肌球蛋白同工型IIA的抗体不会使心肌细胞染色。与心肌细胞不同，这些培养物中的成纤维细胞用针对非肌肉肌球蛋白IIA和IIB的抗体染色。片状伪足前沿附近的前肌原纤维与肌联蛋白或肌联丝蛋白的抗体无反应，而新生肌原纤维和成熟肌原纤维则有反应。在前肌原纤维和新生肌原纤维中，带状α - 辅肌动蛋白染色的间距范围为0.3至1.4微米，在成熟肌原纤维中达到完整间距（1.8 - 2.5微米）。基于这些观察结果，我们提出了一个前肌原纤维模型，其中非肌肉肌球蛋白IIB、肌联蛋白和肌联丝蛋白在肌原纤维形成中起关键作用。该模型提出，前肌原纤维和新生肌原纤维由微小肌节组成，其长度增加，推测是通过肌动蛋白丝的同时伸长、非肌肉肌球蛋白II丝的丢失、致密体或Z体融合形成宽Z带以及肌肉肌球蛋白II丝的捕获和排列以形成成熟肌原纤维的完整间距。
