Imanaka-Yoshida K, Sanger J M, Sanger J W
Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia 19104-6058.
Cell Motil Cytoskeleton. 1993;26(4):301-12. doi: 10.1002/cm.970260405.
The purpose of this study was to determine how quickly contractile proteins are incorporated into the myofibrils of freshly isolated cardiomyocytes and to determine whether there are regions of the cells that are more dynamic than others in their ability to incorporate the proteins. Paired cardiomyocytes joined at intercalated discs and single cells were isolated from adult rats, and microinjected 3 hours later with fluorescently labeled actin, alpha-actinin, myosin light chains and vinculin. The cells were fixed and permeabilized at various period, 5 seconds and longer, after microinjection. Actin became incorporated throughout the I-Bands in as short a time as 5 seconds. The free edges of the cells, which were formerly intercalated discs, exhibited concentrations of actin greater than that incorporated in the I-Bands. This extra concentration of actin was not detected, however, at intact intercalated discs connecting paired cells. Alpha-actinin was incorporated immediately into Z-Bands and intercalated discs. Vinculin, also, was localized at the Z-Bands and at intercalated discs, but in contrast to alpha-actinin, there was a higher concentration of vinculin in the region of the intact intercalated discs. Both alpha-actinin and vinculin were concentrated at the free ends of the cells that were formerly parts of intercalated discs. Myosin light chains were observed to incorporate into the A-Bands in periods as short as 5 seconds. These results suggest that the myofibrils of adult cardiomyocytes may be capable of rapid isoform transitions along the length of the myofibrils. The rapid accumulation of fluorescent actin, alpha-actinin, and vinculin in membrane sites that were previously parts of intercalated discs, may reflect the response to locomotory activity that is initiated in these areas as cells spread in culture. A similar response after an injury in the intact heart could allow repair to occur.
本研究的目的是确定收缩蛋白多快能被整合到新分离的心肌细胞的肌原纤维中，并确定细胞中是否存在某些区域，在整合这些蛋白的能力方面比其他区域更具动态性。从成年大鼠中分离出在闰盘处相连的成对心肌细胞和单个细胞，并在3小时后用荧光标记的肌动蛋白、α-辅肌动蛋白、肌球蛋白轻链和纽蛋白进行显微注射。在显微注射后的不同时间段（5秒及更长时间）对细胞进行固定和通透处理。肌动蛋白在短短5秒内就整合到整个I带中。细胞以前是闰盘的自由边缘，其肌动蛋白浓度高于整合到I带中的浓度。然而，在连接成对细胞的完整闰盘处未检测到这种额外的肌动蛋白浓度。α-辅肌动蛋白立即整合到Z带和闰盘中。纽蛋白也定位于Z带和闰盘，但与α-辅肌动蛋白不同，在完整闰盘区域纽蛋白浓度更高。α-辅肌动蛋白和纽蛋白都集中在以前是闰盘一部分的细胞自由端。观察到肌球蛋白轻链在短短5秒内就整合到A带中。这些结果表明，成年心肌细胞的肌原纤维可能能够沿肌原纤维长度进行快速的同工型转变。荧光肌动蛋白、α-辅肌动蛋白和纽蛋白在以前是闰盘一部分的膜位点中的快速积累，可能反映了细胞在培养中铺展时在这些区域引发的对运动活性的反应。完整心脏受伤后的类似反应可能会促进修复。
