Gourdie R G, Green C R, Severs N J
Department of Anatomy and Developmental Biology, University College London, UK.
J Cell Sci. 1991 May;99 ( Pt 1):41-55. doi: 10.1242/jcs.99.1.41.
A polyclonal antiserum, raised against a synthetic peptide matching part of the sequence of connexin43 (a rat cardiac gap-junctional protein), was used in combination with laser scanning confocal microscopy to investigate gap junction distribution in cardiac tissues from a range of mammalian species. Comparison of the localised punctate staining patterns obtained in ventricular tissue with the distribution of intercalated disks as viewed by conventional light microscopy and electron microscopy, and with the staining observed by standard light-microscope immunofluorescence using the same anti-serum, demonstrated highly specific labelling of clearly resolved individual gap junctions. Laser scanning confocal microscopy of ventricular myocardium showed the immunostained gap junctions to be confined to well-defined intercalated disks bisecting the long axis of the muscle fibre, whereas in the atrial myocardium, gap junctions were commonly distributed widely over the lateral surfaces of the myocyte body. Rat atrial gap junctions were significantly larger (as measured by the longest axial lengths of fluorescent spots), and showed a narrower spread of sizes, than their counterparts in the ventricle. Ventricular myocardium from six mammalian species including man gave similar immunostaining patterns, indicating conservation both of the epitope(s) detected by the antiserum, and of the general organisation of the cell-to-cell pathways for electrical propagation, in the mammalian heart. Optical section series obtained by laser scanning confocal microscopy permitted the quantification and mapping of the three-dimensional distribution of gap junctions in ventricular intercalated disks with high clarity over substantial specimen depths. A consistent feature of gap junction organisation within disks of ventricular myocardium in all species studied was the presence of a conspicuous ring of large gap junctions around the periphery of the disk. Immunostained gap junctions lying within the interior zone delineated by the peripheral junctions generally occurred at lower numerical densities and were significantly smaller. In all species, less than 3% of all immunolabelled gap junctions measured were greater than 2 microns in maximal length, though a small proportion (0.06%) exceeded 4 microns. The numerical density of immunolabelled gap junctions in the disk was similar between species; however, within species there was a significant decrease in numerical density with increasing disk size. The new features of intercalated disk structure revealed in this study may have an important part to play in the intercellular communication and electrical propagation properties of the mammalian heart.
一种针对与连接蛋白43（一种大鼠心脏间隙连接蛋白）部分序列匹配的合成肽产生的多克隆抗血清，与激光扫描共聚焦显微镜结合使用，以研究一系列哺乳动物心脏组织中的间隙连接分布。将心室组织中获得的局部点状染色模式与传统光学显微镜和电子显微镜观察到的闰盘分布进行比较，并与使用相同抗血清的标准光学显微镜免疫荧光观察到的染色进行比较，结果表明对清晰分辨的单个间隙连接具有高度特异性标记。心室心肌的激光扫描共聚焦显微镜显示，免疫染色的间隙连接局限于平分肌纤维长轴的明确闰盘，而在心房心肌中，间隙连接通常广泛分布于心肌细胞体的侧面。大鼠心房间隙连接（通过荧光斑点的最长轴向长度测量）明显大于心室中的对应物，并且尺寸分布范围更窄。包括人类在内的六种哺乳动物的心室心肌给出了相似的免疫染色模式，表明抗血清检测到的表位以及哺乳动物心脏中细胞间电传播途径的总体组织具有保守性。通过激光扫描共聚焦显微镜获得的光学切片系列允许在相当大的标本深度上以高清晰度对心室闰盘中间隙连接的三维分布进行定量和映射。在所有研究物种的心室心肌闰盘内，间隙连接组织的一个一致特征是在闰盘周边存在一个明显的大间隙连接环。位于周边连接所划定的内部区域内的免疫染色间隙连接通常以较低的数量密度出现，并且明显较小。在所有物种中，测量的所有免疫标记间隙连接中，最大长度大于2微米的不到3%，尽管有一小部分（0.06%）超过4微米。不同物种之间闰盘中免疫标记间隙连接的数量密度相似；然而，在同一物种内，随着闰盘尺寸的增加，数量密度显著降低。本研究中揭示的闰盘结构的新特征可能在哺乳动物心脏的细胞间通讯和电传播特性中发挥重要作用。
