CARDIAC ION TRANSPORT AND ULTRASTRUCTURE

心脏离子转运和超微结构

• 批准号: 3334351
• 负责人: ERNEST PAGE
• 金额: $ 22.22万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-05-01 至 1988-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3334351
• 关键词: X ray crystallography; electron microscopy; freeze etching; gel electrophoresis; heart; heart Purkinje's fiber; heart contraction; image enhancement; immunoelectron microscopy; intercellular connection; ion transport; membrane permeability; membrane potentials; membrane reconstitution /synthesis; membrane structure; monoclonal antibody; myocardial ischemia /hypoxia; myocardium; myofibrils; myogenesis; sarcoplasmic reticulum; vascular smooth muscle

We propose to apply powerful ultrastructural techniques to in situ and isolated gap junctional membranes (GJ) from mammalian heart muscle in order to correlate changes in internal membrance structure and in GJ channel distribution with phsiological and developmental changes in GJ permeability. The structural and analytical methods to be applied to isolated GJ include low-irradiation electron microscopy of negatively stained GJ following by optical diffraction, image processing and reconstruction; X-ray diffraction; freeze fracture with rotary replication, quantitative analysis of particle size, spacing and distribution, and stereo-imaging by tilting with a goniometer stage; immunoelectron microscopy of thin-sectioned GJ using monoclonal antibodies against GJ protein determinants at the cytoplasmic face of the GJ, plus ferritin-labeled secondary antibody; and two-dimenstional electrophoresis with the Cleveland technique. Effects of incubation at different ionized Ca2+ concentrations and pH, differences between GJ isolated from atria and ventricles, and GJ differences between stages of development will be thus studied. Complementary freeze fracture and immunocytological studies on in situ GJ from atrial, ventricular, Purkinje fiber, and embryonic ventricular muscle are designed to correlate data on isolated GJ with that from relatively intact cardiac tissues. The physiological and biological issues addressed by this research are (a) change in membrane channel structure when GJ permeability changes; (b) channel dimensions, submit structure and subunit protein composition of the channel-containing units (connexons); (c) distribution, assembly, and disassembly of GJ channels and GJ precursors during cardiac development; and (d) whether GJ structure, composition, and (perhaps) permeability differ in atria, ventricles, and conduction cells, and between different stages of ventricular development. Since GJ permeability in cardiac tissues is a critical variable for both action potential conduction and the sealing off of injured cells, this research is relevant to disturbances of cardiac conduction and rhythmicity and to sealing off of heart muscle cells that are irreversibly injured by ischemia or other disease processes.

我们建议将强大的超微结构技术应用于原位和 从哺乳动物心肌中分离间隙连接膜（GJ） 关联内膜结构和 GJ 通道的变化 GJ 的分布随生理和发育变化 渗透性。 适用的结构和分析方法 分离的 GJ 包括负电镜的低辐照电子显微镜 GJ 染色后进行光学衍射、图像处理和 重建; X射线衍射；旋转复制冷冻断裂， 颗粒尺寸、间距和分布的定量分析，以及 通过测角仪平台倾斜进行立体成像；免疫电子 使用针对 GJ 的单克隆抗体对 GJ 进行薄切片显微镜检查 GJ 细胞质面的蛋白质决定簇，加上 铁蛋白标记的二抗；和二维电泳 与克利夫兰技术。 不同电离条件下孵育的影响 Ca2+ 浓度和 pH，从心房分离的 GJ 和 心室和 GJ 发育阶段之间的差异将因此 研究过。 补充冷冻断裂和免疫细胞学研究 来自心房、心室、浦肯野纤维和胚胎心室的原位 GJ 肌肉旨在将孤立的 GJ 数据与来自 心脏组织相对完整。 生理和生物学问题 这项研究解决的是（a）膜通道结构的变化 当GJ渗透性改变时； (b) 渠道规模、提交结构和 含有通道的单位（连接子）的亚基蛋白质组成； (c) GJ通道和GJ的分配、组装和拆卸 心脏发育过程中的前兆； (d)是否有GJ结构， 心房、心室和心室的成分和（也许）渗透性不同 传导细胞，以及心室发育的不同阶段之间。 由于心脏组织中的 GJ 通透性对于两者来说都是一个关键变量 动作电位传导和受损细胞的封闭，这 研究与心脏传导和节律紊乱相关 并封闭受到不可逆损伤的心肌细胞 缺血或其他疾病过程。