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 哺乳动物心肌的孤立间隙连接膜（GJ） 将内部膜结构和GJ通道的变化相关联 GJ的PHSIologology和发育变化的分布 渗透性。 用于应用于的结构和分析方法 孤立的GJ包括负面的低辐照电子显微镜 通过光学衍射，图像处理和染色的GJ跟随 重建; X射线衍射；用旋转复制冻结断裂， 粒度，间距和分布的定量分析，以及 立体成像通过斜向阶段倾斜；免疫电子 使用单克隆抗体对GJ的薄片GJ的显微镜检查 GJ的细胞质面的蛋白质决定因素，加上 铁蛋白标记的二抗；和二维电泳 利用克利夫兰技术。 在不同离子化下孵育的影响 Ca2+浓度和pH，从心房分离的GJ之间的差异和 因此，心室和GJ发展阶段之间的差异将是 研究。 互补的冻结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GJ的数据与来自 相对完整的心脏组织。 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渗透性变化时； （b）渠道维度，提交结构和 含通道单位的亚基蛋白组成（连接子）； （c）GJ通道和GJ的分配，组装和拆卸 心脏发育过程中的前体； （d）GJ是否结构 组成，并且（也许）渗透性在心脏，心室和 传导细胞，以及心室发育的不同阶段。 由于心脏组织中的GJ渗透性对于两者都是关键的变量 动作电位传导和受伤细胞的密封，这 研究与心脏传导和节奏性的干扰有关 并密封心肌细胞，这些细胞不可逆地受伤 缺血或其他疾病过程。