MYOSIN-BASED CARDIAC DISEASE--DEVELOPING A RAT MODEL

基于肌球蛋白的心脏病——开发大鼠模型

• 批准号: 2228466
• 负责人: REX L CHISHOLM
• 金额: $ 26.66万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-12-15 至 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2228466
• 关键词: cell bank /registry; chemical chain length; disease /disorder model; electroporation; embryo /fetus; embryo /fetus tissue /cell culture; embryonic stem cell; gene expression; gene mutation; genetic manipulation; genetic models; genetically modified animals; heart contraction; heart disorder; heart function; laboratory rat; microinjections; model design /development; myocardium; myosins; nucleic acid sequence; protein isoforms; recombinant DNA; tissue mosaicism

Myosin is responsible for a variety of contractile processes including contraction of cardiac muscle. Myosin consists of three different subunits, the myosin heavy chain (MHC), and two different light chains, MLC1 and MLC2. Because mutations in any of these subunits have been shown to produce cells with similar phenotypes, it seems likely that mutations in any of the cardiac myosin subunits could result in defects in cardiac contractility. Recently mutations in the cardiac beta-MHC have been shown to produce about 50% of the cases of familial hypertrophic cardiomyopathy. The basis for the remaining cases are thought to be mutations in gene other than the MHC. In this application we propose a comprehensive series of experiments to employ rat ES cells that we have recently generated, as hosts for the molecular genetic manipulation of cardiac MLC gene expression. These manipulations involve the use of gene targeting to disrupt the cardiac MLC isoforms and the use of expression vectors to express cardiac MLC isoforms in both wildtype and mutant rat ES cells. We will examine the resulting cells to determine the consequences of these molecular genetic manipulations. The mutant cells will be employed to generate chimeric rats and the consequences of MLC manipulations on the development and function of the heart will be investigated. Four specific aims are proposed: (1) to further characterize our rat ES cells, (a) establishing their competence to contribute to the germ line of chimeric rats, (b) to establish additional rat ES cell lines, and (c) to optimize rat chimera production using these cells; (2) to employ gene replacement to create rat ES cells which no longer express the ventricular or atrial MLCs and analyze the consequences for the contractile properties of these mutant cell lines; (3) to employ the MLC mutant ES cells as hosts for the expression of wildtype and mutant MLC isoforms to investigate the contribution of MLC isoforms to myosin function; and (4) to use these cell lines to produce rats displaying altered expression of cardiac MLC isoforms. These animals will be used to study the developmental and physiological consequences of myosin mutations. These studies may produce rat models for cardiomyopathies or other myosin-based pathologies. Once established, these animal models may be useful for the development of therapeutic modalities and interventions which might be effective in treating cardiovascular diseases in humans.

肌球蛋白负责多种收缩过程，包括 心肌收缩。 肌球蛋白由三种不同的 亚基、肌球蛋白重链 (MHC) 和两条不同的轻链， MLC1 和 MLC2。因为这些亚基中的任何一个都已显示出突变 为了产生具有相似表型的细胞，突变似乎很可能 任何心肌肌球蛋白亚基中的任何一个都可能导致心脏缺陷 收缩性。最近发现心脏 β-MHC 发生突变 产生约50%的家族性肥厚型心肌病病例。 其余病例的基础被认为是基因突变 除了 MHC 之外。在此应用中，我们提出了一个全面的系列 使用我们最近生成的大鼠 ES 细胞的实验，如 心脏MLC基因分子遗传操作的宿主 表达。这些操作涉及使用基因靶向 破坏心脏 MLC 亚型并使用表达载体 在野生型和突变型大鼠 ES 细胞中表达心脏 MLC 亚型。 我们 将检查产生的细胞以确定这些的后果 分子遗传操作。突变细胞将被用于 产生嵌合大鼠以及 MLC 操作对 将研究心脏的发育和功能。四具体 提出的目标是：(1) 进一步表征我们的大鼠 ES 细胞，(a) 建立他们为嵌合种系做出贡献的能力 大鼠，(b) 建立额外的大鼠 ES 细胞系，(c) 优化 使用这些细胞生产大鼠嵌合体； (2)采用基因替换 创造不再表达心室或心房的大鼠 ES 细胞 MLC 并分析这些材料的收缩特性的后果 突变细胞系； (3) 使用MLC突变型ES细胞作为宿主 野生型和突变型 MLC 亚型的表达以研究 MLC 同种型对肌球蛋白功能的贡献； (4) 使用这些细胞 产生心脏 MLC 表达改变的大鼠的品系 同工型。这些动物将用于研究发育和 肌球蛋白突变的生理后果。这些研究可能会产生 心肌病或其他基于肌球蛋白的病理的大鼠模型。 一次 建立后，这些动物模型可能有助于开发 可能有效的治疗方式和干预措施 治疗人类心血管疾病。