MYOSIN-BASED CARDIAC DISEASE--DEVELOPING A MODEL

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 肌球蛋白重链(MHC)和两个不同的轻链， MLC1和MLC2。因为这些亚基中的任何一个亚基都已经显示出突变 为了产生具有相似表型的细胞，突变似乎很可能 在任何一种心肌中，肌球蛋白亚基都会导致心脏缺陷。 伸缩性。最近发现了心脏β-MHC的突变。 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。 而不是MHC。在本申请中，我们提出了一个全面的系列 使用我们最近产生的大鼠细胞的实验，如 心脏MLC基因分子遗传操作的宿主 表情。这些操作涉及使用基因靶向来 干扰心脏MLC亚型和表达载体的使用 在野生型和突变型大鼠ES细胞中表达心脏MLC亚型。我们 将检查生成的单元格以确定这些结果 分子遗传操作。突变细胞将被用来 产生嵌合体大鼠和MLC操作对 将对心脏的发育和功能进行调查。四个具体的 提出的目标是：(1)进一步描述我们的大鼠ES细胞，(A) 建立他们对嵌合体种系做出贡献的能力 大鼠，(B)建立额外的大鼠ES细胞系，以及(C)优化 使用这些细胞生产大鼠嵌合体；(2)使用基因替换 为了创造不再表达心室或心房的大鼠胚胎细胞 并分析了对这些材料收缩性能的影响。 突变细胞系；(3)利用MLC突变ES细胞作为宿主 野生型和突变型MLC亚型的表达 MLC亚型对肌球蛋白功能的贡献；以及(4)使用这些细胞 产生心脏MLC表达改变的大鼠的品系 异构体。这些动物将被用来研究发育和 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 这些动物模型的建立可能有助于发展 治疗模式和干预措施可能在以下方面有效 治疗人类的心血管疾病。

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.