MYOSIN-BASED CARDIAC DISEASE--DEVELOPING A RAT 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 亚基，肌球蛋白重链（MHC），和两个不同的轻链， MLC 1和MLC 2。因为这些亚基中的突变已经被证明 为了产生具有相似表型的细胞， 任何一种心肌肌球蛋白亚单位的缺失都可能导致心肌细胞的缺陷。 收缩性最近，心脏β-MHC的突变已被证明是 家族性肥厚型心肌病的发病率约为50%。 其余病例的基础被认为是基因突变 除了MHC。在本申请中，我们提出了一个全面的系列 实验中使用了我们最近产生的大鼠ES细胞， 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， 破坏心脏MLC同种型和使用表达载体， 在野生型和突变型大鼠ES细胞中表达心脏MLC同种型。 我们 将检查由此产生的细胞，以确定这些后果 分子遗传操作。突变细胞将用于 产生嵌合体大鼠和MLC操作的后果， 将研究心脏的发育和功能。四个具体 目的是：（1）进一步表征我们的大鼠ES细胞，（a） 建立它们的能力，以促进嵌合体的生殖系， 大鼠，（B）建立另外的大鼠ES细胞系，和（c）优化 利用这些细胞生产大鼠嵌合体;（2）采用基因置换 来制造大鼠胚胎干细胞， MLCs和分析的后果，这些收缩性能 突变细胞系;（3）采用MLC突变ES细胞作为宿主， 表达野生型和突变型MLC同种型，以研究 MLC同种型对肌球蛋白功能的贡献;以及（4）使用这些细胞 细胞系以产生显示心脏MLC表达改变的大鼠 同种型。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 用于心肌病或其他肌球蛋白基病理的大鼠模型。 一旦 建立，这些动物模型可能是有用的， 可能有效的治疗方式和干预措施， 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.