VENTRICULAR MYOSIN LIGHT CHAIN 2 FUNCTION IN NORMAL AND FAILING HEARTS

正常和衰竭心脏中心室肌球蛋白轻链 2 的功能

• 批准号: 6242352
• 负责人: Jeffrey Robbins
• 金额: $ 24.4万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 1997-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6242352
• 关键词: chloramphenicol acetyltransferase; congestive heart failure; gene mutation; genetic promoter element; genetically modified animals; heart contraction; heart function; laboratory mouse; messenger RNA; molecular genetics; myocardium disorder; myosin light chain kinase; phenotype; polymerase chain reaction; protein isoforms; western blottings

The objectives of this proposal are driven by the hypothesis that the regulatory, or phosphorylatable light chain 2 (MLC2) plays a critical role in cardiac function. Furthermore, we hypothesize that there are functional consequences which result from the differential expression patterns of the different MLC2 isoforms; these are reflected in the changing patterns of MLC2 expression that are observed in the ventricles and atria of falling hearts. Our objective is to test these hypotheses directly in the whole animal context using transgenic mice. Using promoters that are able to drive varying levels of transgene expression in the murine cardiac compartment the steady state levels and isoform populations of the MLC2's that are normally present in the murine atrium and ventricle will be modified. There are two major MLC2's that are expressed in the heart, an atrial specific and ventricle specific form. In the first set of experiments, we will test the stoichiometry of the system as well as isoform functionality by overexpressing in both the atria and ventricle, at various levels, the endogenous MLC2 that is normally found in the ventricle. Similar experiments using a construct in the antisense orientation will be performed in order to lower the ratio of MLC2/MLC1, a condition that is observed in idiopathic dilated cardiomyopathy. Finally, experiments will be carried out with a cDNA which encodes an isoform that is normally expressed only in skeletal striated muscle (MLC2f) and not in the heart in order to determine if there are functional correlates to MLC2 isoform diversity. Parallel studies on the MLC isoforms present in human heart tissue derived from patients in early (NYHA class 1 and 2) and lat. (NYHA class 3 and 4) systolic and diastolic heart failure will be carried out using quantitative PCR analyses in order to relate changes in MLC2/MLC1 ratios at the RNA level to altered cardiac function in vivo. A genetic approach using overexpression and ablation studies carried out via transgenesis holds the promise of providing an unambiguous assignation of basic function and the physiologic or pathophysiologic significance of differential MLC2 isoform content. By coupling a molecular genetic approach with the ability to generate stable transgenic lines for analyses of contractile function and performance, a rigorous analysis of both major and minor phenotypic effects resulting from perturbations in MLC content can be made.

该提案的目标是由以下假设驱动的： 调节性或可磷酸化轻链 2 (MLC2) 在 对心脏功能的作用。此外，我们假设有 差异表达导致的功能后果 不同 MLC2 同工型的模式；这些都反映在 在心室中观察到的 MLC2 表达模式的变化 和心房坠落的心。我们的目标是检验这些假设 使用转基因小鼠直接在整个动物环境中进行研究。使用 能够驱动不同水平的转基因表达的启动子 在小鼠心脏室中，稳态水平和亚型 通常存在于小鼠心房中的 MLC2 群体 和心室将被修改。有两个主要的 MLC2： 在心脏中表达，有心房特异性和心室特异性形式。 在第一组实验中，我们将测试化学计量 系统以及亚型功能通过在两个中过度表达 心房和心室的不同水平上，内源性 MLC2 通常发现于心室。使用构造的类似实验 在反义方向将进行，以降低 MLC2/MLC1 的比率，这是在特发性扩张中观察到的情况 心肌病。最后，用cDNA进行实验 它编码通常仅在骨骼中表达的同工型 横纹肌（MLC2f）而不是心脏，以确定是否 MLC2 同工型多样性存在功能相关性。平行线 对人类心脏组织中存在的 MLC 异构体​​的研究 早期（NYHA 1 级和 2 级）和晚期患者。 （NYHA 3 级和 4 级） 收缩性和舒张性心力衰竭将使用 定量 PCR 分析，以便关联 MLC2/MLC1 比率的变化 在RNA水平上改变体内心脏功能。 遗传方法 使用通过转基因进行的过度表达和消融研究 承诺提供基本的明确分配 的功能和生理或病理生理意义 MLC2 同种型含量差异。通过耦合分子遗传学 方法能够产生稳定的转基因系 分析收缩功能和性能，严格分析 由干扰引起的主要和次要表型效应 可以制作MLC内容。