VENTRICULAR MYOSIN LIGHT CHAIN 2 FUNCTION IN NORMAL AND FAILING HEARTS

这项提议的目标是由这样的假设驱动的 调节性或可磷酸化轻链2(MLC2)起着关键作用 在心脏功能中的作用。此外，我们假设有 由差分表达式产生的函数结果 不同MLC2亚型的模式；这些反映在 在脑室观察到的MLC2表达模式的变化 和坠落的心的中庭。我们的目标是检验这些假说 直接在整个动物环境中使用转基因小鼠。vbl.使用 能够驱动不同水平的转基因表达的启动子 在小鼠心脏隔室中的稳态水平和亚型 正常存在于小鼠心房的MLC2‘S种群 而脑室将会被改造。有两个主要的MLC2的S是 在心脏中表达，是一种特定于心房和心室的形式。 在第一组实验中，我们将测试化学计量比 系统和异构体功能，通过在 心房和心室，在不同水平上，内源性MLC2，即 通常在脑室发现。使用构造物的类似实验 将在反义定向中进行，以降低 MLC2/MLC1的比率，在特发性扩张中观察到的一种情况 心肌病。最后，我们将用一个cDNA进行实验。 它编码一种通常只在骨骼中表达的异构体 横纹肌(MLC2f)，而不是在心脏，以确定是否 MLC2亚型多样性与功能相关。平行 人心脏组织中MLC亚型的研究 早期(NYHA 1级和2级)和晚期患者。(纽约房委会3级和4级) 收缩期和舒张期心力衰竭将使用 定量聚合酶链式反应分析以关联MLC2/MLC1比率的变化 在RNA水平上改变体内的心脏功能。一种遗传方法 使用通过转基因进行的过表达和消融研究 承诺提供明确的基本 功能及其生理学或病理生理学意义 MLC2异构体含量不同。通过将一种分子遗传 具有产生稳定转基因株系的能力的方法 收缩功能和性能的分析，严格的分析 由扰动引起的主要和次要的表型效应 可以制作MLC内容。

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.