Vascular Reactivity in Heart Failure

描述(申请人提供)：在充血性心力衰竭(CHF)的临床综合征期间，血管系统的特点是静息血管收缩，对应激反应异常，以及对一氧化氮(NO)介导的血管扩张产生抵抗。导致这些血管系统变化的机制尚不清楚。以前的研究没有讨论血管平滑肌收缩表型的变化是否会导致这些异常。该应用的目的是确定与充血性心力衰竭相关的血管异常的机制。20 kDa肌球蛋白轻链(MLC20)的磷酸化水平依赖于肌球蛋白轻链(MLC)的相对活性，而肌球蛋白轻链(MLC20)的磷酸化水平又依赖于MLC的相对活性。MLC磷酸酶是由三个亚基组成的全酶：约-20 kDa亚基、约-38 kDa催化亚基和肌球蛋白结合亚基(MBS)。四种不同的MBS亚型的特征是中心插入和亮氨酸拉链的存在(133 KDa)或不存在(130 KDa)。初步数据表明，与133 kDa亚型相比，130 kDa亚型具有更强的活性，中心插入决定了平滑肌对激动剂诱导的作用力增强的敏感性。此外，我们的数据表明，亮氨酸拉链的剪接变体表达决定了对NO介导的血管扩张的敏感性。这一应用是基于一种假设，即与CHF相关的血管异常是由于MBS剪接变体表达的变化所致。我们认为静息性血管收缩是由于中央插入片段的剪接变体表达的变化，而对NO介导的血管扩张的抵抗是由于亮氨酸拉链的剪接变体表达的变化。为了验证这一假说，我们将确定(1)在CHF发生前后，平滑肌对激动剂诱导的作用力增强、NO介导的血管扩张以及MBS剪接变体异构体的表达的敏感性；以及(2)如果强制表达MBS的剪接变体亚型改变了对激动剂诱导的作用力增强的敏感性，则MBS对NO介导的平滑肌松弛的敏感性是否改变。我们将使用几种不同的对照血管平滑肌和CHF的大鼠脑梗塞模型，以及培养的血管平滑肌细胞来验证我们的假设。这些研究将阐明组织特异性对激动剂诱导的作用力增强和NO介导的平滑肌松弛敏感性差异的分子机制。

DESCRIPTION (provided by applicant): During the clinical syndrome of congestive heart failure (CHF), the vasculature is characterized by resting vasoconstriction, abnormal responses to stress, and resistance to nitric oxide (NO) mediated vasodilatation. The mechanism that leads to these changes in the vasculature is unknown. Previous studies have not addressed whether changes in the contractile phenotype of vascular smooth muscle cause these abnormalities. The goal of this application is to determine the mechanism for the vascular abnormalities associated with CHF. Smooth muscle activation is dependent on 20 kDa myosin light chain (MLC20) phosphorylation levels, which depend on the relative activities of MLC kinase and MLC phosphatase. The MLC phosphatase is a holoenzyme consisting of three subunits; a approximately -20 kDa subunit, a approximately -38 kDa catalytic subunit and a myosin binding subunit (MBS). Four distinct isoforms of the MBS are characterized by the presence (133 kDa) or absence (130 kDa) of a central insert and a leucine zipper. Preliminary data demonstrate that the 130 kDa isoform has an increased activity compared to the 133 kDa isoform, and that the central insert determines the sensitivity of smooth muscle to agonist induced force enhancement. In addition, our data suggest that splice variant expression of the leucine zipper determines the sensitivity to NO mediated vasodilatation. This application is based on the hypothesis that vascular abnormalities associated with CHF are due to changes in splice variant expression of the MBS. We suggest that the resting vasoconstriction is due to changes in splice variant expression of the central insert, while the resistance to NO mediated vasodilatation is due to changes in splice variant expression of the leucine zipper. To test this hypothesis, we will determine (1) the sensitivity of smooth muscle to agonist induced force enhancement, NO mediated vasodilatation, and the expression of splice variant isoforms of the MBS prior to and after the development of CHF; and (2) if forced expression of the splice variant isoforms of the MBS alters the sensitivity to agonist induced force enhancement mad NO mediated smooth muscle relaxation. We will use several different vascular smooth muscles of control and a rat infarct model of CHF, as well as cultured smooth muscle cells to test our hypothesis. These studies will elucidate the molecular mechanism underlying the tissue specific differences in the sensitivity to agonist induced force enhancement and NO mediated smooth muscle relaxation.