Reactive oxygen species and respiratory muscle dysfunction in heart failure

 描述(申请人提供)：隔膜无力是慢性心力衰竭(CHF)患者的一个重要健康问题，原因是：a)由于呼吸困难，影响他们维持呼吸的能力并限制体力活动，b)触发交感神经激活，可能导致心律失常和死亡，或导致四肢肌肉血管收缩和疲劳，以及c)损害呼吸道清除，使患者容易患肺炎。这些观察结果强调了了解横隔膜异常的机制的重要性，以及确定预防CHF虚弱的生物靶点的必要性。CHF横隔肌无力主要由肌原纤维蛋白改变引起的比力丧失(即收缩功能障碍)引起，而活性氧(ROS)参与了此过程。线粒体电子传递链被认为是肌肉中ROS的主要来源。然而，我们从人的横隔膜活检和动物模型中获得的初步数据表明，主要定位于横隔膜肌膜(NOX2)和线粒体(NOX4)的NADPH氧化酶(NOx)参与了CHF时ROS的升高和横隔膜的虚弱。此外，我们的初步研究表明，肌原纤维蛋白硫醇氧化是CHF引起的横隔肌无力的关键分子事件。基于我们的数据，我们建议完成三个特定的目标：1)确定隔膜NOX2复合体在CHF中ROS过度和虚弱中的作用，2)确定NOX4和线粒体ROS是否是CHF中隔膜虚弱的介质，3)确定ROS诱导的CHF中隔膜收缩功能障碍的机制。为了达到这些目的，我们将使用可诱导的骨骼肌特异性基因敲除小鼠，并在骨骼肌特异性启动子的控制下，胸膜内注射带有shRNA或表达质粒的重组腺相关病毒来靶向Sham和CHF小鼠的横隔膜纤维。我们还将分离Sham和CHF小鼠的横隔膜单纤维，并进行实验以确定比力缺陷是否可以在体外修复。最后，我们将使用全球无标记蛋白质组学和差异半胱氨酸标记来确定Sham和CHF动物横隔膜中特定半胱氨酸残基的蛋白质丰度和氧化还原状态。我们对ROS的来源、分子种类和靶点以及可逆性的关注对于理解病理生理学和为治疗导致CHF患者发病率和死亡率的隔膜无力奠定了基础。

 DESCRIPTION (provided by applicant): Diaphragm weakness is a significant health problem in chronic heart failure (CHF) patients because: a) compromises their ability to sustain ventilation and limits physical activity due to dyspnea, b) triggers sympathetic activation that ca lead to cardiac arrhythmias and death or induces vasoconstriction and fatigue in limb muscles, and c) impairs airway clearance, predisposing patients to pneumonia. These observations highlight the importance of understanding the mechanisms underlying diaphragm abnormalities and the need to identify biological targets for prevention of weakness in CHF. CHF diaphragm weakness is predominantly caused by loss of specific force (i.e. contractile dysfunction) due to alterations in myofibrillar proteins, and reactive oxygen species (ROS) have been implicated in this process. The mitochondria electron transport chain has been considered the main source of ROS in muscle. However, our preliminary data from human diaphragm biopsies and animal models suggest that NADPH oxidases (Nox) localized predominantly in diaphragm sarcolemma (Nox2) and mitochondria (Nox4) are involved in the heightened ROS and diaphragm weakness in CHF. Moreover, our pilot studies suggest that myofibrillar protein thiol oxidation is a key molecular event in CHF induced diaphragm weakness. Based on our data, we propose to complete three specific aims: 1) to determine the role of diaphragmatic Nox2 complex on excess ROS and weakness in CHF, 2) to determine whether Nox4 and mitochondrial ROS are mediators of diaphragm weakness in CHF, 3) to define the mechanism of ROS-induced diaphragm contractile dysfunction in CHF. To address these aims, we will use inducible skeletal muscle specific knockout mice and intra-pleural injection of recombinant adeno-associated virus with shRNA or expression plasmids under control of a skeletal muscle-specific promoter to target diaphragm fibers in Sham and CHF mice. We will also isolate diaphragm single fibers from sham and CHF mice and perform experiments to determine if specific force deficits can be rescued in vitro. Finally, we will use global label free proteomics and differential Cysteine labeling to determine the abundance of proteins and redox status of specific Cysteine residues in diaphragm from Sham and CHF animals. Our focus on ROS sources, molecular species and targets, and reversibility is critical to understand the pathophysiology and set the stage for nove therapies to treat diaphragm weakness that contributes to the morbidity and mortality in CHF patients.