Reactive oxygen species and respiratory muscle dysfunction in heart failure

 描述（由申请人提供）：膈肌无力是慢性心力衰竭（CHF）患者的一个严重健康问题，因为：a）由于呼吸困难而损害其维持通气的能力并限制体力活动，B）触发交感神经激活，可导致心律失常和死亡或诱导血管收缩和肢体肌肉疲劳，以及c）损害气道清除，使患者易患肺炎。这些观察结果强调了了解横膈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隔膜无力主要是由于肌原纤维蛋白的改变导致的比力损失（即收缩功能障碍）引起的，并且活性氧（ROS）与该过程有关。线粒体电子传递链被认为是肌肉中ROS的主要来源。然而，我们从人类膈肌活检和动物模型的初步数据表明，NADPH氧化酶（Nox）主要定位于膈肌肌膜（Nox 2）和线粒体（Nox 4）参与了高活性氧和膈肌无力在CHF。此外，我们的初步研究表明，肌原纤维蛋白巯基氧化是CHF诱导膈肌无力的关键分子事件。基于我们的数据，我们建议完成三个具体的目标：1）确定线粒体Nox 2复合物在CHF中过量ROS和虚弱中的作用，2）确定Nox 4和线粒体ROS是否是CHF中膈肌虚弱的介质，3）确定ROS诱导的CHF中膈肌收缩功能障碍的机制。为了实现这些目标，我们将使用诱导型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，胸膜内注射重组腺相关病毒与shRNA或表达质粒，以靶向假手术和CHF小鼠的膈肌纤维。我们还将从假手术和CHF小鼠中分离隔膜单纤维，并进行实验以确定是否可以在体外挽救特定的力缺陷。最后，我们将使用全局无标记蛋白质组学和差异半胱氨酸标记来确定假手术和CHF动物膈肌中蛋白质的丰度和特定半胱氨酸残基的氧化还原状态。我们对ROS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至关重要，并为治疗导致CHF患者发病率和死亡率的膈肌无力的新疗法奠定基础。

 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.