Nox2-Calcium Signaling in Skeletal Muscle

描述(申请人提供)：在生理条件下产生的活性氧(ROS)是骨骼肌产生正常力量所必需的。然而，在剧烈运动中观察到高水平的ROS，并促进收缩功能障碍，导致肌肉无力和疲劳。越来越多的证据表明，亚细胞部位特异性ROS的产生控制着ROS的有益和有害影响。NADPH氧化酶(NOX2)是一种产生ROS的酶复合体。PI正在进行的研究发现，在剧烈的收缩活动中，NOX2产生的ROS增加会导致疲劳。我们有强有力的证据表明，Src酪氨酸激酶作为氧化还原开关来激活NOX2。这一理论的中心假设是，NOX2和Src通过前馈途径发挥作用，导致ROS产生过多和收缩功能障碍。我们将使用缺乏NOX2的转基因小鼠，基因编码的定点氧化还原传感器和一种新的工具来同时测量活的骨骼肌纤维中的力和钙瞬变，以解决以下特定目标：1.确定收缩活动增加骨骼肌依赖NOX2的ROS产生的机制，2.确定Src是否作为氧化还原开关来调节依赖活动的ROS的产生，以及3.评估NOX2和Src在胞浆[Ca~(2+)]i瞬变和力量产生中的作用。这项拟议的研究将确定ROS产生的亚细胞位置，并阐明控制ROS调节骨骼肌微域钙信号的信号通路。此外，它还将为开发有针对性的抗氧化疗法奠定基础，以对抗肌肉疲劳和疾病中ROS过量产生的有害影响。

DESCRIPTION (provided by applicant): Generation of reactive oxygen species (ROS) under physiological conditions is required for normal force production in skeletal muscle. However, high levels of ROS are observed during intense physical exercise and promote contractile dysfunction, resulting in muscle weakness and fatigue. There is growing evidence that sub-cellular site-specific ROS production governs the beneficial vs. damaging effects of ROS. NADPH oxidase (Nox2) is an enzyme complex that generates ROS. Ongoing work by PI has found that increased ROS generation from Nox2 during intense contractile activity contributes to fatigue. We have strong evidence that Src tyrosine kinase acts as a redox switch to activate Nox2. The central hypothesis of this proposal is that Nox2 and Src act through a feed-forward pathway leading to excessive ROS production and contractile dysfunction. We will use genetically modified mice lacking Nox2, genetically encoded site-specific redox sensors and a novel tool to measure force and Ca2+ transients simultaneously in living skeletal muscle fibers to address the following specific aims: 1. Define the mechanisms by which contractile activity increases Nox2 dependent ROS production in skeletal muscle, 2. Determine whether Src serves as a redox switch to modulate activity dependent ROS production, and 3. Assess the role of Nox2 and Src on cytosolic [Ca2+ ]i transients and force generation. The proposed research will identify the sub-cellular sites of ROS production and elucidate the signaling pathways that control ROS regulation of microdomain Ca2+ signaling in skeletal muscle. Furthermore, it will lay the foundation for the development of targeted antioxidant therapy to combat the deleterious effects of overproduction of ROS seen in muscle fatigue and disease.