Redox Mechanisms of Respiratory Muscle Stress Adaptation

描述（由申请人提供）：骨骼肌产生活性氧（ROS）以响应各种应激刺激，包括热应激、渗透应激、强烈刺激和缺氧。这些信号似乎在功能上很重要，但在大多数正常生理条件下不会造成损伤或损害。我们假设，在这种情况下，ROS在旨在帮助细胞承受压力的信号网络中发挥重要作用。目前建议的重点将是在骨骼肌中从高到低O2的过渡中产生的ROS。这种现象与缺氧诱导的细胞氧化还原状态（NADH/NAD+）的转变一致，但我们不知道该信号是否来自氧化还原的变化或一些其他缺氧诱导的细胞反应。我们也不知道这种信号的亚细胞起源或什么表型产生它。该提案的AIM 1将鉴定代谢应激期间产生的ROS形成的主要细胞和亚细胞起源，并且它将确定ROS生成系统对PO 2变化与NADH/NAD+变化的敏感性。为了实现这一目标，我们设计了新的成像方法，包括多光子和荧光寿命，和一种新的荧光探针定位超氧化物接近膜。我们还将通过测量和独立操纵PO 2和细胞氧化还原状态来确定临界刺激方式和强度。在AIM 2中，我们将研究缺氧诱导的ROS的功能意义。我们推测，应激诱导的ROS促进能量动员，抑制能量消耗。首先，我们将评估AMP依赖性蛋白激酶和糖酵解通量作为应激诱导的ROS的可能靶点的潜在作用。其次，我们将确定ROS如何影响Ca+2释放和力之间的关系，以及ROS和细胞氧化还原状态在改变Ca+2诱导力中的潜在作用。这项基础科学研究将为基础骨骼肌生物学提供新的见解，这些生物学将应用于与O2运输限制相关的各种肌肉疾病。

DESCRIPTION (provided by applicant): Skeletal muscles produce reactive oxygen species (ROS) in response to a variety of stress stimuli, including thermal stress, osmotic stress, intense stimulation and hypoxia. These signals appear to be functionally significant but do not cause injury or damage under most normal physiologic conditions. We hypothesize that ROS, in this setting, play important roles in signaling networks designed to assist cells to withstand stress. The focus of the current proposal will be on the ROS produced in the transition from high to low O2 in skeletal muscle. This phenomenon is coincident with the hypoxia-induced shift in the redox state of the cell (NADH/NAD+), but we do not know if the signal arises from changes in redox or some other hypoxia- induced cellular response. We also do not know the sub-cellular origins of this signal or what phenotype produces it. AIM 1 of the proposal will identify the primary cellular and subcellular origins of ROS formation produced during metabolic stress and it will determine the sensitivity of the ROS-generating system to changes in PO2 vs. shifts in NADH/NAD+. To address this aim we have designed new imaging methods, including multiphoton and fluorescence lifetime, and a new fluorescent probe for localization of superoxide close to membranes. We will also determine the critical stimulus modality and intensity by measuring, and independently manipulating PO2 and cell redox state. In AIM 2, we will study the functional significance of hypoxia-induced ROS. We hypothesize that stress-induced ROS promotes energy mobilization and inhibits energy expenditure. First, we will evaluate the potential role of AMP-dependent protein kinase and glycolytic flux as a possible target for stress-induced ROS. Second, we will determine how ROS influences the relationships between Ca+2 release and force and the potential roles ROS and cell redox state have in altering Ca+2-induced force. This basic science investigation will give new insights into fundamental skeletal muscle biology that will have applications to a variety of muscle disorders related to O2 transport limitation.