Redox Mechanisms of Respiratory Muscle Stress Adaptation

描述(申请人提供)：骨骼肌产生活性氧物种(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伤害或损害。我们假设，在这种情况下，ROS在旨在帮助细胞抵御压力的信号网络中扮演着重要的角色。目前的提案将重点放在骨骼肌从高氧到低氧的转变过程中产生的ROS。这种现象与缺氧引起的细胞氧化还原状态(NADH/NAD+)的改变是一致的，但我们不知道这种信号是由氧化还原反应的变化还是其他一些缺氧诱导的细胞反应引起的。我们也不知道这种信号的亚细胞来源或产生它的表型。该提案的目标1将确定代谢应激期间产生的ROS形成的主要细胞和亚细胞来源，并将确定ROS生成系统对PO2变化与NADH/NAD+变化的敏感性。为了解决这个问题，我们设计了新的成像方法，包括多光子和荧光寿命，并设计了一种新的荧光探针来定位靠近膜的超氧化物。我们还将通过测量和独立操作PO2和细胞氧化还原状态来确定关键的刺激形式和强度。在AIM 2中，我们将研究低氧诱导ROS的功能意义。我们假设，应激诱导的ROS促进能量动员并抑制能量消耗。首先，我们将评估AMP依赖的蛋白激酶和糖酵解通量作为应激诱导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，这将应用于与氧气运输限制有关的各种肌肉疾病。

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.