Oxygen dependence of ROS generation in contracting single isolated myofibers

描述(申请人提供)：虽然多年来人们已经知道骨骼肌中的线粒体和非线粒体来源会产生活性氧物种(ROS)，但ROS产生的意义直到最近才变得清晰。ROS的产生与衰老过程和许多疾病状态有关，从癌症到心脏病，再到与炎症过程相关的病理。此外，最近的研究表明，ROS不仅对某些细胞功能有害(在极端条件下诱导细胞凋亡)，而且在许多细胞信号事件中起着重要的中介作用，并被认为调节与细胞氧化应激保护和必要的细胞运动适应相关的基因表达的重要变化。然而，在骨骼肌收缩和不同的氧合条件下调节ROS代谢的因素仍然不完全清楚。特别是，在缺氧过程中ROS产生的特殊行为，即尽管氧气紧张程度降低，但矛盾地诱导出更高的ROS产生，直到最近才被清楚地证明。最后，虽然已经证明骨骼肌中ROS的产生对收缩能力和肌肉功能有显著的影响，但对于骨骼肌中ROS的产生如何依赖于线粒体呼吸速率、功输出、细胞内氧合、抗氧化缓冲和肌肉纤维类型仍不清楚。这些不确定性很大程度上是由于与整个肌肉模型内的不均一性有关的混杂因素、不受控制的细胞外环境、可变的肌肉纤维招募模式等。本提案的目的是使用一个分离的小鼠骨骼肌纤维模型，在该模型中，细胞外环境可以被精确控制，细胞内环境可以使用非侵入性成像技术进行仔细的监测，以测试与描绘调控ROS产生的因素、细胞内ROS形成的位置以及在不同氧合条件下收缩慢收缩和快收缩纤维中ROS调节基因表达的假设。在这两种纤维类型的良好控制实验中，细胞外PO2在收缩过程中发生变化，将测量线粒体呼吸和膜电位、ROS的产生和清除、细胞内PO2、收缩功能、胞浆和线粒体[Ca~(2+)]、与ROS产生相关的大量细胞内信号、细胞完整性的抗氧化保护以及基因表达的调节(使用QPCR)。这一应用的优势在于我们的单肌纤维模型的使用，从而减少了解释完整肌肉、细胞培养或分离线粒体模型的结果的许多困难，并提供了在正常生理细胞内环境下从可存活的、健康的完整肌纤维生成的数据。这一应用为研究调节骨骼肌中ROS代谢的因素提供了一个难得的机会，从而对理解和潜在地改善人类健康具有重要意义。与公共卫生相关。直到最近才清楚的是，在运动和低氧条件下产生的活性氧对肌肉功能和适应能力有重大影响。这项研究的目的是阐明在运动和低氧条件下影响活性氧产生的调节因素，以及这些变化如何影响肌肉表现和对运动的适应性反应。

DESCRIPTION (provided by applicant): While it has been known for a number of years that mitochondria and non-mitochondrial sources within skeletal muscle produce reactive oxygen species (ROS), the significance of ROS generation has only become clear recently. ROS generation has become implicated in the aging process and numerous disease states, from cancer to heart disease to pathologies related to inflammatory processes. In addition, it has recently become clear that ROS are not only deleterious to some cell functions (inducing apoptosis under extreme conditions) but serve as essential mediators in a number of cell signaling events and have been proposed to modulate important changes in gene expression related to cellular protection from oxidative stress and essential cellular adaptations to exercise. Yet, the factors that modulate ROS metabolism during skeletal muscle contractions and varied conditions of oxygenation remain incompletely understood. In particular, the peculiar behavior of ROS generation during hypoxia, in which higher ROS generation is paradoxically induced despite reduced O2 tensions, has only recently been clearly demonstrated. Finally, while it has been shown that ROS generation in skeletal muscle has significant effects on contractility and muscle function, it remains unclear as to the manner in which generation of ROS in skeletal muscle is dependent on the rate of mitochondrial respiration, work output, intracellular oxygenation, antioxidant buffering, and muscle fiber type. Much of these uncertainties are due to confounding factors related to inhomogeneities within whole muscle models, uncontrolled extracellular environments, variable muscle fiber recruitment patterns, etc. The purpose of this proposal is to use an isolated single mouse skeletal myofiber model, in which the extracellular environment can be precisely controlled and the intracellular environment carefully monitored using non-invasive imaging techniques, to test hypotheses related to delineating the factors regulating ROS generation, the locations of intracellular ROS formation, and ROS modulated gene expression in contracting slow- and fast-twitch fibers under varied oxygenation conditions. In well-controlled experiments in both fiber types with the extracellular PO2 varied during contractions, measurements will be made of mitochondrial respiration and membrane potential, ROS generation and removal, intracellular PO2, contractile function, cytosolic and mitochondrial [Ca2+], numerous intracellular signals related to ROS production, antioxidant protection of cellular integrity, and regulation of gene expression (using QPCR). The strength of this application is in the use of our single muscle fiber model, thereby reducing many of the difficulties related to interpreting results from whole muscle, cell culture, or isolated mitochondria models, and providing data generated from viable, healthy intact myofibers with a normal physiological intracellular environment. This application provides a singular opportunity to study the factors that regulate ROS metabolism in skeletal muscle and thereby has significant implications for understanding and potentially improving human health. PUBLIC HEALTH RELEVANCE. It has only recently become clear that the production of reactive oxygen species during exercise and during low oxygenation conditions has a significant impact on muscle function and adaptation. It is the goal of this proposed research to elucidate the regulatory factors that influence reactive oxygen species production during exercise and low oxygen conditions and how these changes affect muscle performance and the adaptive response to exercise.