Oxygen dependence of ROS generation in contracting single isolated myofibers

收缩单根分离肌纤维中活性氧生成的氧依赖性

基本信息

批准号：
8053752
负责人：
MICHAEL C HOGAN
金额：
$ 32.3万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-09-30 至 2013-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8053752
关键词：
Address Affect Aging Aging-Related Process Antimycin A Antioxidants Apoptosis Area Behavior Buffers Cell Culture Techniques Cell Death Cell Hypoxia Cell physiology Cells Cellular biology Complex Confocal Microscopy Contracts Data Dependence Disease Electron Transport Electron Transport Complex III Electrons Environment Event Excision Exercise Fast-Twitch Muscle Fibers Fatigue Fiber Fluorescence Fluorescent Probes Frequencies Gene Expression Gene Expression Regulation Generations Goals HSP72 protein Health Heart Diseases Heterogeneity Human Hydrogen Peroxide Hydroxyl Radical Hypoxia Image Imaging Techniques Inflammatory Investigation Laboratories Lead Life Link Location Malignant Neoplasms Measurement Measures Mediator of activation protein Membrane Potentials Messenger RNA Metabolic Metabolism Mitochondria Modeling Monitor Mus Muscle Muscle Cells Muscle Contraction Muscle Fibers Muscle function NG-Nitroarginine Methyl Ester Nitric Oxide Nitric Oxide Synthase Normal Cell Output Oxidants Oxidases Oxidative Phosphorylation Oxidative Stress Oxygen Oxygen measurement, partial pressure, arterial PPAR gamma Pathology Pattern Performance Physiological Positioning Attribute Preparation Process Production Property Proteins Protocols documentation Reactive Oxygen Species Reporting Research Respiration Rest Role Rotenone Signal Pathway Signal Transduction Skeletal Muscle Slow-Twitch Muscle Fibers Source Specificity Superoxide Dismutase Superoxides Techniques Testing Time Uncertainty Vascular Endothelial Growth Factors Work arginine methyl ester cell injury density dihydroethidium diphenyleneiodonium ebselen extracellular improved inhibitor/antagonist mRNA Expression mitochondrial membrane novel receptor research study respiratory response rhod-2 skeletal uptake

项目摘要

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.

描述（由申请人提供）：虽然已经知道骨骼肌内的线粒体和非点部分来源多年来产生活性氧（ROS），但ROS产生的意义仅在最近才明确。 ROS的产生已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有关的病理。此外，最近已经很清楚，ROS不仅对某些细胞功能有害（在极端条件下诱导凋亡），而且还可以作为许多细胞信号事件中的必不可少的介体，并且已提议调节与氧化应激相关的基因表达的重要变化，从而免受氧化应激和必要的细胞适应运动。然而，在骨骼肌收缩期间调节ROS代谢的因素和多种氧合条件尚不完全了解。特别是，直到最近才清楚地证明，在缺氧过程中，ROS产生在缺氧过程中的特殊行为是矛盾的，尽管O2张力减少了。最后，虽然已经证明骨骼肌中的ROS产生对收缩力和肌肉功能具有重大影响，但尚不清楚骨骼肌中ROS的产生依赖于线粒体呼吸的速度，工作输出，细胞内氧合，抗氧化剂的液化和肌肉纤维类型。这些不确定性的大部分是由于与整个肌肉模型中的不均匀性，不受控制的细胞外环境，可变的肌肉纤维募集模式等相关的混杂因素所致。该提议的目的是使用孤立的单小鼠骨骼肌纤维模型，在该模型中，使用细胞外环境可以精心控制的验证，并仔细地监控了内部控制的环境。与描述调节ROS产生的因素，细胞内ROS形成的位置以及ROS调节基因在不同的氧合条件下的慢速和快速交换纤维中的基因表达有关。 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）。这种应用的强度在于使用我们的单肌纤维模型，从而减少了与解释整个肌肉，细胞培养或孤立的线粒体模型的解释结果相关的许多困难，并提供了具有正常生理内细胞内环境的可行，健康完整的肌纤维产生的数据。该应用为研究调节骨骼肌中ROS代谢的因素提供了一个单一的机会，从而对理解和潜在改善人类健康具有重要意义。公共卫生相关性。直到最近才清楚地表明，在运动过程和低氧化条件下，活性氧的产生对肌肉功能和适应性产生了重大影响。这项拟议的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性能以及对运动的适应性反应。