Oxygen dependence of ROS generation in contracting myofibers

收缩肌纤维中ROS产生的氧依赖性

• 批准号: 7613214
• 负责人: MICHAEL C HOGAN
• 金额: $ 41.02万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-08 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7613214
• 关键词: Acetylcysteine; Address; Affect; Antioxidants; Carbohydrates; Cell physiology; Cells; Chronic; Chronic Obstructive Airway Disease; Confocal Microscopy; Contracts; Data; Dependence; Disease; Dithiothreitol; Electric Stimulation; Exercise; Exhibits; Fast-Twitch Muscle Fibers; Fatigue; Fatty Acids; Fiber; Fluorescence; Fluorescent Probes; Functional disorder; Generations; Glycolysis; Health; Heterogeneity; Homeostasis; Hydrogen Peroxide; Hydroxyl Radical; Hypoxia; Image; Impairment; Inflammatory; Investigation; Location; Measures; Mediating; Membrane Potentials; Metabolic; Metabolism; Microcirculation; Mitochondria; Modeling; Mus; Muscle; Muscle Cells; Muscle Fibers; Myopathy; Organ; Oxidants; Oxidative Stress; Oxygen; Patients; Pattern; Performance; Peripheral; Play; Pneumonia; Positioning Attribute; Preparation; Principal Investigator; Process; Production; Property; Protocols documentation; Publications; Quality of life; Ramp; Regulatory Pathway; Research Personnel; Respiration; Role; Ru 360; Skeletal Muscle; Source; Specificity; Superoxides; Supplementation; Testing; Transgenic Model; Work; attenuation; cytokine; density; dihydroethidium; ebselen; extracellular; mitochondrial membrane; originality; preference; programs; research study; respiratory; rhod-2; tempol; uptake

A deleterious systemic effect of chronic obstructive pulmonary disease (COPD) is peripheral muscle dysfunction, a condition that contributes to the exercise intolerance and reduced quality of life of COPD patients. The causes of the peripheral muscle dysfunction have yet to be fully elucidated. It has recently become clear that the hypoxia of COPD, oxidative stress within active muscle, and circulating inflammatory cytokines (particularly TNF-a) are potential mechanisms that may play a role in the muscle myopathy found in many COPD patients. However, there remains a paucity of data at the cellular level concerning the manner in which O2 availability to contracting skeletal muscle affects the interactions between intracellular PO2 (PjO2) and cell homeostasis, metabolic state, reactive O2 species (ROS) generation, and contractile function. We now use an isolated, intact single skeletal muscle fiber model in which slow- or fast-twitch fibers can be isolated from mice. By utilizing this model, intrinsic properties of contracting isolated single myofibers can be investigated without confounding factors related to the microcirculation, fiber recruitment patterns, and fiber type heterogeneities seen in whole muscle. We are proposing to use single isolated myofibers, from both healthy control mice and a model of pulmonary inflammation, to elucidate the regulatory pathways that are affected by P|O2 and thereby alter cellular function. Specifically, we are proposing to conduct experiments related to the general hypothesis that several important cellular regulatory factors (i.e. pH, Ca2+ handling, PI,fuel preference, ROS generation, etc.) are modualted by P|O2 (even at levels that do not inhibit oxidative phophorylation) which thereby modifies contractile function of the muscle fiber in a manner that is fiber type dependent. Our experimental preparation will allow us to precisely control the extracellular milieu, metabolic and respiratory rate will be varied by electrical stimulation, intracellular PO2 measured, specific pharmacological blockades will be induced, and intracellular fluorescent imaging (pH, Ca2+, ROS, PI,etc) will be conducted. The originality and significance of the proposed experiments will be to investigate the O2 dependence of regulatory pathways that determine oxidative stress and single myofiber function, which has important implications related to health at the cellular, organ, and whole body level, particularly during hypoxia or disease states involving hypoxia such as COPD.

慢性阻塞性肺疾病（COPD）的一个有害的全身效应是外周肌肉 功能障碍，一种导致运动不耐受和COPD生活质量下降的疾病 患者外周肌肉功能障碍的原因尚未完全阐明。它最近 COPD的缺氧、活动性肌肉内的氧化应激和循环炎性细胞因子的表达， 细胞因子（特别是TNF-α）是可能在发现的肌肉肌病中起作用的潜在机制， 许多COPD患者。然而，在细胞水平上， 收缩骨骼肌的O2可用性影响细胞内 PO 2（PjO 2）和细胞内稳态、代谢状态、活性氧（ROS）产生和收缩 功能我们现在使用一个分离的、完整的单个骨骼肌纤维模型，在该模型中，慢或快收缩纤维 可以从小鼠中分离。通过利用该模型，收缩的孤立的单肌纤维的内在特性 可以在没有与微循环，纤维募集模式， 以及在整个肌肉中观察到的纤维类型异质性。我们建议使用单个分离的肌纤维， 从健康对照小鼠和肺部炎症模型，以阐明调节途径 受P的影响|从而改变细胞功能。具体来说，我们建议进行 实验涉及几种重要的细胞调节因子（即pH， Ca 2+处理、PI、燃料偏好、ROS生成等）由P调制|O2（即使在这样的水平下 不抑制氧化磷酸化），从而改变肌纤维的收缩功能 其方式取决于纤维类型。我们的实验准备将使我们能够精确地控制 细胞外环境、代谢和呼吸速率将通过电刺激、细胞内PO 2 将诱导测量的、特异性的药理学阻断，并且细胞内荧光成像（pH， Ca 2+、ROS、PI等）。所提出的实验的独创性和意义将是 研究确定氧化应激和单个肌纤维的调节途径的O2依赖性 功能，这对细胞、器官和全身水平的健康具有重要影响， 特别是在缺氧或涉及缺氧的疾病状态如COPD期间。