Oxygen dependence of ROS generation in contracting myofibers

慢性阻塞性肺疾病(COPD)的一个有害的全身影响是外周肌肉 功能障碍，这是导致COPD运动不耐受和生活质量下降的一种情况 病人。外周肌功能障碍的原因尚未完全阐明。最近是这样的 现已明确，COPD的低氧、活跃肌肉内的氧化应激和循环炎症 细胞因子(尤其是肿瘤坏死因子-a)是可能在肌肉肌病中发挥作用的潜在机制。 在许多慢性阻塞性肺疾病患者中。然而，在细胞水平上仍然缺乏关于 骨骼肌收缩时氧气供应影响细胞内相互作用的方式 PO2(PjO2)与细胞动态平衡、代谢状态、活性氧(ROS)生成和收缩 功能。我们现在使用一种分离的、完整的单一骨骼肌纤维模型，在该模型中，慢或快抽动纤维 可以从老鼠身上分离出来。利用这一模型，分离的单个肌纤维收缩的内在特性 可以在没有与微循环、纤维募集模式、 整个肌肉中可见纤维类型的异质性。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，阐明调节途径 会受到P|O2的影响，从而改变细胞功能。具体地说，我们正在提议进行 实验涉及几个重要的细胞调节因子(即pH， Ca2+处理、PI、燃料偏好、ROS生成等)被P|O2模化(即使在 不抑制氧化磷酸化)，从而改变肌肉纤维的收缩功能 以依赖于纤维类型的方式。我们的实验准备将使我们能够精确地控制 细胞外环境、代谢和呼吸频率会因电刺激、细胞内PO2而改变。 被测量的、特定的药物阻断将被诱导，并且细胞内荧光成像(pH， Ca~(2+)、ROS、PI等)。拟议实验的原创性和意义将是 研究决定氧化应激和单个肌纤维的调节通路的氧依赖性 功能，这对细胞、器官和全身层面的健康具有重要意义， 特别是在缺氧或涉及缺氧的疾病状态(如COPD)期间。

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.