O2 DEPENDENCE OF ISOLATED SINGLE SKELETAL MUSCLE CELLS

对于细胞的维护，没有几个过程比这更重要 完整性是指细胞对氧气的利用率和利用率 呼吸。就像氧气对维持细胞一样重要，而且 归根结底，生物的动态平衡，几乎没有具体的信息 它量化了细胞内O2水平和细胞之间的关系 功能。本项目的目标是确定氧气的依赖性 单个骨骼的细胞呼吸、新陈代谢和功能 肌肉纤维。具体地说，我们将测试细胞 功能受到细胞内[O2]的直接和间接影响 远高于被认为是孤立的速率限制 线粒体。单一，膜完好，骨骼肌纤维将 从非洲爪哇的髂腓肌中分离出来，放入 一个玻璃毛细管室，细胞外环境可以在其中 精确控制的代谢率和呼吸率可以通过 可以进行刺激、氧气摄取测量和光学成像。 光学成像系统将被用来测量各种 在细胞氧合状态改变下的细胞内过程。为了测试 我们的主要假设是，细胞内的PO2将在不同的 条件和细胞氧合与呼吸之间的关系， 糖酵解率、氧化磷酸化调节因子和 收缩功能的调节将被确定。因为有三个 不同的纤维类型，具有低、中、高线粒体 内容，可以从这块肌肉中分离出来，关系 线粒体密度(和分布)和上述过程 将会被检查。此外，还提出了实验，验证了这一点。 肌红蛋白在细胞内氧气中重要性的假说 运输、潜在的细胞内氧气感受器和细胞损伤的原因 与氧气不足有关。通过利用孤立的单个 骨骼肌细胞，工作细胞的固有属性可以 没有微循环和纤维混杂因素的调查- 对纤维的异质性。的独创性和重要意义 建议的实验驻留在几个已建立的 研究受[O2]影响的单细胞功能的技术。 此外，聚集在一起的人员的独特研究专长 这个应用程序为研究这些问题提供了难得的机会 流程。建议的研究将提供有价值的资料。 定义细胞功能对氧的依赖；这直接 对细胞、器官和有机体健康的影响，特别是 在氧剥夺诱导的疾病状态中。

Few processes are more important for the maintenance of cellular integrity as the availability and utilization of O2 for cellular respiration. As critical as O2 is for maintaining cellular, and ultimately organismal homeostasis, there is little specific information that quantifles the relationship between intracellular O2 levels and cell function. The objective of this project is to determine the O2 dependence of cell respiration, metabolism, and function in isolated single skeletal muscle fibers. Specifically, we will test the hypothesis that cellular function is affected, both directly and indirectly, by intracellular [O2] levels well above those considered rate limiting for isolated mitochondria. Single, membrane intact, skeletal muscle fibers will be isolated from the iliofibularis muscle of Xenopus laevis and placed into a glass capillary chamber in which the extracellular milieu can be precisely controlled, metabolic and respiratory rate can be varied by stimulation, O2 uptake measured, and optical imaging can be conducted. An optical imaging system will be employed to measure various intracellular processes under altered states of cell oxygenation. To test our primary hypothesis, intracellular PO2 will be measured under varied conditions and the relationships between cell PO2 and respiration, glycolytic rate, the regulators of oxidative phosphorylation, and the regulation of contractile function will be determined. Because three different fiber types, with low, intermediate, and high mitochondrial content, can be isolated from this muscle, the relationship between mitochondrial density (and distribution) and the processes listed above will be examined. In addition, experiments are proposed that test hypotheses concerning the importance of myoglobin in intracellular O2 transport, potential intracellular O2 sensors, and causes of cell damage related to inadequate oxygenation. By utilizing an isolated single skeletal muscle cell, intrinsic properties of the working cell can be investigated without confounding factors of microcirculation and fiber- to-fiber heterogeneities. The originality and significance of the proposed experiments reside in the integration of several established techniques to investigate single cell function as it is affected by [O2]. In addition, the unique research expertise of the personnel assembled in this application provides a singular opportunity to study these processes. The proposed studies will provide valuable information defining the O2 dependence of cellular function; which has direct implications concerning cell, organ, and organismal health, particularly during disease states induced by O2 deprivation.