O2 dependence of isolated single skeletal muscle fibers

尽管氧气对维持细胞以及最终的机体内环境平衡至关重要，但几乎没有具体的信息来量化细胞内氧气水平和细胞功能之间的关系。该项目的目标是继续进行实验，以确定单个骨骼肌纤维细胞呼吸、新陈代谢和功能对氧气的依赖性。具体地说，我们将测试单个骨骼肌纤维的一般假设。具体地说，我们将测试一般的假设，即细胞功能受到细胞内[O2]水平远高于那些被认为是速率限制的水平的直接和间接影响。用于分离的线粒体。单个、膜完整的非洲爪哇骨骼肌纤维将被分离出来，放入一个可以精确控制细胞外环境、可以通过刺激改变新陈代谢和呼吸频率、测量Q2摄取和进行光学成像的小室中。为了验证我们最初的假设，我们将在不同的条件下测量细胞内P02(使用磷光寿命技术)，并确定细胞P02与呼吸、糖酵解速率、氧化磷酸化调节因子以及收缩功能调节之间的关系。线粒体密度(和分布)与上述过程之间的关系将在三种不同的非洲爪哇纤维类型中进行研究。此外，还提出了关于肌红蛋白在细胞内O2运输中的重要性、潜在的细胞内O2传感器、与氧合不足有关的细胞损伤原因以及收缩能源的测试假设，这些假设与氧合变化有关。通过利用分离的单个骨骼肌细胞，可以在没有微循环和纤维类型异质性的混杂因素的情况下研究工作细胞的内在特性。提议的实验的原创性和意义在于整合了几种现有的和新的技术来研究细胞功能，因为它受到[O2]的影响，而在该计划项目应用中聚集的人员的独特研究专业知识为研究这些过程提供了一个独特的机会。拟议的研究将提供有价值的信息，确定细胞功能对氧气的依赖；这对细胞、器官和有机体的健康有直接影响，特别是在涉及氧气缺乏的疾病状态下。

As critical as O2 is for maintaining cellular, and ultimately organismal homeostasis, there is little specific information that quantifies the relationship between intracellular O2 levels and cell function. The objective of this project is to continue experimentation designed to determine the O2 dependence of cell respiration, metabolism, and function in isolated single skeletal muscle fibers. Specifically, we will test the general hypotheses single skeletal muscle fibers. Specifically, we will test the general hypotheses that cellular function is affected, both directly and indirectly, by intracellular [O2] levels considerably above those considered rate limiting. for isolated mitochondria. Single, membrane intact, skeletal muscle fibers will be isolated from Xenopus laevis and placed into a chamber in which the extracellular milieu can be precisely controlled, metabolic and respiratory rate can be varied by stimulation, Q2 uptake measured, and optical imaging can be conducted. To test our primary hypothesis, intracellular P02 will be measured (using a phosphorescence lifetime technique) under varied conditions and the relationships between cell P02 and respiratory, glycolytic rate, the regulators of oxidative phosphorylation, and the regulation of contractile function will be determined. The relationship between mitochondrial density (and distribution) and the processes listed above will be examined in three different Xenopus fiber types. In addition, experiments are proposed that test hypotheses concerning the importance of myoglobin in intracellular O2 transport, potential intracellular O2 sensors, causes of cell damage related to inadequate oxygenation, and energy sources for contractile work with altered 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 type heterogeneities. The originality and significance of the proposed experiments residue in the integration of several established and new techniques to investigate cell function as it is affected by [O2], and the unique research expertise of the personnel assembled in this program project 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 involving O2 deprivation.