O2 dependence of isolated single skeletal muscle fibers

由于O2对于维持细胞和最终生物体内平衡至关重要，因此很少有具体信息可以量化细胞内O2水平与细胞功能之间的关系。本项目的目的是继续实验，旨在确定O2依赖的细胞呼吸，代谢和功能在孤立的单一骨骼肌纤维。具体来说，我们将测试一般假设单骨骼肌纤维。具体来说，我们将测试的一般假设，细胞功能的影响，直接和间接，细胞内[O2]水平大大高于那些被认为是限速。分离的线粒体。将从非洲爪蟾中分离出单个膜完整的骨骼肌纤维，并将其置于一个室中，在该室中可以精确控制细胞外环境，可以通过刺激改变代谢和呼吸速率，测量Q2摄取，并可以进行光学成像。为了检验我们的主要假设，将在不同条件下测量（使用磷光寿命技术）细胞内P02，并将确定细胞P02与呼吸、糖酵解速率、氧化磷酸化的调节剂和收缩功能的调节之间的关系。将在三种不同的爪蟾纤维类型中检查线粒体密度（和分布）与上述过程之间的关系。此外，实验提出，测试假设肌红蛋白在细胞内O2运输，潜在的细胞内O2传感器的重要性，与氧合不足有关的细胞损伤的原因，和能量来源的收缩工作与改变氧合。通过利用分离的单个骨骼肌细胞，可以研究工作细胞的内在特性，而没有微循环和纤维类型异质性的混杂因素。建议的实验的原创性和意义残留在几个既定的和新的技术的整合，以调查细胞功能，因为它是由[O2]的影响，并在此程序项目应用程序中组装的人员的独特的研究专业知识提供了一个独特的机会来研究这些过程。拟议的研究将提供有价值的信息，定义细胞功能的O2依赖性;这对细胞，器官和生物体健康有直接影响，特别是在涉及O2剥夺的疾病状态下。

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.