O2 dependence of isolated single skeletal muscle fibers

尽管O2对于维持细胞和最终的有机体稳态至关重要，但很少有具体的信息可以量化细胞内O2水平与细胞功能之间的关系。该项目的目的是继续实验设计，以确定细胞呼吸，代谢和功能的O2依赖性在分离的单个骨骼肌纤维。具体来说，我们将测试单一骨骼肌纤维的一般假设。具体来说，我们将测试一般假设，即细胞内[O2]水平明显高于被认为的速率限制，直接或间接地影响细胞功能。对于分离的线粒体。将从非洲爪蟾中分离出单个、膜完整的骨骼肌纤维，并将其放入一个可以精确控制细胞外环境、通过刺激改变代谢和呼吸速率、测量Q2摄取并进行光学成像的室中。为了验证我们的主要假设，将在不同条件下测量细胞内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和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爪蟾纤维类型中进行研究。此外，研究人员还提出了一些实验来验证肌红蛋白在细胞内氧运输中的重要性、潜在的细胞内氧传感器、与充氧不足相关的细胞损伤原因以及充氧改变时收缩运动的能量来源等假设。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，而不受微循环和纤维类型异质性的干扰。提出的实验的独创性和意义在于整合了几种已建立的和新的技术来研究细胞功能，因为它受到[O2]的影响，并且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来定义细胞功能对氧的依赖性；这对细胞、器官和机体健康有直接影响，特别是在涉及氧气剥夺的疾病状态下。

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.