O2 Dependence of Isolated Single Skeletal Muscle Cells

描述（由申请人提供）：低氧血症既是许多人类病理的病因，也是其结果。低氧血症对细胞功能的有害影响可能是深远的。特别是，缺氧对细胞内和细胞外来源的碳水化合物利用的影响可能受到组织氧合程度的强烈影响，这些变化可能对骨骼肌功能产生重大影响——即使细胞内PO2可能远高于足够的线粒体呼吸所必需的水平。然而，在各种肌纤维类型的氧可用性降低的情况下，导致骨骼肌代谢和收缩功能改变的具体机制的信息缺乏。了解特定纤维类型在缺氧期间骨骼肌功能障碍的相关机制是重要的，因为随着某些病理（如糖尿病）和衰老，整个肌肉中的纤维类型组成会发生改变。本项目的主要目标是在细胞水平上，利用一个分离的单一骨骼肌纤维模型（来自青蛙和老鼠的慢速和快动纤维），在高强度和/或耐力工作期间，检验与缺氧诱导骨骼肌代谢和功能改变机制相关的一系列假设。使用该模型，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，可以监测收缩性能，测量细胞内PO2和O2摄取，并且不同类型的荧光成像可以无创地监测细胞内事件（即pH， Ca2+处理，葡萄糖摄取等）。这项提议的一个特别令人兴奋的方面是使用转基因小鼠的单个纤维来解决一些假设。通过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，以确定缺氧和葡萄糖可用性在细胞水平上改变肌肉代谢和功能的机制。拟议的研究将提供一个独特的机会来解决长期存在的问题，即不同骨骼肌纤维类型的细胞代谢和功能对O2的依赖性；这对细胞、器官和机体健康有直接影响，特别是在涉及代谢紊乱和氧气剥夺的疾病状态下。

DESCRIPTION (provided by applicant): Hypoxemia is both a causative agent for, and a result of, numerous human pathologies. The deleterious effects of hypoxemia on cellular function can be profound. In particular, the effect of hypoxia on carbohydrate utilization from both intra- and extracellular sources can be strongly influenced by the degree of tissue oxygenation, and these changes can have significant consequences on skeletal muscle function-even when the intracellular PO2 may be well above that necessary for adequate mitochondrial respiration. However, there is a paucity of information concerning the specific mechanisms that result in alterations in skeletal muscle metabolism and contractile function during conditions of reduced O2 availability in the various muscle fiber types. Understanding the mechanisms related to skeletal muscle dysfunction during hypoxia in the specific fiber types is significant because of the alterations in fiber type composition in whole muscle that occurs with certain pathologies (such as diabetes) and aging. The principle objective of this project is to examine on a cellular level a number of hypotheses related to the mechanisms of hypoxia-induced alterations in skeletal muscle metabolism and function using an isolated single skeletal muscle fiber model (slow- and fast-twitch fibers from frogs and mice) during high-intensity and/or endurance work. Using this model, the extracellular milieu can be precisely controlled, metabolic and respiratory rates can be varied by stimulation, contractile performance can monitored, intracellular PO2 and O2 uptake measured, and different types of fluorescent imaging can non-invasively monitor intracellular events (i.e. pH, Ca2+ handling, glucose uptake, etc.). A particularly exciting aspect of this proposal is the use of single fibers from transgenic mice to address some of the hypotheses. By using 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 reside in the integration of several established and new techniques to determine the mechanisms by which hypoxia and glucose availability alters muscle metabolism and function on a cellular level. The proposed studies will provide a singular opportunity to address long-standing questions concerning the O2 dependence of cellular metabolism and function in the different skeletal muscle fiber types; which has direct implications concerning cell, organ, and organismal health, particularly during disease states involving metabolic disorders and O2 deprivation.