O2 Dependence of Isolated Single Skeletal Muscle Cells

描述(由申请人提供)：低氧血症既是多种人类疾病的致病因素，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，低氧对来自细胞内外来源的碳水化合物利用的影响会受到组织氧合程度的强烈影响，这些变化可能会对骨骼肌功能产生重大影响--即使细胞内的PO2可能远远高于充分的线粒体呼吸所需的氧分压。然而，在不同类型的肌肉纤维中，在氧气利用率降低的情况下，导致骨骼肌代谢和收缩功能改变的具体机制的信息很少。了解特定纤维类型在缺氧时骨骼肌功能障碍的相关机制具有重要意义，因为在某些病理(如糖尿病)和衰老过程中，整个肌肉的纤维类型组成会发生变化。该项目的主要目标是在高强度和/或耐力运动中，使用分离的单一骨骼肌纤维模型(来自青蛙和小鼠的慢和快抽动纤维)，在细胞水平上检验与低氧导致骨骼肌代谢和功能改变的机制有关的一些假说。使用该模型，可以精确地控制细胞外环境，可以通过刺激改变代谢和呼吸频率，可以监测收缩性能，可以测量细胞内的PO2和O2摄取，不同类型的荧光成像可以非侵入性地监测细胞内的事件(即pH、钙处理、葡萄糖摄取等)。这一提议的一个特别令人兴奋的方面是使用转基因小鼠的单一纤维来解决一些假设。通过使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。提出的实验的原创性和意义在于整合了几种已有的和新的技术，以确定缺氧和葡萄糖供应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难得的机会来解决长期存在的关于不同骨骼肌纤维类型的细胞代谢和功能对氧的依赖的问题；这对细胞、器官和组织的健康具有直接的影响，特别是在涉及代谢障碍和氧气缺乏的疾病状态下。

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.