Dickinson J M, Lee J D, Sullivan B E, Harber M P, Trappe S W, Trappe T A
Human Performance Laboratory, Ball State University, Muncie, IN 47306, USA.
J Appl Physiol (1985). 2010 May;108(5):1410-6. doi: 10.1152/japplphysiol.00905.2009. Epub 2010 Mar 4.
The aim of this study was to develop an approach to directly assess protein fractional synthesis rate (FSR) in isolated human muscle fibers in a fiber type-specific fashion. Individual muscle fibers were isolated from biopsies of the vastus lateralis (VL) and soleus (SOL) obtained from eight young men during a primed, continuous infusion of [5,5,5-(2)H3]leucine performed under basal conditions. To determine mixed protein FSR, a portion of each fiber was used to identify fiber type, fibers of the same type were pooled, and the [5,5,5-(2)H3]leucine enrichment was determined via GC-MS. Processing isolated slow-twitch [myosin heavy chain (MHC) I] and fast-twitch (MHC IIa) fibers for mixed protein bound [5,5,5-(2)H3]leucine enrichment yielded mass ion chromatographic peaks that were similar in shape, abundance, and measurement reliability as tissue homogenates. In the VL, MHC I fibers exhibited a 33% faster (P<0.05) mixed protein FSR compared with MHC IIa fibers (0.068+/-0.006 vs. 0.051+/-0.003%/h). MHC I fibers from the SOL (0.060+/-0.005%/h) and MHC I fibers from the VL displayed similar (P>0.05) mixed protein FSR. Feasibility of processing isolated human muscle fibers for analysis of myofibrillar protein [5,5,5-(2)H3]leucine enrichment was also confirmed in non-fiber-typed pooled fibers from the VL. These methods can be applied to the study of fiber type-specific responses in human skeletal muscle. The need for this level of investigation is underscored by the different contributions of each fiber type to whole muscle function and the numerous distinct adaptive functional and metabolic changes in MHC I and MHC II fibers originating from the same muscle.
本研究的目的是开发一种方法，以纤维类型特异性方式直接评估分离的人肌纤维中的蛋白质分数合成率（FSR）。在基础条件下进行[5,5,5-(2)H3]亮氨酸的预充、持续输注期间，从八名年轻男性的外侧股四头肌（VL）和比目鱼肌（SOL）活检组织中分离出单个肌纤维。为了确定混合蛋白FSR，将每根纤维的一部分用于鉴定纤维类型，将相同类型的纤维汇集在一起，并通过气相色谱-质谱联用仪（GC-MS）测定[5,5,5-(2)H3]亮氨酸的富集情况。处理分离的慢肌纤维[肌球蛋白重链（MHC）I]和快肌纤维（MHC IIa）以测定混合蛋白结合的[5,5,5-(2)H3]亮氨酸富集情况，得到的质量离子色谱峰在形状、丰度和测量可靠性方面与组织匀浆相似。在VL中，MHC I纤维的混合蛋白FSR比MHC IIa纤维快33%（P<0.05）（分别为0.068±0.006与0.051±0.003%/小时）。来自SOL的MHC I纤维（0.060±0.005%/小时）和来自VL的MHC I纤维显示出相似的（P>0.05）混合蛋白FSR。在来自VL的非纤维类型汇集纤维中，也证实了处理分离的人肌纤维以分析肌原纤维蛋白[5,5,5-(2)H3]亮氨酸富集情况的可行性。这些方法可应用于研究人类骨骼肌中纤维类型特异性反应。每种纤维类型对全肌功能的不同贡献以及源自同一肌肉的MHC I和MHC II纤维中众多独特的适应性功能和代谢变化强调了进行这种水平研究的必要性。
