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Mechanical isolation, and measurement of force and myoplasmic free [Ca2+] in fully intact single skeletal muscle fibers.
Nature Protocols ( IF 14.8 ) Pub Date : 2017-Sep-01 , DOI: 10.1038/nprot.2017.056
Arthur J Cheng , Håkan Westerblad

Mechanical dissection of single intact mammalian skeletal muscle fibers permits real-time measurement of intracellular properties and contractile function of living fibers. A major advantage of mechanical over enzymatic fiber dissociation is that single fibers can be isolated with their tendons remaining attached, which allows contractile forces (in the normal expected range of 300-450 kN/m2) to be measured during electrical stimulation. Furthermore, the sarcolemma of single fibers remains fully intact after mechanical dissection, and hence the living fibers can be studied with intact intracellular milieu and normal function and metabolic properties, as well as ionic control. Given that Ca2+ is the principal regulator of the contractile force, measurements of myoplasmic free [Ca2+] ([Ca2+]i) can be used to further delineate the intrinsic mechanisms underlying changes in skeletal muscle function. [Ca2+]i measurements are most commonly performed in intact single fibers using ratiometric fluorescent indicators such as indo-1 or fura-2. These Ca2+ indicators are introduced into the fiber by pressure injection or by using the membrane-permeable indo-1 AM, and [Ca2+]i is measured by calculating a ratio of the fluorescence at specific wavelengths emitted for the Ca2+-free and Ca2+-bound forms of the dye. We describe here the procedures for mechanical dissection, and for force and [Ca2+]i measurement in intact single fibers from mouse flexor digitorum brevis (FDB) muscle, which is the most commonly used muscle in studies using intact single fibers. This technique can also be used to isolate intact single fibers from various muscles and from various species. As an alternative to Ca2+ indicators, single fibers can also be loaded with fluorescent indicators to measure, for instance, reactive oxygen species, pH, and [Mg2+], or they can be injected with proteins to change functional properties. The entire protocol, from dissection to the start of an experiment on a single fiber, takes ∼3 h.

中文翻译:

机械隔离,并测量完全完整的单个骨骼肌纤维中的力和肌浆游离[Ca2 +]。

机械解剖单个完整的哺乳动物骨骼肌纤维可以实时测量活体纤维的细胞内特性和收缩功能。机械解离酶促纤维解离的主要优势在于,单根纤维可以在其肌腱保持附着的状态下进行分离,从而可以在电刺激过程中测量收缩力(正常预期范围为300-450 kN / m 2)。此外,单根纤维的肌瘤在机械解剖后仍保持完整,因此可以研究具有完整细胞内环境,正常功能和代谢特性以及离子控制的活性纤维。假设Ca 2+是收缩力的主要调节剂,则测量肌浆游离[Ca2+ ]([Ca 2+ ] i)可用于进一步描述骨骼肌功能变化的内在机制。[Ca 2+ ] i测量最常见的是使用比例荧光指示剂(如indo-1或fura-2)在完整的单纤维中进行。这些Ca 2+指示剂通过压力注入或使用可透过膜的indo-1 AM引入到纤维中,[Ca 2+ ] i通过计算Ca 2+在特定波长发射的荧光比率来测量。-和Ca 2+结合形式的染料。我们在这里描述了从小鼠屈指短肌(FDB)肌肉获得的完整单纤维中的机械解剖,力和[Ca 2+ ] i测量的程序,这是使用完整单纤维的研究中最常用的肌肉。该技术还可用于从各种肌肉和各种物种中分离完整的单纤维。作为Ca 2+指示剂的替代方法,单纤维也可以加载荧光指示剂,以测量例如活性氧,pH和[Mg 2+ ],或者可以向它们注入蛋白质以改变功能特性。从解剖到在一条光纤上进行实验的整个过程需要大约3个小时。
更新日期:2017-09-06
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