Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
Nat Protoc. 2017 Sep;12(9):1763-1776. doi: 10.1038/nprot.2017.056. Epub 2017 Aug 3.
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/m) 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 Ca is the principal regulator of the contractile force, measurements of myoplasmic free [Ca] ([Ca]) can be used to further delineate the intrinsic mechanisms underlying changes in skeletal muscle function. [Ca] measurements are most commonly performed in intact single fibers using ratiometric fluorescent indicators such as indo-1 or fura-2. These Ca indicators are introduced into the fiber by pressure injection or by using the membrane-permeable indo-1 AM, and [Ca] is measured by calculating a ratio of the fluorescence at specific wavelengths emitted for the Ca-free and Ca-bound forms of the dye. We describe here the procedures for mechanical dissection, and for force and [Ca] 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 Ca indicators, single fibers can also be loaded with fluorescent indicators to measure, for instance, reactive oxygen species, pH, and [Mg], 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.
机械分离单个完整的哺乳动物骨骼肌纤维可实时测量活纤维的细胞内特性和收缩功能。与酶法纤维解离相比，机械分离的一个主要优势是可以在保持肌腱附着的情况下分离单个纤维，从而可以在电刺激过程中测量收缩力（在正常预期的 300-450 kN/m 范围内）。此外，机械分离后单个纤维的肌膜仍然保持完整，因此可以用完整的细胞内环境、正常的功能和代谢特性以及离子控制来研究活纤维。由于 Ca 是收缩力的主要调节剂，因此可以测量细胞质游离 [Ca]（[Ca]），以进一步阐明骨骼肌功能变化的内在机制。[Ca]的测量最常用于完整的单个纤维中，使用比率荧光指示剂，如 indo-1 或 fura-2。这些 Ca 指示剂通过压力注射或使用膜通透 indo-1 AM 引入纤维中，并通过计算染料的 Ca 自由和 Ca 结合形式发出的特定波长的荧光比值来测量 [Ca]。我们在这里描述了从小鼠屈趾短肌（FDB）中机械分离、测量完整单个纤维的力和 [Ca]的程序，这是最常用于完整单个纤维研究的肌肉。该技术还可用于从各种肌肉和各种物种中分离完整的单个纤维。作为 Ca 指示剂的替代方法，也可以将单个纤维加载荧光指示剂来测量例如活性氧、pH 和 [Mg]，或者可以将蛋白质注入纤维以改变功能特性。从分离到单个纤维实验开始的整个过程大约需要 3 小时。
