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Mechanical isolation, and measurement of force and myoplasmic free [Ca2+] in fully intact single skeletal muscle fibers

Nature Protocols volume 12, pages 1763–1776 (2017)Cite this article

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Abstract

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.

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Figure 1: Dissection of the flexor digitorum brevis (FDB) muscle and digit.
Figure 2: Dissection of a single FDB fiber.
Figure 3: Optimal micropipette tip shape.
Figure 4: Establishment of Rmin and Rmax values in fibers injected with indo-1.
Figure 5: Force and [Ca2+]i in a fiber tetanically stimulated (70 Hz, 350-ms duration) at optimal length (Lo) and after being stretched.
Figure 6: The concentration of indo-1 loaded into the fiber affects [Ca2+]i and force measurement.

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Acknowledgements

J. Lännergren (Karolinska Institutet) was essential in designing the dissection procedures and the custom-made equipment for dissection, stimulation, and force recording described in this protocol. This work was supported by a Swedish Research Council grant to H.W. and by Swedish Research Council for Sport Science grants to A.J.C. and H.W.

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  1. Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden

    Arthur J Cheng & Håkan Westerblad

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  1. Arthur J Cheng
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A.J.C. performed the experiments and data collection. A.J.C. and H.W. were equally involved in drafting the manuscript.

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Correspondence to Arthur J Cheng.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Dissection trough schematic.

General dimensions of the dissection trough are shown in the illustration (red arrows). The muscle bundle is held by inserting the tendons into the split ends of the nylon rod located in the middle of the chamber (blue). The nylon rod is pulled into a tight-fitting stainless steel tube, thus closing the split end onto the tendon. A polyethylene tube is inserted into the frame to fit the stainless steel tube tightly into the acrylic frame to prevent leakage of Tyrode, while at the same time allowing rotation and longitudinal movements. Silicon grease can be added between the stainless steel tube and the tube insert to further prevent leakage of Tyrode. The shelf is useful for stabilizing the dissection tools during dissection, and it can also be used as a shelf to mount the dissected single fiber into T-clips. The glass bottom allows for darkfield illumination. Tyrode is typically filled to the top surface of the chamber.

Supplementary Figure 2 Stimulation pen.

General dimensions of a custom-made stimulation pen are shown in the illustration. Two common insulated multi-strand copper wires are inserted into the body of a ballpoint pen and are intertwined with the teflon-coated platinum wires. Note that it is not possible to solder copper wire to platinum wire using typical soldering metal. The teflon-coating on the platinum wire ends must be removed to conduct an electrical current. Epoxy is filled into the tip to prevent the wires from moving.

Supplementary information

Combo PDF

Supplementary Figures 1 and 2. (PDF 234 kb)

Single fiber dissection from the mouse FDB muscle.

This video describes how to dissect an intact single fiber starting from the whole isolated FDB muscle. (MP4 27001 kb)

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Cheng, A., Westerblad, H. Mechanical isolation, and measurement of force and myoplasmic free [Ca2+] in fully intact single skeletal muscle fibers. Nat Protoc 12, 1763–1776 (2017). https://doi.org/10.1038/nprot.2017.056

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