Assessment of the Contractile Properties of Permeabilized Skeletal Muscle Fibers

Abstract

Permeabilized individual skeletal muscle fibers offer the opportunity to evaluate contractile behavior in a system that is greatly simplified, yet physiologically relevant. Here we describe the steps required to prepare, permeabilize and preserve small samples of skeletal muscle. We then detail the procedures used to isolate individual fiber segments and attach them to an experimental apparatus for the purpose of controlling activation and measuring force generation. We also describe our technique for estimating the cross-sectional area of fiber segments. The area measurement is necessary for normalizing the absolute force to obtain specific force, a measure of the intrinsic force-generating capability of the contractile system.

Keywords: Contractility; Cross-sectional area; Isometric force; Muscle physiology; Permeabilized muscle fiber; Single muscle fiber; Skeletal muscle; Specific force.

Fig. 1

Preparation of fiber bundles and extraction of individual fibers. (a) Fresh tissue is placed in a dissecting dish and (b) divided into small bundles of parallel fibers in preparation for permeabilization and freezer storage. (c) On the day of an experiment, a bundle of permeabilized fibers is removed from freezer storage and pinned to a dissection dish. Individual fibers are extracted from the bundle by pulling gently on one end of a fiber with the pulling force applied along the long axis of the fiber. Reproduced from Roche et al. 2015 [4] with permission from JoVE

Fig. 2

Fiber transfer scoop formed by trimming a 100 μL disposable pipette tip. Reproduced from Roche et al. 2015 [4] with permission from JoVE

Fig. 3

Loop of USP 10–0 monofilament nylon suture used to attach the fiber to the experimental apparatus. Note that the knot type is double-overhand, not single-overhand. Reproduced from Roche et al. 2015 [4] with permission from JoVE

Fig. 4

Sequence for attaching fiber to force transducer and servomotor. (a) Thread two suture loops onto the force transducer and servomotor attachment extensions. (b) Transfer fiber to chamber using transfer scoop (see Fig. 2). (c) Secure fiber using two outermost suture ties. (d) Secure fiber using two innermost suture ties. Reproduced from Roche et al. 2015 [4] with permission from JoVE

Fig. 5

Laser light diffracted by the fiber is used to adjust sarcomere length. The laser target screen is calibrated for a specific laser wavelength and “D”, the distance from fiber to screen (see text for explanation). Reproduced from Roche et al. 2015 [4] with permission from JoVE

Fig. 6

Representative recording of maximum isometric force generation. Upon immersion in a high [Ca²⁺] solution, force increases rapidly (top trace) to a steady level (“max”). The servomotor is then controlled to introduce a large, brief reduction in fiber length followed by return to original length (bottom trace). The brief shortening serves to indicate the output of the force transducer when the fiber is slack and force is zero. The large fluctuations near the beginning and end of the force recording are caused by the stepper motors that move the chamber system and by the force transducer extension passing through the liquid-air interface during solution changes. They are not indicative of fiber tension during those times

Fig. 7

Estimation of fiber cross-sectional area. Fiber cross-sectional area is estimated by measuring the “top-view” and “side-view” diameters at several locations (indicated by double-headed arrows), computing the cross-sectional area at each location under the assumption of an elliptical cross section, and then averaging the individual estimates to obtain global fiber cross-sectional area (see text for explanation). Reproduced from Roche et al. 2015 [4] with permission from JoVE