Thin filament-reconstituted skinned muscle fibers for the study of muscle physiology

Abstract

We review the use of thin filament-reconstituted muscle fibers in the study of muscle physiology. Thin filament extraction and reconstitution protocol is a powerful technique to study the role of each component of the thin filament. It is also useful for studying the properties of genetically modified molecules such as actin and tropomyosin. We also review the combination of this protocol with sinusoidal analysis, which will provide a solid technique for determining the effect of regulatory proteins on actomyosin interaction and concomitant cross-bridge kinetics. We suggest that thin filament-reconstituted muscle fibers are an ideal system for studying muscle physiology especially when gene modifications of actin or tropomyosin are involved.

Figure 1

Schematic diagram illustrating the thin filament removal and reconstitution protocol. By applying gelsolin to skinned muscle fibers (a), thin filament free muscle fibers (b) can be obtained. By adding actin monomer to these thin filament free muscle fibers, actin-filament-reconstituted muscle fibers without regulatory proteins (c) can be obtained. Thin filament can be reconstituted by adding regulatory proteins to these actin-filament-reconstituted muscle fibers.

Figure 2

Confocal fluorescent micrograph of muscle fibers at each step of thin filament removal and reconstitution. (a) Control cardiac muscle fibers. (b) Gelsolin-treated muscle fibers. (c) Actin-filament-reconstituted muscle fibers. Actin filament was stained with rhodamine-phalloidin. Scale bar: 5 μm.

Figure 3

Kinetic model of actomyosin (AM) ATPase. The main pathway is shown in black and the minor pathway in grey. Upper row is in attached state and lower row is in detached state. A: actin; M: myosin. The asterisks show the difference in the conformation of the molecule.

Figure 4

(a) Phase-contrast image of cardiac myofibril treated with gelsolin. Myofibril was held with two glass needles. Thick needle was moved to the left to stretch the myofibril and force was measured by the bending of the thin needle. Scale bar: 10 μm. (b) Stress-strain relationship of myofibril treated with gelsolin. Two examples are shown. Strain is expressed relative to the slack length.