Thin Filament‐Reconstituted Skinned Muscle Fibers for the Study of Muscle Physiology

Sayaka Higuchi,Norio Fukuda,Satoshi Kurihara,Yoshikazu Tsukasaki,Hideaki Fujita

doi:10.1155/2011/486021

Sayaka Higuchi, Norio Fukuda + Show 3 more

Open Access

https://doi.org/10.1155/2011/486021

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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.

Highlights

Striated muscle has a highly ordered structure where muscle proteins are arranged in an array of filaments [1, 2]
In the in vitro motility assay system, the higher-order structure of thin filaments is reconstituted but myosin or the motor domain of myosin is randomly placed on glass-coverslip, which differs from the physiological condition
Study of the Effect of Regulatory Proteins in Muscle Physiology Using Thin FilamentReconstituted Skinned Muscle Fibers. Unlike small proteins such as troponin or myosin light chains, tropomyosin is bound to seven actin monomers and it is extremely difficult to remove without damaging other muscle proteins

Summary

Introduction

Striated muscle has a highly ordered structure where muscle proteins are arranged in an array of filaments [1, 2]. A new in vitro assay system was developed which used an isolated A-band and reconstituted actin filament to measure the active force developed in the thick filament lattice structure [8] This approach enables the study of the physiology of muscle proteins preserving higher-order structure of muscle but, because the preparation of A-band is labor consuming and the setup is relatively complicated, this technique did not become widely used. Thin filament-reconstituted muscle fibers are an ideal model for studying the physiology of thin filament proteins [21] Unlike small proteins such as troponin or myosin light chains, tropomyosin is bound to seven actin monomers and it is extremely difficult to remove without damaging other muscle proteins. The prediction made by the actin-filament-reconstituted model was shown to reflect the properties of muscle

Combination with Sinusoidal Analysis

Studying Titin Using Thin Filament-Free Muscle Fibers

Findings

Conclusions