Use of thin filament reconstituted muscle fibres to probe the mechanism of force generation

Abstract

The technique of selective removal of the thin filament by gelsolin in bovine cardiac muscle fibres, and reconstitution of the thin filament from isolated proteins is reviewed, and papers that used reconstituted preparations are discussed. By comparing the results obtained in the absence/presence of regulatory proteins tropomyosin (Tm) and troponin (Tn), it is concluded that the role of Tm and Tn in force generation is not only to expose the binding site of actin to myosin, but also to modify actin for better stereospecific and hydrophobic interaction with myosin. This conclusion is further supported by experiments that used a truncated Tm mutant and the temperature study of reconstituted fibres. The conclusion is consistent with the hypothesis that there are three states in the thin filament: blocked state, closed state, and open state. Tm is the major player to produce these effects, with Tn playing the role of Ca 2+ sensing and signal transmission mechanism. Experiments that changed the number of negative charges at the N-terminal finger of actin demonstrates that this part of actin is essential to promote the strong interaction between actin and myosin molecules, in addition to the well-known weak interaction that positions the myosin head at the active site of actin prior to force generation. KeywordsActin; Regulatory proteins; Tropomyosin; Troponin; Myosin; Gelsolin; Cross-bridge kinetics; Sinusoidal analysis; BDM Thin filament reconstituted muscle fibre systemFor the purpose of understanding the cross-bridge interaction with actin and the role of regulatory proteins in this interaction, we reconstituted the thin filament in cardiac muscle fibres. This method is schematically represented in Fig. 1. The thin filament is first removed by gelsolin (Fig. 1A to 1B), followed by sequential reconstitution with G-actin (Fig. 1C), and regulatory proteins, tropomyosin (Tm) and troponin (Tn) (Fig. 1D). Gelsolin is an actin and thin filament severing protein, hence its action is specific and it does not disrupt other sarcomeric structure. However, overtreatment with gelsolin would disrupt the Z-line structure, because actin is one of the main constituents in the Z-line. The thin filament reconstitution method was initially developed at Ishiwata's laboratory and the key properties of the Correspondence to: Masataka Kawai, masataka-kawai@uiowa.edu. reconstituted fibres, such as the recovery of isometric tension, the tension-pCa relationship, and the function of spontaneous oscillatory contraction, were examined on skeletal fibres (Funatsu et al. 1994), followed by cardiac fibres (Fujita et al. 1996; Ishiwata 1998, 1999; for brief review see Ishiwata et al. 1998). In these works, it was demonstrated that bovine cardiac fibres were successful as the reconstituted system, in which actin, Tm and Tn can be replaced with those prepared from other muscles, for example, rabbit skeletal muscle proteins (Fujita et al. 1996;Fujita and Ishiwata 1999). The reconstitution method in cardiac fibres was successfully applied at ...

Cardiac Troponin T, a Sarcomeric AKAP, Tethers Protein Kinase A at the Myofilaments

“…Gelsolin is a Ca 2ϩ -dependent actin filament-severing molecule (31). It selectively removes cardiac thin filaments leaving behind relatively intact thick, intermediate and titin filaments, anchored at the Z lines (32)(33)(34). Fig.…”

Regulatory mechanism of smooth muscle contraction studied with gelsolin-treated strips of taenia caeci in guinea pig

“…6. Note that we did not apply 2,3-butanedione monoxime (BDM) during gelsolin treatment as has been done in reconstitution experiments on striated muscles (9,10,29), since BDM had little effect on the skinned smooth muscle (59). As summarized in Fig.…”

“…The removal and reconstitution of the thin filaments in smooth muscle were done according to the technique previously developed for striated muscle with slight modifications (9,10,12,13,29). That is, to remove the thin filaments from smooth muscle cells, the skinned muscle tissue was immersed into the contracting solution containing 0.4 mg/ml gelsolin on ice in the cold room for 90 min.…”

“…The potential role of the thin filament-associated regulatory proteins such as tropomyosin (Tm), caldesmon (CaD), and calponin (CaP) have been described in several types of smooth muscles (1,16,34,41,45,58). However, considerable controversy remains on the putative physiological significance of these proteins.An experimental approach of selective removal and reconstitution of the components in striated muscle has been proved to be a powerful technique for studying the dynamics of reconstituted actin filament assembly and functional roles of thin filament-associated proteins in the reconstituted skeletal (13) and cardiac muscles (9,10,29). In this study, we have created a novel system of selective removal and reconstitution of the components to examine the physiological functions of the thin filament-associated proteins Tm and CaD in the regulation of smooth muscle contraction.…”

“…An experimental approach of selective removal and reconstitution of the components in striated muscle has been proved to be a powerful technique for studying the dynamics of reconstituted actin filament assembly and functional roles of thin filament-associated proteins in the reconstituted skeletal (13) and cardiac muscles (9,10,29). In this study, we have created a novel system of selective removal and reconstitution of the components to examine the physiological functions of the thin filament-associated proteins Tm and CaD in the regulation of smooth muscle contraction.…”