28 research outputs found
Mechanical and kinetic effects of shortened tropomyosin reconstituted into myofibrils
The effects of tropomyosin on muscle mechanics and kinetics were examined in skeletal myofibrils using a novel method to remove tropomyosin (Tm) and troponin (Tn) and then replace these proteins with altered versions. Extraction employed a low ionic strength rigor solution, followed by sequential reconstitution at physiological ionic strength with Tm then Tn. SDS-PAGE analysis was consistent with full reconstitution, and fluorescence imaging after reconstitution using Oregon-green-labeled Tm indicated the expected localization. Myofibrils remained mechanically viable: maximum isometric forces of myofibrils after sTm/sTn reconstitution (control) were comparable (~84%) to the forces generated by non-reconstituted preparations, and the reconstitution minimally affected the rate of isometric activation (kact), calcium sensitivity (pCa50), and cooperativity (nH). Reconstitutions using various combinations of cardiac and skeletal Tm and Tn indicated that isoforms of both Tm and Tn influence calcium sensitivity of force development in opposite directions, but the isoforms do not otherwise alter cross-bridge kinetics. Myofibrils reconstituted with Î23Tm, a deletion mutant lacking the second and third of Tmâs seven quasi-repeats, exhibited greatly depressed maximal force, moderately slower kact rates and reduced nH. Î23Tm similarly decreased the cooperativity of calcium binding to the troponin regulatory sites of isolated thin filaments in solution. The mechanisms behind these effects of Î23Tm also were investigated using Pi and ADP jumps. Pi and ADP kinetics were indistinguishable in Î23Tm myofibrils compared to controls. The results suggest that the deleted region of tropomyosin is important for cooperative thin filament activation by calcium
Effects of tropomyosin internal deletion Î23Tm on isometric tension and the cross-bridge kinetics in bovine myocardium
Tropomyosin (Tm) spans seven actin monomers and contains seven quasi-repeating, loosely similar regions, 1â7. Deletion of regions 2â3 decreases the in vitro sliding speed of synthetic filaments of actin-Tm-Troponin (Tn), and weakens Tm binding to the actin-myosin subfragment 1 (S1) complex (acto-S1). The thin filament was selectively removed from bovine myocardium by gelsolin, and the actin filament was reconstituted, followed by further reconstitution with Tm and Tn. In this reconstitution, full-length Tm (control) was compared with Tm internal deletion mutant Î23Tm, which lacks residues 47â123 (regions 2â3). The effects of phosphate, MgATP, MgADP and Ca2+ were studied in Tm-reconstituted myocardium and Î23Tm-reconstituted myocardium at pH 7.00 and 25 °C. In Î23Tm, both isometric tension and stiffness were about 40 % of the control. The Hill factor with Î23Tm, deduced from the pCa-tension plot, was 1.4 times that of the control, but the Ca2+ sensitivity was the same. Sinusoidal analysis indicated that the cross-bridge number in force-generating states was not decreased with Î23Tm. We conclude that the thin filament cooperativity is increased with Î23Tm, presumably because of the increased density of the Ca2+-binding sites. We further conclude that tension per cross-bridge is 40 % of control and stiffness per cross-bridge is 40 % of control in Î23Tm. These results are consistent with the idea that Tm modifies the actin-myosin interface so as to increase the stereospecific interaction between moieties of actin and myosin. In Î23Tm, the interface may not have a perfect stereospecific match so that the tension- and stiffness-generating capacity is greatly diminished
A mutation in the α tropomyosin gene TPM3 associated with autosomal dominant nemaline myopathy
Nemaline myopathies are diseases characterized by the presence in muscle fibres of pathognomonic rod bodies. These are composed largely of alphaâactinin and actin. We have identified a missense mutation in the alphaâtropomyosin gene, TPM3, which segregates completely with the disease in a family whose autosomal dominant nemaline myopathy we had previously localized to chromosome 1p13âq25. The mutation substitutes an arginine residue for a highly conserved methionine in a putative actinâbinding site near the N terminus of the alphaâtropomyosin. The mutation may strengthen tropomyosin â actin binding, leading to rod body formation, by adding a further basic residue to the postulated actinâbinding motif