title: The immediate effect of HCM causing actin mutants E99K and A230V on actin-Tm-myosin interaction in thin-filament reconstituted myocardium.
abstract: Human cardiac actin mutants E99K and A230V were expressed with baculovirus/insect cells and used to reconstitute the thin-filament of bovine cardiac (BVC) muscle fibers, together with tropomyosin (Tm) and troponin (Tn) purified from bovine ventricles. Effects of [Ca(2+)], [ATP], and [phosphate] on tension and its transients were studied at 25°C. In the absence of Tm/Tn, both mutants significantly decreased the tension of actin filament reconstituted fibers (WT: 0.75±0.06 T0, E99K: 0.58±0.04 T0, A230V: 0.58±0.03 T0), where T0 is active tension of native fibers (T0=26.9±1.1kPa, N=41), indicating diminished actin-myosin interactions. However, in the presence of Tm and Tn, WT, E99K, and A230V recovered tension (0.85±0.06 T0, 0.89±0.06 T0, and 0.85±0.05 T0, respectively), demonstrating the compensatory effect of Tm/Tn. Ca(2+) sensitivity (pCa50) increased (5.59±0.02, 5.80±0.03, 5.77±0.03, respectively) and cooperativity (nH) decreased (2.6±0.3, 1.87±0.21, 1.60±0.11, respectively). The kinetic constants of the cross-bridge cycle were deduced using sinusoidal analysis. E99K did not show any significant changes in any of the kinetic constants compared to those of WT. A230V caused a decrease in K1 (ATP association constant), k2 and k-2 (rate constants of the cross-bridge detachment step). The cross-bridge distribution was similar among WT, E99K, and A230V. In conclusion, our experiments demonstrate that the first step of HCM pathogenesis with E99K is increased pCa50 and decreased nH, which result in larger tension during partial activation to cause a diastolic problem. The effect on nH is more severe with A230V. In addition, A230V has a problem of decreased cross-bridge kinetics, which affects the normal functions of the cross-bridge cycle and may contribute to the first step of the HCM pathogenesis.
authors.affiliation: Department of Anatomy and Cell Biology, and Internal Medicine, The University of Iowa, Iowa City, IA 52242-1109, USA. Electronic address: Fan-Bai@uiowa.edu.
authors.forename: Fan
authors.initials: F
authors.lastname: Bai
authors.affiliation: Department of Anatomy and Cell Biology, and Internal Medicine, The University of Iowa, Iowa City, IA 52242-1109, USA. Electronic address: casterha@gmail.com.
authors.forename: Hannah M
authors.initials: HM
authors.lastname: Caster
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, College of Biological Science, Guelph, Ontario N1G 2W1, Canada. Electronic address: jdawso01@uoguelph.ca.
authors.forename: John F
authors.initials: JF
authors.lastname: Dawson
authors.affiliation: Department of Anatomy and Cell Biology, and Internal Medicine, The University of Iowa, Iowa City, IA 52242-1109, USA. Electronic address: Masataka-Kawai@uiowa.edu.
authors.forename: Masataka
authors.initials: M
authors.lastname: Kawai
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001696:Biomechanical Phenomena; D002118:Calcium / Q000378:metabolism; D002312:Cardiomyopathy, Hypertrophic / Q000378:metabolism* / Q000473:pathology; D002417:Cattle; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D058105:Polymerization; D011485:Protein Binding; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000302:isolation & purification; D014335:Tropomyosin / Q000378:metabolism*; D014336:Troponin
chemical_list: D000199:Actins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D014335:Tropomyosin; D014336:Troponin; D009218:Myosins; D002118:Calcium
keywords: Ca(2+) sensitivity; Cross-bridge; Elementary steps; Kinetics; Sinusoidal analysis; Tropomyosin