Journal article
The immediate effect of HCM causing actin mutants E99K and A230V on actin–Tm–myosin interaction in thin-filament reconstituted myocardium
Journal of molecular and cellular cardiology, Vol.79, pp.123-132
02/2015
DOI: 10.1016/j.yjmcc.2014.10.014
PMID: 25451174
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 [Ca2+], [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. Ca2+ 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.
•Active tension of reconstituted fibers without Tm or Tn diminished with mutant actins (E99K, A230V).•When reconstituted further with Tm and Tn, active tension was not any different between mutants and WT actins.•Tm/Tn exhibited a significantly higher positive allostery on E99K and A230V actins than on WT actin.•Ca2+ sensitivity increased and cooperativity decreased with mutant actins, which may cause HCM phenotype.•Cross-bridge kinetics of E99K actin were similar to WT; ATP binding & subsequent dissociation steps were diminished in A230V.
Details
- Title: Subtitle
- The immediate effect of HCM causing actin mutants E99K and A230V on actin–Tm–myosin interaction in thin-filament reconstituted myocardium
- Creators
- Fan Bai - Department of Anatomy and Cell Biology, and Internal Medicine, The University of Iowa, Iowa City, IA 52242-1109, USAHannah M Caster - Department of Anatomy and Cell Biology, and Internal Medicine, The University of Iowa, Iowa City, IA 52242-1109, USAJohn F Dawson - Department of Molecular and Cellular Biology, University of Guelph, College of Biological Science, Guelph, Ontario N1G 2W1, CanadaMasataka Kawai - Department of Anatomy and Cell Biology, and Internal Medicine, The University of Iowa, Iowa City, IA 52242-1109, USA
- Resource Type
- Journal article
- Publication Details
- Journal of molecular and cellular cardiology, Vol.79, pp.123-132
- Publisher
- Elsevier Ltd
- DOI
- 10.1016/j.yjmcc.2014.10.014
- PMID
- 25451174
- ISSN
- 0022-2828
- eISSN
- 1095-8584
- Grant note
- DOI: 10.13039/100000002, name: NIH, award: HL70041; DOI: 10.13039/501100001145, name: AHA, award: 13GRNT16810043; DOI: 10.13039/100004411, name: HSFC, award: NA 6469
- Language
- English
- Date published
- 02/2015
- Academic Unit
- Anatomy and Cell Biology; Internal Medicine
- Record Identifier
- 9984025365002771
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