Subdomain location of mutations in cardiac actin correlate with type of functional change.

Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
PLoS ONE (Impact Factor: 3.53). 05/2012; 7(5):e36821. DOI: 10.1371/journal.pone.0036821
Source: PubMed

ABSTRACT Determining the molecular mechanisms that lead to the development of heart failure will help us gain better insight into the most costly health problem in the Western world. To understand the roles that the actin protein plays in the development of heart failure, we have taken a systematic approach toward characterizing human cardiac actin mutants that have been associated with either hypertrophic or dilated cardiomyopathy. Seven known cardiac actin mutants were expressed in a baculovirus system, and their intrinsic properties were studied. In general, the changes to the properties of the actin proteins themselves were subtle. The R312H variant exhibited reduced stability, with a T(m) of 53.6 °C compared to 56.8 °C for WT actin, accompanied with increased polymerization critical concentration and Pi release rate, and a marked increase in nucleotide release rates. Substitution of methionine for leucine at amino acid 305 showed no impact on the stability, nucleotide release rates, or DNase-I inhibition ability of the actin monomer; however, during polymerization, a 2-fold increase in Pi release was observed. Increases to both the T(m) and DNase-I inhibition activity suggested interactions between E99K actin molecules under monomer-promoting conditions. Y166C actin had a higher critical concentration resulting in a lower Pi release rate due to reduced filament-forming potential. The locations of mutations on the ACTC protein correlated with the molecular effects; in general, mutations in subdomain 3 affected the stability of the ACTC protein or affect the polymerization of actin filaments, while mutations in subdomains 1 and 4 more likely affect protein-protein interactions.


Available from: Melissa L Chow, Apr 08, 2014
  • [Show abstract] [Hide abstract]
    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. Copyright © 2014 Elsevier Ltd. All rights reserved.
    Journal of Molecular and Cellular Cardiology 11/2014; 79C:123-132. DOI:10.1016/j.yjmcc.2014.10.014 · 5.22 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In striated muscle tropomyosin (Tm) extends along the length of F-actin-containing thin filaments. Its location governs access of myosin binding sites on actin and, hence, force production. Intermolecular electrostatic associations are believed to mediate critical interactions between the proteins. For example, actin residues K326, K328, and R147 were predicted to establish contacts with E181 of Tm. Moreover, K328 also potentially forms direct interactions with E286 of myosin when the motor is strongly bound. Recently, LC-MS/MS analysis of the cardiac acetyl-lysine proteome revealed K326 and K328 of actin were acetylated, a post-translational modification (PTM) that masks the residues' inherent positive charges. Here, we tested the hypothesis that by removing the vital actin charges at residues 326 and 328, the PTM would perturb Tm positioning and/or strong myosin binding as manifested by altered skeletal muscle function and structure in the Drosophila melanogaster model system. Transgenic flies were created that permit tissue-specific expression of K326Q, K328Q, or K326Q/K328Q acetyl-mimetic actin and of wild-type actin via the UAS-GAL4 bipartite expression system. Compared to wild-type actin, muscle-restricted expression of mutant actin had a dose-dependent effect on flight ability. Moreover, excessive K328Q and K326Q/K328Q actin overexpression induced indirect flight muscle degeneration, a phenotype consistent with hypercontraction observed in other Drosophila myofibrillar mutants. Based on F-actin-Tm and F-actin-Tm-myosin models and on our physiological data, we conclude that acetylating K326 and K328 of actin alters electrostatic associations with Tm and/or myosin and thereby augments contractile properties. Our findings highlight the utility of Drosophila as a model that permits efficient targeted design and assessment of molecular and tissue-specific responses to muscle protein modifications, in vivo.
    Frontiers in Physiology 04/2015; 6:116. DOI:10.3389/fphys.2015.00116
  • Source
    Biophysical Journal 01/2015; 108(2):421a-422a. DOI:10.1016/j.bpj.2014.11.2308 · 3.83 Impact Factor