Human actin mutations associated with hypertrophic and dilated cardiomyopathies demonstrate distinct thin filament regulatory properties in vitro

Department of Molecular Physiology & Biophysics, University of Vermont, College of Medicine, 149 Beaumont Drive, Burlington, VT 05405, USA.
Journal of Molecular and Cellular Cardiology (Impact Factor: 4.66). 09/2009; 48(2):286-92. DOI: 10.1016/j.yjmcc.2009.09.014
Source: PubMed


Two cardiomyopathic mutations were expressed in human cardiac actin, using a Baculovirus/insect cell system; E99K is associated with hypertrophic cardiomyopathy whereas R312H is associated with dilated cardiomyopathy. The hypothesis that the divergent phenotypes of these two cardiomyopathies are associated with fundamental differences in the molecular mechanics and thin filament regulation of the underlying actin mutation was tested using the in vitro motility and laser trap assays. In the presence of troponin (Tn) and tropomyosin (Tm), beta-cardiac myosin moved both E99K and R312H thin filaments at significantly (p<0.05) slower velocities than wild type (WT) at maximal Ca(++). At submaximal Ca(++), R312H thin filaments demonstrated significantly increased Ca(++) sensitivity (pCa(50)) when compared to WT. Velocity as a function of ATP concentration revealed similar ATP binding rates but slowed ADP release rates for the two actin mutants compared to WT. Single molecule laser trap experiments performed using both unregulated (i.e. actin) and regulated thin filaments in the absence of Ca(++) revealed that neither actin mutation significantly affected the myosin's unitary step size (d) or duration of strong actin binding (t(on)) at 20 microM ATP. However, the frequency of individual strong-binding events in the presence of Tn and Tm, was significantly lower for E99K than WT at comparable myosin surface concentrations. The cooperativity of a second myosin head binding to the thin filament was also impaired by E99K. In conclusion, E99K inhibits the activation of the thin filament by myosin strong-binding whereas R312H demonstrates enhanced calcium activation.

15 Reads
  • Source
    • "Interestingly, as discussed previously, the E99A/ E100A double mutant was identified in the Wertman alanine scanning mutagenesis screen in yeast where the two together produced a deleterious phenotype [Wertman et al., 1992; Drubin et al., 1993; Miller et al., 1996]. Both the E99K and R312H dilated cardiomyopathy mutations in baculovirus-expressed cardiac actin were also studied [Bookwalter and Trybus, 2006; Debold et al., 2010] for their effects on tropomyosin and troponin regulation. Both mutations resulted in decreased filament motility rates compared with WT actin, and both mutations affected calcium regulation of thin filament activity both at sub-saturating and saturating calcium concentrations. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mutations in all six actins in humans have now been shown to cause diseases. However, a number of factors have made it difficult to gain insight into how the changes in actin functions brought about by these pathogenic mutations result in the disease phenotype. These include the presence of multiple actins in the same cell, limited accessibility to pure mutant material, and complexities associated with the structures and their component cells that manifest the diseases. To try to circumvent these difficulties, investigators have turned to the use of model systems. This review describes these various approaches, the initial results obtained using them, and the insight they have provided into allosteric mechanisms that govern actin function. Although results so far have not explained a particular disease phenotype at the molecular level, they have provided valuable insight into actin function at the mechanistic level which can be utilized in the future to delineate the molecular bases of these different actinopathies. © 2014 Wiley Periodicals, Inc.
    Cytoskeleton 04/2014; 71(4). DOI:10.1002/cm.21169 · 3.12 Impact Factor
  • Source
    • "The degree of crowding of the filament with other motors may also affect individual motor behavior (Seitz and Surrey, 2006). Analogously, in the muscle, tropomyosin and troponin decorate actin to form a ''regulated thin filament,'' and the presence of these regulatory proteins has been shown to affect myosin motors' in vitro behaviors, such as velocity and force production, but the effects and mechanisms should be characterized more thoroughly (Fraser and Marston, 1995; Homsher et al., 1996, 2000; Gordon et al., 1998; Bing et al., 2000; Clemmens and Regnier, 2004; Kad et al., 2005; Debold et al., 2010; Suzuki and Ishiwata, 2011). The field will benefit from a continued and more detailed analysis of the responses of motors to various filament geometries and accessory proteins. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Single-molecule analysis is a powerful modern form of biochemistry, in which individual kinetic steps of a catalytic cycle of an enzyme can be explored in exquisite detail. Both single-molecule fluorescence and single-molecule force techniques have been widely used to characterize a number of protein systems. We focus here on molecular motors as a paradigm. We describe two areas where we expect to see exciting developments in the near future: first, characterizing the coupling of force production to chemical and mechanical changes in motors, and second, understanding how multiple motors work together in the environment of the cell.
    Developmental Cell 12/2012; 23(6):1084-91. DOI:10.1016/j.devcel.2012.10.002 · 9.71 Impact Factor
  • Source
    • "While myosin is detached (or more precisely weakly bound) from actin, ATP is hydrolyzed, a biochemical event coupled to resetting of the lever arm, ensuring that the next binding event causes another productive displacement. It is important to point out that more complex models have been proposed based on recent in vitro findings including additional AM.ADP states that could be coupled to a structural change in the position of the lever arm (Capitanio et al., 2006) and strongly strongly bound pre-powerstroke states (Takagi et al., 2004) but this simple model is consistent with much of the data from in vitro experiments (Palmiter et al., 1999; Baker et al., 2002; Debold et al., 2008, 2010) and provides the best starting point for understanding the effects of elevated levels of Pi, H+, and ADP on actomyosin. In fact, years of investigations using skinned muscle fibers have led to several hypotheses regarding how elevated levels of Pi, H+ and ADP during fatigue could directly inhibit specific steps in the cross-bridge cycle (Cooke, 2007). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Muscle fatigue is a complex multi-factorial process in humans that is dependent on the mode and intensity of the contractile activity. The depression in force and/or velocity associated with fatigue can be the result of a failure at any level, from the initial events in the motor cortex of the brain to the formation of an actomyosin cross-bridge in the muscle cell. Factors distal to the neuromuscular junction appear to be most important for fatigue resulting from intense contractile activity. Given that all the force and motion generated by muscle ultimately derives from the cyclical interaction of actin and myosin, researchers have focused heavily on the impact of the accumulation of intracellular metabolites (e.g. Pi, H+ and ADP) on the function these contractile proteins. These efforts demonstrate when elevated to the levels reached during intense contractile activity these metabolites can significantly alter a muscles’ force and motion generating capacity. At saturating Ca++ levels, elevated Pi appears to be the primary cause for the loss in maximal isometric force, while increased [H+] and possibly ADP act to slow unloaded shortening velocity. These studies, performed on isolated muscle preparations, provided strong evidence that these metabolites play a causative role in muscular fatigue, however the precise mechanisms through which these metabolites might affect the individual function of the contractile proteins remains unclear because intact muscle is a highly complex structure with the observed contractile properties representing the collective action of billions of myosin molecules. Fortunately more recent experiments on isolated proteins, including some single molecule measurements, are giving us unprecedented insight into the molecular processes that may be at work during fatigue. For example, using isolated actin and myosin in an in vitro motility assay, researchers have demonstrated decreasing pH from a resting level (~7.0) to a level reache
    Frontiers in Physiology 06/2012; 3:151. DOI:10.3389/fphys.2012.00151 · 3.53 Impact Factor
Show more


15 Reads
Available from