title: Contractility parameters of human β-cardiac myosin with the hypertrophic cardiomyopathy mutation R403Q show loss of motor function.
abstract: Hypertrophic cardiomyopathy (HCM) is the most frequently occurring inherited cardiovascular disease. It is caused by mutations in genes encoding the force-generating machinery of the cardiac sarcomere, including human β-cardiac myosin. We present a detailed characterization of the most debated HCM-causing mutation in human β-cardiac myosin, R403Q. Despite numerous studies, most performed with nonhuman or noncardiac myosin, there is no consensus about the mechanism of action of this mutation on the function of the enzyme. We use recombinant human β-cardiac myosin and new methodologies to characterize in vitro contractility parameters of the R403Q myosin compared to wild type. We extend our studies beyond pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin. We find that, with pure actin, the intrinsic force generated by R403Q is ~15% lower than that generated by wild type. The unloaded velocity is, however, ~10% higher for R403Q myosin, resulting in a load-dependent velocity curve that has the characteristics of lower contractility at higher external loads compared to wild type. With regulated actin filaments, there is no increase in the unloaded velocity and the contractility of the R403Q myosin is lower than that of wild type at all loads. Unlike that with pure actin, the actin-activated adenosine triphosphatase activity for R403Q myosin with Ca(2+)-regulated actin filaments is ~30% lower than that for wild type, predicting a lower unloaded duty ratio of the motor. Overall, the contractility parameters studied fit with a loss of human β-cardiac myosin contractility as a result of the R403Q mutation.
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: Suman
authors.initials: S
authors.lastname: Nag
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: Ruth F
authors.initials: RF
authors.lastname: Sommese
authors.affiliation: School of Biosciences, University of Kent, Canterbury CT2 7NJ, UK.
authors.forename: Zoltan
authors.initials: Z
authors.lastname: Ujfalusi
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, BioFrontiers Institute, University of Colorado, Boulder, CO 80309, USA.
authors.forename: Ariana
authors.initials: A
authors.lastname: Combs
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, BioFrontiers Institute, University of Colorado, Boulder, CO 80309, USA.
authors.forename: Stephen
authors.initials: S
authors.lastname: Langer
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: Shirley
authors.initials: S
authors.lastname: Sutton
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, BioFrontiers Institute, University of Colorado, Boulder, CO 80309, USA.
authors.forename: Leslie A
authors.initials: LA
authors.lastname: Leinwand
authors.affiliation: School of Biosciences, University of Kent, Canterbury CT2 7NJ, UK.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Geeves
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA. ; Department of Pediatrics (Cardiology), Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Ruppel
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: James A
authors.initials: JA
authors.lastname: Spudich
keywords: Chemical bonding; Crystallography; High pressure; Metallic alloy; electron density; maximum entropy method