title: Effect of a myosin regulatory light chain mutation K104E on actin-myosin interactions.
abstract: Familial hypertrophic cardiomyopathy (FHC) is the most common cause of sudden cardiac death in young individuals. Molecular mechanisms underlying this disorder are largely unknown; this study aims at revealing how disruptions in actin-myosin interactions can play a role in this disorder. Cross-bridge (XB) kinetics and the degree of order were examined in contracting myofibrils from the ex vivo left ventricles of transgenic (Tg) mice expressing FHC regulatory light chain (RLC) mutation K104E. Because the degree of order and the kinetics are best studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs in an ex vivo ventricle was minimized to ∼20. Autofluorescence and photobleaching were minimized by labeling the myosin lever arm with a relatively long-lived red-emitting dye containing a chromophore system encapsulated in a cyclic macromolecule. Mutated XBs were significantly better ordered during steady-state contraction and during rigor, but the mutation had no effect on the degree of order in relaxed myofibrils. The K104E mutation increased the rate of XB binding to thin filaments and the rate of execution of the power stroke. The stopped-flow experiments revealed a significantly faster observed dissociation rate in Tg-K104E vs. Tg-wild-type (WT) myosin and a smaller second-order ATP-binding rate for the K104E compared with WT myosin. Collectively, our data indicate that the mutation-induced changes in the interaction of myosin with actin during the contraction-relaxation cycle may contribute to altered contractility and the development of FHC.
authors.affiliation: Department of Cell Biology & Immunology and Center for Commercialization of Fluorescence Technologies, University of North Texas, Health Science Center, Fort Worth, Texas; and.
authors.forename: D
authors.initials: D
authors.lastname: Duggal
authors.affiliation: Department of Cell Biology & Immunology and Center for Commercialization of Fluorescence Technologies, University of North Texas, Health Science Center, Fort Worth, Texas; and.
authors.forename: J
authors.initials: J
authors.lastname: Nagwekar
authors.affiliation: Department of Cell Biology & Immunology and Center for Commercialization of Fluorescence Technologies, University of North Texas, Health Science Center, Fort Worth, Texas; and.
authors.forename: R
authors.initials: R
authors.lastname: Rich
authors.affiliation: Department of Molecular & Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida; and.
authors.forename: W
authors.initials: W
authors.lastname: Huang
authors.affiliation: Department of Cell Biology & Immunology and Center for Commercialization of Fluorescence Technologies, University of North Texas, Health Science Center, Fort Worth, Texas; and.
authors.forename: K
authors.initials: K
authors.lastname: Midde
authors.affiliation: Department of Cell Biology & Immunology and Center for Commercialization of Fluorescence Technologies, University of North Texas, Health Science Center, Fort Worth, Texas; and.
authors.forename: R
authors.initials: R
authors.lastname: Fudala
authors.affiliation: Department of Cell Biology & Immunology and Center for Commercialization of Fluorescence Technologies, University of North Texas, Health Science Center, Fort Worth, Texas; and Department of Pharmacology and Neuroscience, Institute of Aging and Alzheimer's Disease Research, Institute of Cancer Research, Fort Worth, Texas.
authors.forename: H
authors.initials: H
authors.lastname: Das
authors.affiliation: Department of Cell Biology & Immunology and Center for Commercialization of Fluorescence Technologies, University of North Texas, Health Science Center, Fort Worth, Texas; and.
authors.forename: I
authors.initials: I
authors.lastname: Gryczynski
authors.affiliation: Department of Molecular & Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida; and.
authors.forename: D
authors.initials: D
authors.lastname: Szczesna-Cordary
authors.affiliation: Department of Cell Biology & Immunology and Center for Commercialization of Fluorescence Technologies, University of North Texas, Health Science Center, Fort Worth, Texas; and Julian.Borejdo@unthsc.edu.
authors.forename: J
authors.initials: J
authors.lastname: Borejdo
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D024741:Cardiomyopathy, Hypertrophic, Familial / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism; D051379:Mice; D020125:Mutation, Missense; D009200:Myocardial Contraction; D009210:Myofibrils / Q000378:metabolism / Q000502:physiology; D018994:Myosin Light Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D024723:Ventricular Myosins / Q000235:genetics / Q000378:metabolism*
chemical_list: D018994:Myosin Light Chains; D000255:Adenosine Triphosphate; D024723:Ventricular Myosins
keywords: left ventricle; mutation of regulatory light chain; polarization of fluorescence