title: ADP-stimulated contraction: A predictor of thin-filament activation in cardiac disease.
abstract: Diastolic dysfunction is general to all idiopathic dilated (IDCM) and hypertrophic cardiomyopathy (HCM) patients. Relaxation deficits may result from increased actin-myosin formation during diastole due to altered tropomyosin position, which blocks myosin binding to actin in the absence of Ca(2+). We investigated whether ADP-stimulated force development (without Ca(2+)) can be used to reveal changes in actin-myosin blockade in human cardiomyopathy cardiomyocytes. Cardiac samples from HCM patients, harboring thick-filament (MYH7mut, MYBPC3mut) and thin-filament (TNNT2mut, TNNI3mut) mutations, and IDCM were compared with sarcomere mutation-negative HCM (HCMsmn) and nonfailing donors. Myofilament ADP sensitivity was higher in IDCM and HCM compared with donors, whereas it was lower for MYBPC3. Increased ADP sensitivity in IDCM, HCMsmn, and MYH7mut was caused by low phosphorylation of myofilament proteins, as it was normalized to donors by protein kinase A (PKA) treatment. Troponin exchange experiments in a TNNT2mut sample corrected the abnormal actin-myosin blockade. In MYBPC3trunc samples, ADP sensitivity highly correlated with cardiac myosin-binding protein-C (cMyBP-C) protein level. Incubation of cardiomyocytes with cMyBP-C antibody against the actin-binding N-terminal region reduced ADP sensitivity, indicative of cMyBP-C's role in actin-myosin regulation. In the presence of Ca(2+), ADP increased myofilament force development and sarcomere stiffness. Enhanced sarcomere stiffness in sarcomere mutation-positive HCM samples was irrespective of the phosphorylation background. In conclusion, ADP-stimulated contraction can be used as a tool to study how protein phosphorylation and mutant proteins alter accessibility of myosin binding on actin. In the presence of Ca(2+), pathologic [ADP] and low PKA-phosphorylation, high actin-myosin formation could contribute to the impaired myocardial relaxation observed in cardiomyopathies.
authors.affiliation: Department of Physiology, Institute for Cardiovascular Research, Vrije Universiteit Medisch Centrum, 1081BT Amsterdam, The Netherlands; v.sequeiraoliveira@vumc.nl.
authors.forename: Vasco
authors.initials: V
authors.lastname: Sequeira
authors.affiliation: Department of Physiology, Institute for Cardiovascular Research, Vrije Universiteit Medisch Centrum, 1081BT Amsterdam, The Netherlands;
authors.forename: Aref
authors.initials: A
authors.lastname: Najafi
authors.affiliation: Department of Physiology, Institute for Cardiovascular Research, Vrije Universiteit Medisch Centrum, 1081BT Amsterdam, The Netherlands;
authors.forename: Paul J M
authors.initials: PJ
authors.lastname: Wijnker
authors.affiliation: Muscle Research Unit, Bosch Institute, University of Sydney, Sydney, NSW 2006, Australia;
authors.forename: Cristobal G
authors.identifier: http://orcid.org/0000-0003-1052-7490
authors.initials: CG
authors.lastname: Dos Remedios
authors.affiliation: Cardiology, Erasmus Medical Center, 3000CA Rotterdam, The Netherlands;
authors.forename: Michelle
authors.initials: M
authors.lastname: Michels
authors.affiliation: Department of Physiology, Institute for Cardiovascular Research, Vrije Universiteit Medisch Centrum, 1081BT Amsterdam, The Netherlands;
authors.forename: Diederik W D
authors.initials: DW
authors.lastname: Kuster
authors.affiliation: Department of Physiology, Institute for Cardiovascular Research, Vrije Universiteit Medisch Centrum, 1081BT Amsterdam, The Netherlands; Interuniversity Cardiology Institute of The Netherlands-Netherlands Heart Institute, 3501DG Utrecht, The Netherlands.
authors.forename: Jolanda
authors.initials: J
authors.lastname: van der Velden
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006331:Heart Diseases / Q000378:metabolism*; D006801:Humans; D009200:Myocardial Contraction / Q000187:drug effects*; D010766:Phosphorylation
chemical_list: D000244:Adenosine Diphosphate; D017868:Cyclic AMP-Dependent Protein Kinases
keywords: ADP; cardiomyopathy; hypertrophy; idiopathic dilated; three-state model