Journal Article

ADP-stimulated contraction

Sequeira, Vasco, van der Velden, Jolanda, Kuster, Diederik W. D., Wijnker, Paul J. M., dos Remedios, Cristobal G., Michels, Michelle, Najafi, Aref

2015

Overview

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 Ca²⁺. We investigated whether ADP-stimulated force development (without Ca²⁺) can be used to reveal changes in actin–myosin blockade in human cardiomyopathy cardiomyocytes. Cardiac samples from HCM patients, harboring thick-filament (MYH7 mut,MYBPC3 mut) and thin-filament (TNNT2 mut,TNNI3 mut) 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 forMYBPC3. Increased ADP sensitivity in IDCM, HCMsmn, andMYH7 mutwas caused by low phosphorylation of myofilament proteins, as it was normalized to donors by protein kinase A (PKA) treatment. Troponin exchange experiments in aTNNT2 mutsample corrected the abnormal actin–myosin blockade. InMYBPC3 truncsamples, 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 Ca²⁺, 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 Ca²⁺, pathologic [ADP] and low PKA-phosphorylation, high actin–myosin formation could contribute to the impaired myocardial relaxation observed in cardiomyopathies.

• Publisher: National Academy of Sciences,National Acad Sciences
• Subject: Adenosine diphosphate / Adenosine Diphosphate - pharmacology / Binding sites / Biological Sciences / Cardiomyocytes / Cardiomyopathy / Cardiovascular disease / Cyclic AMP-Dependent Protein Kinases - metabolism / Heart Diseases - metabolism / Humans / Kinases / Mutation / Myocardial Contraction - drug effects / Phosphorylation / PNAS Plus