ADP-Stimulated Contraction: A Predictor of Thin-Filament Activation in Cardiac Disease

Abstract

Background Diastolic dysfunction is general to all hypertrophic cardiomyopathy (HCM) patients. Relaxation deficits may result from inappropriate formation of force-producing cross-bridges during diastole, due to changes in tropomyosin's position, which normally blocks actomyosin interactions in the absence of Ca2+. We investigated if actomyosin blockade is altered in human disease. By measuring cardiomyocyte force-production in ADP-containing solutions (without Ca2+), the ability of myosin-ADP to actin was assessed. Methods Force measurements were performed in single membrane-permeabilized cardiomyocytes at sarcomere length of 2.2 μm in the absence of Ca2+, but in the presence of mM levels of ADP. Exogenous protein kinase A (PKA)-treatment was performed to determine whether myofilaments are sensitive to kinase treatment. Cardiac samples used were harvested from HCM patients, harboring thick- (MYH7mut, MYBPC3mut) and thin-filament (TNNT2mut, TNNI3mut) mutations, and were compared with sarcomere mutation-negative HCM (HCMsmn) and non-failing donors. Results Myofilament ADP-sensitivity was higher in HCM samples compared with donors, except for MYBPC3mut. Increased ADP-sensitivity in HCMsmn and MYH7mut was caused by low phosphorylation of myofilament targets, as it was normalized to donors by PKA. In MYBPC3mut samples, ADP sensitivity highly correlated with cMyBP-C protein level. Incubation of failing cardiomyocytes with cMyBP-C antibody against the thin-filament binding N-terminal region reduced ADP-sensitivity. Troponin exchange experiments of the TNNT2mut sample corrected the abnormal actomyosin blockade. Conclusions Our data provides a mechanism of how phosphorylation alterations and/or expression of mutant proteins increase actomyosin interactions, that precede Ca2+ rise, and limit diastolic relaxation in HCM.