Cardiomyopathy Linked Mutations in Alpha Tropomyosin Influence Blocked State Stability but not Myosin Strong Binding

Abstract

Coiled-coil tropomyosin binds super-helically along F-actin. Together with the troponin complex, tropomyosin inhibits myosin-binding. McKillop and Geeves (1993) introduced a three-state model for thin filament regulation with tropomyosin occupying three positions on the actin filament; blocked, closed and open. In the absence of myosin, tropomyosin primarily occupies the closed and blocked-states with the blocked-state being stabilized by Ca2+-free troponin. The probability of tropomyosin occupying any one of these two states is dependent on factors including cytosolic Ca2+-concentration (altering troponin conformation), mechanical strain, ionic strength, pH, and mutations that alter tropomyosin affinity for actin. Recently, we examined the impact of cardiomyopathy-linked tropomyosin mutations on the energy landscape of the tropomyosin/actin interaction. Our results demonstrated that mutations associated with hypertrophic cardiomyopathy showed reduced interaction-energy with actin resulting in a destabilization of the blocked-state. Using an in vitro motility assay here, we tested the ability of the mutant tropomyosins to regulate acto-myosin interactions, as well as the impact of strong binding cross-bridges on reconstituted thin filaments containing E40K and E62Q mutations in human alpha-tropomyosin (which lead to dilated (DCM) and hypertrophic (HCM) cardiomyopathies, respectively). Regulated thin filament velocity measurements showed an increase in low-Ca2+ contractility of E62Q reconstituted thin filaments when compared to wild-type and E40K reconstituted thin filaments. The ability of myosin to activate reconstituted mutant thin filaments at low-Ca2+ was not significantly different between the three tropomyosins, indicating that the enhanced motile fraction observed with E62Q is likely due to a destabilization of the blocked-state rather than alterations in myosin-dependent activation. The motility data is consistent with molecular dynamics simulations of the mutant tropomyosins in the presence of actin which show significant, yet subtle and complex alterations to the electrostatic interactions between tropomyosin and actin.