Cardiomyopathy Mutations Impact the Actin-Activated Power Stroke of Human Cardiac Myosin.

2021 
Abstract Cardiac muscle contraction is driven by the molecular motor myosin that uses the energy from ATP hydrolysis to generate a power stroke when interacting with actin filaments, while it is unclear how this mechanism is impaired by mutations in myosin that can lead to heart failure. We have applied a FRET strategy to investigate structural changes in the lever arm domain of human β-cardiac myosin subfragment 1 (M2β-S1). We exchanged the human ventricular regulatory light chain labeled at a single cysteine (V105C) with Alexa 488 onto M2β-S1, which served as a donor for Cy3ATP bound to the active site. We monitored the FRET signal during the actin-activated product release steps using transient kinetic measurements. We propose that the fast phase measured with our FRET probes represents the macroscopic rate constant associated with actin-activated rotation of the lever arm during the power stroke in M2β-S1. Our results demonstrated M2β-S1 has a slower actin-activated power stroke compared with fast skeletal muscle myosin and myosin V. Measurements at different temperatures comparing the rate constants of the actin-activated power stroke and phosphate release are consistent with a model in which the power stroke occurs before phosphate release, and the two steps are tightly coupled. We suggest that the actin-activated power stroke is highly reversible but followed by a highly irreversible phosphate release step, in the absence of load and free phosphate. We demonstrated that HCM (R723G) and DCM (F764L) associated mutations both reduced actin-activation of the power stroke in M2β-S1. We also demonstrate that both mutations alter in vitro actin gliding in the presence and absence of load. Thus, examining the structural kinetics of the power stroke in M2β-S1 has revealed critical mutation-associated defects in the myosin ATPase pathway, suggesting these measurements will be extremely important for establishing structure-based mechanisms of contractile dysfunction.
    • Correction
    • Source
    • Cite
    • Save
    • Machine Reading By IdeaReader
    76
    References
    1
    Citations
    NaN
    KQI
    []