N-Terminal Fragment of Cardiac Myosin Binding Protein-C (CMYBP-C) Reduces Actomyosin Power Output in the Laser Trap Assay

Abstract

Mutations in cMyBP-C cause hypertrophic cardiomyopathy, but cMyBP-C's modulation of cardiac contractility is poorly understood. The N-terminus of cMyBP-C binds actin and myosin S2, while the C-terminus binds the myosin rod. Thus, cMyBP-C may act as a tether between the thick and thin filament, imposing a load against which myosin must operate. We used a laser trap force clamp assay (Debold et al. B.J., 2005) to characterize actomyosin's force velocity relationship (F:V) in the absence and presence of a bacterially expressed N-terminal cMyBP-C fragment, C0C3. In this assay a small ensemble of skeletal myosin (∼8 heads) translocates a skeletal actin filament under a series of constant loads (1-8 pN), resulting in a classic hyperbolic F:V with a maximum isometric force of 10 ± 2 pN. Adding C0C3 at a 1:1 ratio to myosin depressed actomyosin's F:V: i.e. 48% reduction in maximum unloaded velocity with no effect on maximum force. This reduced actomyosin's maximum power output by 40%. The depressed velocities suggest C0C3 may impose as much as 2 pN of opposing load to the myosin ensemble. As limited numbers of myosin translocate the actin filament under load, a finite probability exists that no myosin will be attached, causing rapid (∼2 ms duration) backward movements (34 ± 1 nm) of the actin filament, i.e. slips. The frequency of slips rises with increasing load, which is diminished significantly in the presence of C0C3, suggesting C0C3 prevents slips by its attachment to the actin filament. Based on the depression of the F:V and fewer actin filament slips in the presence of C0C3, we propose that the N-terminus of cMyBP-C can tether actin and impose an effective viscous load in the sarcomere to modulate cardiac power output.