Engineering Synthetic DNA Nanotube Thick Filaments to Dissect Beta-Cardiac Myosin and Cardiac Myosin-Binding Protein C Interactions

Abstract

Cardiac myosin-binding protein C (cMyBP-C) modulates cardiac contractility by sensitizing the thin filament to calcium while slowing shortening velocity. cMyBP-C's proposed mechanisms of action depend on its interactions with binding partners, including the myosin head region and/or to the thin filament. Although evidence exists to support both binding partner interactions, how each contributes to the collective modulation of actomyosin ensemble function is unknown. Therefore, we developed an in vitro artificial “thick filament” assay that allows us to dictate cMyBP-C binding partner interactions by precisely controlling the spacing between alternating recombinant human β-cardiac myosin HMM and N-terminal cMyBP-C fragments (C0-C2), on the filament surface. The spacing between HMM and C0-C2 on ten-helix DNA nanotubes (up to 10µm in length) was either 14 or 28 nm, which correspond to spacing found in native thick filaments. Protein attachment specificity was achieved by SNAP tags labeled with unique oligo strands. With only HMM on the nanotube surface, no significant difference in mean actin filament velocity was observed when HMM was spaced at 14 nm (467 nm/s ±90 (SD) vs. 28 nm (395 nm/s ±83 (SD); consistent with our previous results using β-cardiac myosin S1 (Hariadi et al.,Nature Nanotechnology, 2015). In contrast, alternating HMM and C0-C2 every 14 nm on the nanotube reduced the mean actin velocity nearly 4-fold relative to HMM alone. Interestingly, as much as a 10-fold velocity reduction was observed when calcium-regulated thin filaments were introduced at pCa5. Thus, the nanotube assay recapitulates the slowing of actomyosin motility by cMyBP-C. The synthetic thick filament provides a platform for precisely manipulating cMyBP-C's spatially dependent interactions thereby helping define the cMyBP-C binding partner interactions that are critical to cMyBP-C's modulatory capacity within the muscle sarcomere.