Molecular mechanics of cardiac myosin-binding protein C in native thick filaments

Abstract

The heart's pumping capacity results from highly regulated interactions of actomyosin molecular motors. Mutations in the gene for a potential regulator of these motors, cardiac myosin-binding protein C (cMyBP-C), cause hypertrophic cardiomyopathy. However, cMyBP-C's ability to modulate cardiac contractility is not well understood. Using single-particle fluorescence imaging techniques, transgenic protein expression, proteomics, and modeling, we found that cMyBP-C slowed actomyosin motion generation in native cardiac thick filaments. This mechanical effect was localized to where cMyBP-C resides within the thick filament (i.e., the C-zones) and was modulated by phosphorylation and site-specific proteolytic degradation. These results provide molecular insight into why cMyBP-C should be considered a member of a tripartite complex with actin and myosin that allows fine tuning of cardiac muscle contraction.

Fig. 1

Native cardiac thick filaments and cMyBP-C. (A) Cardiac muscle sarcomere with interdigitating thick and thin filaments with cMyBP-C localized to thick filament C-zones. (B) Illustration of one half of thick filament with an actin filament traveling towards bare zone as in experiments. (C) Schematic diagram of cMyBPC's Ig-like (oval) and fibronectin (rectangle) domains, with 4 phosphorylation sites (P) in motif linker, and 29kDa fragment (dashed box). Sarcomeric protein domain interactions identified. (D) Native cardiac thick filament imaged by electron microscopy and TIRFM (inset) by effectively labeling the myosin heads with fluorescent ATP (6).

Fig. 2

Effect of cMyBP-C on actin motility. (A) TIRFM image series of actin shard moving along a native thick filament. (B) Displacement-time plots for 5 actin filaments on wild-type thick filaments demonstrated two velocity phases (fast, blue; slow, red). Inset for 20 filaments with distance traveled during slow velocity phase identified. (C) As in B except actin filaments exhibited constant velocities. (D) Frequency-velocity histograms and Gaussian fits for actin trajectories as in B (N = 58; fast phase, blue; slow phase, red). (E) Frequency-velocity histogram and Gaussian fit for actin trajectories as in C with constant velocities (N = 21). (F) Spatial relations for an analytic model where actin filaments (green) moved over a thick filament with myosin crowns at the same azimuthal position separated by 43 nm and cMyBP-C localized in the C-zone (red highlighted crowns 3–11; (8)). Actin detached upon reaching the bare zone (crown 0). (G) Model-generated displacement-time plots for a 250 nm actin filament on a thick filament with 78% intact cMyBP-C, as in B. Fast phase = 1.98 ± 0.03 μm/s (SEM), slow phase = 1.15 ± 0.02 μm/s (SEM), N = 5 (inset, N = 20, slow phase travel distance identified). (H) As in G but for a theoretical thick filament with 10% intact cMyBP-C that showed only a constant velocity (2.06 ± 0.01 μm/s (SEM), N = 5; inset, 20 runs). (I) Inhibition of actin sliding velocity by C0C1f and C0C3 fragments (mean ± SD) in motility assay. (J) Effect of motif phosphorylation on actin filament velocity inhibition by C0C3 in motility assay or by cMyBP-C in native thick filaments. Percent phosphorylation was defined as the average percent phosphorylation at S273, S282, S302, S307. Percent inhibition of motility assay data was normalized to thick filament inhibition data, where inhibition is the percent velocity reduction compared to control without cMyBP-C or fragment.