Dissecting β-Cardiac Myosin and Cardiac Myosin-Binding Protein C Interactions using a Nanosurf Assay
Authors: Touma Anja M. , Tang Wanjian , Rasicci David V. , Vang Duha , Rai Ashim , Previs Samantha B. , Warshaw David M. , Yengo Christopher M. , Sivaramakrishnan Sivaraj 
Published: March 2022

ABSTRACTCardiac myosin-binding protein C (cMyBP-C) regulates cardiac contractility by slowing shortening velocity and sensitizing the thin filament to calcium. cMyBP-C has been shown to interact with the proximal myosin S2 tail and the thin filament. However, the relative contribution of these interactions to the collective modulation of actomyosin ensemble function remains unclear. Hence, we developed a “nanosurf” assay as a model system to interrogate cMyBP-C interactions with actin and/or myosin. Synthetic thick filaments were generated using recombinant human β-cardiac myosin subfragments (HMM or S1) attached to DNA nanotubes, with 14 or 28 nm spacing, corresponding to the 14.3 nm myosin spacing found in native thick filaments. In vitro motility assays with myosin bound to the surrounding surface, exhibit enhanced thin filament interactions with synthetic thick filaments. No significant differences were observed in mean thin filament velocities between 14 and 28 nm spacing, consistent with our previous results for myosin V, VI, and β-cardiac myosin S1. Our nanosurf assay demonstrates the slowing of actomyosin motility by cMyBP-C. Alternating β-cardiac myosin HMM and cMyBP-C N-terminal fragments, C0-C2 or C1-C2, every 14 nm on the nanotube, reduced the mean thin filament velocity 4-6 fold relative to myosin alone. Interestingly, similar inhibition was observed using a β-cardiac myosin S1 construct, which lacks the S2 region proposed to interact with cMyBP-C, suggesting the actin-cMyBP-C interactions may dominate the inhibitory mechanism. No significant inhibition of thin filament velocity was observed with a C0-C1f fragment, lacking the majority of the M-domain, supporting the importance of this domain for inhibitory interaction(s). A phosphomimetic C0-C2 fragment showed a 3-fold higher velocity compared to its phosphonull counterpart, further highlighting phosphorylation-dependent regulation via the M-domain. Together, we have established the nanosurf assay as a tool to precisely manipulate spatially dependent cMyBP-C binding partner interactions, shedding light on the molecular regulation of β-cardiac myosin contractility.STATEMENT OF SIGNIFICANCECardiac myosin-binding protein C (cMyBP-C) is the most frequently mutated protein associated with hypertrophic cardiomyopathy (HCM), a common cause of sudden cardiac death. Despite the importance of cMyBP-C in cardiac contractility, the mechanisms underlying this regulation are unclear due to experimental challenges in studying the complex, transient, weak interactions of cMyBP-C with the contractile proteins of the sarcomere. In this study, we created a nanosurf synthetic DNA thick filament assay to dissect the cMyBP-C interactions with actin and human β-cardiac myosin. We demonstrate actomyosin inhibition by cMyBP-C fragments regardless of recombinant human β-cardiac myosin subfragment (HMM or S1) and highlight the importance of the cMyBP-C M-domain using cMyBP-C fragments and phosphomimetics.