Cardiac myosin-binding protein C (cMyBP-C) modulates cardiac contractility through putative interactions with the myosin S2 tail and/or the thin filament. The relative contribution of these binding-partner interactions to cMyBP-C modulatory function remains unclear. Hence, we developed a “nanosurfer” assay as a model system to interrogate these cMyBP-C binding-partner interactions. 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 in native thick filaments. The nanosurfer assay consists of DNA nanotubes added to the in vitro motility assay so that myosins on the motility surface effectively deliver thin filaments to the DNA nanotubes, enhancing thin filament gliding probability on the DNA nanotubes. Thin filament velocities on nanotubes with either 14- or 28-nm myosin spacing were no different. We then characterized the effects of cMyBP-C on thin filament motility by alternating HMM and cMyBP-C N-terminal fragments (C0–C2 or C1–C2) on nanotubes every 14 nm. Both C0–C2 and C1–C2 reduced thin filament velocity four- to sixfold relative to HMM alone. Similar inhibition occurred using the myosin S1 construct, which lacks the myosin S2 region proposed to interact with cMyBP-C, suggesting that the cMyBP-C N terminus must interact with other myosin head domains and/or actin to slow thin filament velocity. Thin filament velocity was unaffected by the C0–C1f fragment, which lacks the majority of the M-domain, supporting the importance of this domain for inhibitory interaction(s). A C0–C2 fragment with phospho-mimetic replacement in the M-domain showed markedly less inhibition of thin filament velocity compared with its phospho-null counterpart, highlighting the modulatory role of M-domain phosphorylation on cMyBP-C function. Therefore, the nanosurfer assay provides a platform to precisely manipulate spatially dependent cMyBP-C binding-partner interactions, shedding light on the molecular regulation of β-cardiac myosin contractility.

心肌肌球蛋白结合蛋白 C (cMyBP-C) 通过与肌球蛋白 S2 尾部和/或细丝的假定相互作用来调节心肌收缩力。这些结合伙伴相互作用对 cMyBP-C 调节功能的相对贡献仍不清楚。因此，我们开发了一种“nanosurfer”测定作为模型系统来询问这些 cMyBP-C 结合伙伴相互作用。使用附着在 DNA 纳米管上的重组人β-心肌肌球蛋白亚片段（HMM 或 S1）生成合成粗丝，间距为 14 或 28 nm，对应于天然粗丝中的 14.3 nm 肌球蛋白间距。 Nanosurfer实验由添加到体外运动实验中的DNA纳米管组成，这样运动表面的肌球蛋白可以有效地将细丝传递到DNA纳米管，从而提高细丝在DNA纳米管上滑动的概率。肌球蛋白间距为 14 或 28 nm 的纳米管上的细丝速度没有差异。然后，我们通过每 14 nm 在纳米管上交替使用 HMM 和 cMyBP-C N 端片段（C0–C2 或 C1–C2）来表征 cMyBP-C 对细丝运动的影响。相对于单独的 HMM，C0-C2 和 C1-C2 都将细丝速度降低了四到六倍。使用肌球蛋白 S1 构建体也发生了类似的抑制，该构建体缺乏与 cMyBP-C 相互作用的肌球蛋白 S2 区域，这表明 cMyBP-C N 末端必须与其他肌球蛋白头结构域和/或肌动蛋白相互作用以减慢细丝速度。细丝速度不受 C0-C1f 片段的影响，该片段缺乏大部分 M 结构域，这支持了该结构域对于抑制相互作用的重要性。 与磷酸无效的对应物相比，M 结构域中具有磷酸模拟替换的 C0-C2 片段对细丝速度的抑制显着减少，突出了 M 结构域磷酸化对 cMyBP-C 功能的调节作用。因此，nanosurfer 测定提供了一个精确操纵空间依赖性 cMyBP-C 结合伴侣相互作用的平台，揭示了β-心肌肌球蛋白收缩性的分子调节。