Rationale: Cardiac myosin binding protein-C (cMyBP-C) is a critical regulator of heart contraction, but the mechanisms by which cMyBP-C affects actin and myosin are only partly understood. A primary obstacle is that cMyBP-C localization on thick filaments may be a key factor defining its interactions, but most in vitro studies cannot duplicate the unique spatial arrangement of cMyBP-C within the sarcomere. Objective: The goal of this study was to validate a novel hybrid genetic/protein engineering approach for rapid manipulation of cMyBP-C in sarcomeres in situ. Methods and Results: We designed a novel "cut and paste" approach for removal and replacement of cMyBP-C N'-terminal domains (C0-C7) in detergent-permeabilized cardiomyocytes from gene-edited "Spy-C" mice. Spy-C mice express a tobacco etch virus protease (TEVp) cleavage site and a "SpyTag" between cMyBP-C domains C7 and C8. A "cut" is achieved using TEVp which cleaves cMyBP-C to create a soluble N'-terminal gC0C7 fragment and an insoluble C'-terminal SpyTag (st)-C8-C10 fragment that remains associated with thick filaments. "Paste" of new recombinant (r)C0C7 domains is achieved by a covalent bond formed between SpyCatcher (-sc) (encoded at the C'-termini of recombinant proteins) and SpyTag. Results show that loss of gC0C7 reduced myofilament Ca2+ sensitivity and increased cross bridge cycling (ktr) at submaximal [Ca2+]. Acute loss of gC0C7 also induced auto-oscillatory contractions at submaximal [Ca2+]. Ligation of rC0C7-sc returned pCa50 and ktr to control values and abolished oscillations, but phosphorylated (p)-rC0C7-sc did not completely rescue these effects. Conclusions: We describe a robust new approach for acute removal and replacement of cMyBP-C in situ. The method revealed a novel role for cMyBP-C N'-terminal domains to damp sarcomere-driven contractile waves (so called "SPOC"). Because phosphorylated (p)-rC0C7-sc was less effective at damping contractile oscillations, results suggest that SPOC may contribute to enhanced contractility in response to inotropic stimuli.

中文翻译：

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原位替换cMyBP-C的新型“剪切和粘贴”方法揭示了cMyBP-C在调节收缩振荡中的新作​​用。

原理：心肌肌球蛋白结合蛋白C（cMyBP-C）是心脏收缩的关键调节剂，但仅部分了解了cMyBP-C影响肌动蛋白和肌球蛋白的机制。一个主要的障碍是，cMyBP-C在粗丝上的定位可能是决定其相互作用的关键因素，但是大多数体外研究无法复制肌节内cMyBP-C的独特空间排列。目的：本研究的目的是验证一种新颖的遗传/蛋白质杂合工程方法，用于在原位肉瘤中快速处理cMyBP-C。方法和结果：我们设计了一种新颖的“剪切和粘贴”方法，用于从基因编辑的“ Spy-C”小鼠的去污剂渗透性心肌细胞中去除和替换cMyBP-C N'-末端结构域（C0-C7）。Spy-C小鼠在cMyBP-C域C7和C8之间表达了烟草蚀刻病毒蛋白酶（TEVp）切割位点和“ SpyTag”。使用TEVp切割cMyBP-C以产生可溶的N'-末端gCOC7片段和不溶的C'-末端SpyTag（st）-C8-C10片段，该片段仍与粗细丝相关联，从而实现“切割”。新的重组（r）C0C7域的“粘贴”是通过SpyCatcher（-sc）（在重组蛋白的C'-末端编码）和SpyTag之间形成的共价键实现的。结果表明，在低于最大[Ca2 +]的情况下，gCOC7的丢失降低了肌丝Ca2 +的敏感性，并增加了跨桥循环（ktr）。gC0C7的急性丧失也引起了低于[Ca2 +]的自振荡收缩。rC0C7-sc的连接使pCa50和ktr返回控制值并消除了振荡，但是磷酸化的（p）-rC0C7-sc不能完全挽救这些作用。结论：我们描述了一种健壮的新方法，用于急性原位cMyBP-C的去除和替代。该方法揭示了cMyBP-C N'-末端结构域对抑制肌节驱动的收缩波（所谓的“ SPOC”）的新作用。由于磷酸化的（p）-rC0C7-sc在抑制收缩振荡方面效果较差，因此结果表明，SPOC可能对正性肌力刺激产生增强的收缩性。