Cut and paste of myosin binding protein-C in skeletal muscles

项目总结 这个项目的总体目标是了解肌球蛋白结合蛋白-C(MyBP-C)是如何调节 骨骼肌的收缩和松弛。3个不同的MyBP-C并列基因，由3个独立的 MYBPC1、MYBPC2和MYBPC3基因在慢抽动骨骼肌、快抽动骨骼肌中表达 分别是肌肉和心肌。在三种类型中，心脏旁路是最强烈的 研究是因为MYBPC3基因突变是肥厚型心肌病最常见的原因 (HCM)。然而，像MYBPC3一样，现在很明显MYBPC1和MYBPC2的突变是 越来越多地与先天性骨骼肌疾病有关，如远端关节融合等 最近出现了一类新的家族性肌肉震颤。尽管如此，要理清纷繁复杂的关系仍是一个挑战 MyBP-C的两个骨骼平行基因的不同功能作用部分是因为大多数骨骼肌 含有慢纤维和快纤维类型的混合物，因为在慢抽动纤维中表达的MyBP-C1， 经历广泛的选择性剪接，导致一系列差异表达的蛋白质 具有独特的功能效果。因此，几乎不可能区分功能性的 在工作肌肉的背景下，每个变量的意义。为了克服这些挑战，PI的 Lab最近开发了一种新的“剪切和粘贴”方法来选择性地瞄准和替换不同的 MyBP-C在肌肉肌节中类似。该方法最初是为MYBPC3开发的，依赖于使用 表达烟草蚀刻病毒蛋白水解酶(TEVp)共同位点和“SpyTag”的基因编辑的小鼠 在心脏MyBP-C内的序列，以便当洗涤剂渗透性的心肌细胞被 TEVp MyBP-C被选择性切割。接下来，MyBP-C可以被任何重组蛋白取代 (包含任何所需的序列修改)，只要重组蛋白编码一个 “SpyCatcher”序列是因为SpyCatcher和SpyTag来自瞬间的共价键。这里, 我们扩大了方法，创造了“SpyC1”小鼠，使我们能够有选择地研究缓慢的骨骼 MyBP-C(Aim 1)和“SnoopC2”小鼠研究快速骨骼MyBP-C(Aim 2)。初步数据使用 剪切和粘贴方法已经对MyBP-C功能障碍如何导致 肌肉震颤和这些研究的其他结果将确定 每个MyBP-C并列，以及MyBP-C的突变如何导致疾病。

PROJECT SUMMARY The overall goal of this project is to understand how myosin binding protein-C (MyBP-C) regulates contraction and relaxation in skeletal muscles. 3 distinct paralogs of MyBP-C encoded by 3 separate genes, MYBPC1, MYBPC2, and MYBPC3, are expressed in slow twitch skeletal muscle, fast twitch muscle, and cardiac muscle, respectively. Of the three, the cardiac paralog has been most intensively studied because mutations in MYBPC3 are the most common cause of hypertrophic cardiomyopathy (HCM). However, like MYBPC3, it is now clear that mutations in MYBPC1 and MYBPC2 are increasingly linked to congenital skeletal muscle diseases such as distal arthrogryposis and more recently to a new class of familial muscle tremors. Despite this, it has been challenging to disentangle the distinct functional roles of the 2 skeletal paralogs of MyBP-C in part because most skeletal muscles contain a mixture of slow and fast fiber types and because MyBP-C1, expressed in slow twitch fibers, undergoes extensive alternative splicing resulting in a family of differentially expressed proteins each with unique functional effects. As a result, it has been nearly impossible to distinguish the functional significance of each variant in the context of working muscles. To overcome these challenges, the PI’s lab recently developed a novel “cut and paste” approach to selectively target and replace different MyBP-C paralogs in muscle sarcomeres. The method, first developed for MYBPC3, relies on the use of gene-edited mice that express a tobacco etch virus protease (TEVp) consensus site and a “SpyTag” sequence within cardiac MyBP-C so that when detergent-permeabilized myocytes are treated with TEVp MyBP-C is selectively cleaved. Next, MyBP-C can be replaced with any recombinant protein (containing any desired sequence modification) as long as the recombinant protein encodes a “SpyCatcher” sequence because SpyCatcher and SpyTag from an instantaneous covalent bond. Here, we expanded the method by creating “SpyC1” mice that allow us to selectively study slow skeletal MyBP-C (Aim 1) and “SnoopC2” mice to study fast skeletal MyBP-C (Aim 2). Preliminary data using the cut and paste method has already yielded new insights into how MyBP-C dysfunction can lead to muscle tremors and additional results from these studies will determine the functional significance of each MyBP-C paralog and how mutations in MyBP-C cause disease.