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

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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.