PROJECT SUMMARYThe overall goal of this project is to understand how myosin binding protein-C (MyBP-C) regulatescontraction and relaxation in skeletal muscles. 3 distinct paralogs of MyBP-C encoded by 3 separategenes MYBPC1 MYBPC2 and MYBPC3 are expressed in slow twitch skeletal muscle fast twitchmuscle and cardiac muscle respectively. Of the three the cardiac paralog has been most intensivelystudied 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 areincreasingly linked to congenital skeletal muscle diseases such as distal arthrogryposis and morerecently to a new class of familial muscle tremors. Despite this it has been challenging to disentanglethe distinct functional roles of the 2 skeletal paralogs of MyBP-C in part because most skeletal musclescontain a mixture of slow and fast fiber types and because MyBP-C1 expressed in slow twitch fibersundergoes extensive alternative splicing resulting in a family of differentially expressed proteins eachwith unique functional effects. As a result it has been nearly impossible to distinguish the functionalsignificance of each variant in the context of working muscles. To overcome these challenges the PIslab recently developed a novel cut and paste approach to selectively target and replace differentMyBP-C paralogs in muscle sarcomeres. The method first developed for MYBPC3 relies on the useof gene-edited mice that express a tobacco etch virus protease (TEVp) consensus site and a SpyTagsequence within cardiac MyBP-C so that when detergent-permeabilized myocytes are treated withTEVp 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 aSpyCatcher sequence because SpyCatcher and SpyTag from an instantaneous covalent bond. Herewe expanded the method by creating SpyC1 mice that allow us to selectively study slow skeletalMyBP-C (Aim 1) and SnoopC2 mice to study fast skeletal MyBP-C (Aim 2). Preliminary data usingthe cut and paste method has already yielded new insights into how MyBP-C dysfunction can lead tomuscle tremors and additional results from these studies will determine the functional significance ofeach MyBP-C paralog and how mutations in MyBP-C cause disease.