Project Grant R21AR084767

SKELETAL MYOSIN-BINDING PROTEIN C: DEFINING FUNCTION ACROSS SCALES USING A ZEBRAFISH MODEL SYSTEM. - MYOSIN-BINDING PROTEIN C (MYBP-C), A THICK FILAMENT ASSOCIATED PROTEIN OF VERTEBRATE STRIATED MUSCLE, IS A KEY MODULATOR OF MUSCLE CONTRACTILITY. MULTIPLE DISTINCT SKELETAL MUSCLE MYBP-C ISOFORMS ARE ENCODED BY TWO GENES IN MAMMALS (I.E., MYBPC1 (SLOW-TYPE) AND MYBPC2 (FAST-TYPE)) WITH MUTATIONS TO THESE GENES NOW LINKED TO HUMAN SKELETAL MYOPATHIES, SUCH AS DISTAL ARTHROGRYPOSIS. IN VITRO REDUCTIONIST APPROACHES HAVE PROPOSED MECHANISMS BY WHICH THE MYBP-C N TERMINUS MODULATES MUSCLE CONTRACTILITY THROUGH ITS BINDING- PARTNER INTERACTIONS WITH THE ACTIN-THIN FILAMENT AND THE MYOSIN HEAD REGION. SPECIFICALLY, MYBP-C IS BELIEVED TO SENSITIZE THE THIN FILAMENT TO CALCIUM, STABILIZE THE MYOSIN SUPER-RELAXED STATE, AND/OR ACT AS A MOLECULAR "BRAKE" TO SLOW MYOFILAMENT SLIDING. HOWEVER, SINCE MULTIPLE MYBP-C ISOFORMS ARE CO-EXPRESSED IN MAMMALIAN MUSCLE, IT HAS BEEN IMPOSSIBLE TO DEFINE WHICH OF THESE MODULATORY ROLES ARE LINKED TO SPECIFIC MYBP-C ISOFORMS WITHIN THE CONTEXT OF AN INTACT MUSCLE, LET ALONE HOW THEY MAY BE ALTERED BY GENETIC MUTATIONS. HERE WE PROPOSE A NOVEL APPROACH IN ZEBRAFISH TO GENERATE 'DESIGNER' MUSCLES EXCLUSIVELY EXPRESSING A SINGLE, TRANSGENE-ENCODED MYBP-C ISOFORM AS DESIRED. OUR PRELIMINARY DATA INDICATE THAT MYBP-H, AN MYBP-C FAMILY MEMBER, COMPRISES ~95% OF MYOSIN BINDING PROTEIN IN LARVAL ZEBRAFISH SWIMMING MUSCLES. THEREFORE, AIM 1 MAKES USE OF THE RECENTLY DEVELOPED CRISPR/CAS9 'GENEWELD' METHOD, TO GENERATE PRECISE INTEGRATION ALLELES BY HOMOLOGY MEDIATED END JOINING (HMEJ) THAT WILL SIMULTANEOUSLY: I) INTERRUPT ENDOGENOUS MYBP-H EXPRESSION, AND; II) PLACE A DNA CASSETTE, ENCODING ONE OF TWO MOST FUNCTIONALLY EXTREME MYBP-C ISOFORMS, UNDER REGULATORY CONTROL OF THE MOST HIGHLY EXPRESSED ENDOGENOUS MYBP GENE LOCUS. BY THIS APPROACH, WE PROPOSE TO CREATE ZEBRAFISH WITH "DESIGNER MYBP-C" MUSCLES. QUANTITATIVE PROTEOMICS WILL ENABLE US TO DETERMINE WHETHER TRANSGENIC MYBP-C ACCUMULATES TO WILDTYPE LEVELS, WHILE IMMUNOFLUORESCENCE OF FLAG-TAGGED TRANSGENIC MYBP-C WILL BE USED TO CONFIRM PROPER SUBCELLULAR LOCALIZATION. IN AIM 2 WE USE BIOPHYSICAL ASSAYS PREVIOUSLY DEVELOPED IN THE WARSHAW LAB TO DEFINE THE FUNCTIONAL IMPACT OF TRANSGENIC MYBP-C ISOFORMS ACROSS MULTIPLE SCALES. SPECIFICALLY, NATIVE MYOSIN THICK FILAMENTS WILL BE USED TO ASSESS MYBP-C "BRAKING" ACTION, WHILE MYOFIBRILS WILL BE USED TO ASSESS THE PRESENCE OF THE SUPER-RELAXED MYOSIN STATE. DATA FROM THESE SIMPLIFIED MUSCLE SYSTEMS OBTAINED FROM THE PROPOSED "DESIGNER MYBP-C" ZEBRAFISH WILL BE CORRELATED WITH INTACT LARVAL MUSCLE MECHANICS. THUS, THIS PROJECT WILL PROVIDE SIGNIFICANT INSIGHT INTO HOW AN INDIVIDUAL MYBP-C ISOFORM MODULATES BOTH MOLECULAR AND CELLULAR CONTRACTILITY IN THE CONTEXT OF INTACT MUSCLE. THIS "DESIGNER MYBP-C" ZEBRAFISH MODEL SYSTEM WILL CREATE A PLATFORM FOR FUTURE MECHANISTIC STUDIES OF MYBP-C MUTATIONS ASSOCIATED WITH HUMAN SKELETAL MYOPATHIES AS A FIRST STEP TO THERAPEUTIC DESIGN.