Sarcomere Assembly Myosin Filament Formation and Myosin-binding Protein C and Regulates Four and a H

FourandahalfLIMprotein1(FHL1/SLIM1)ishighlyexpressedin skeletal and cardiac muscle; however, the function of FHL1remainsunknown.Yeasttwo-hybridscreeningidentifiedslowtypeskeletal myosin-binding protein C as an FHL1 binding partner.Myosin-binding protein C is the major myosin-associated proteinin striated muscle that enhances the lateral association and stabili-zation of myosin thick filaments and regulates actomyosin interac-tions.TheinteractionbetweenFHL1andmyosin-bindingproteinCwas confirmed using co-immunoprecipitation of recombinant andendogenous proteins. Recombinant FHL2 and FHL3 also boundmyosin-binding protein C. FHL1 impaired co-sedimentation ofmyosin-binding protein C with reconstituted myosin filaments,suggesting FHL1 may compete with myosin for binding to myosin-bindingproteinC.Inintactskeletalmuscleandisolatedmyofibrils,FHL1 localized to the I-band, M-line, and sarcolemma, co-localiz-ingwithmyosin-bindingproteinCatthesarcolemmainintactskel-etal muscle. Furthermore, in isolated myofibrils FHL1 staining atthe M-line appeared to extend partially into the C-zone of theA-band,whereitco-localizedwithmyosin-bindingproteinC.Over-expression of FHL1 in differentiating C2C12 cells induced “sac-like”myotubeformation(myosac),associatedwithimpairedZ-lineandmyosinthickfilamentassembly.Thisphenotypewasrescuedbyco-expression of myosin-binding protein C. FHL1 knockdownusing RNAi resulted in impaired myosin thick filament formationassociatedwithreducedincorporationofmyosin-bindingproteinCintothesarcomere.ThisstudyidentifiedFHL1asanovelregulatorof myosin-binding protein C activity and indicates a role for FHL1in sarcomere assembly.In striated muscle LIM proteins play critical roles in scaffolding sar-comeric and signaling proteins (1–8). LIM proteins are defined by thepresence of one or more LIM domains, a cysteine-rich double zincfinger protein-binding motif denoted by the sequence (C