Interactions between cardiac myosin binding protein-C and actin contribute to the regulation of muscle contraction.

摘要

Myosin binding protein-C (MyBP-C) is a regulatory and accessory protein associated with thick filaments in vertebrate striated muscles and has been linked to familial hypertrophic cardiomyopathy (FHC). While the C-terminus of cardiac (c) MyBP-C has been shown to bind to light meromyosin (LMM) and titin, the N-terminus appears to exert a regulatory role on actomyosin kinetics through phosphorylation dependent interactions between the M-domain and myosin subfragment-2 (S2) by limiting cross-bridge formation and cycling. MyBP-C has also been shown to bind to F-actin; however, specific actin-binding domains of MyBP-C have not been characterized and the functional role of actin binding is unknown. The aim of this thesis was to determine if interactions between the cMyBP-C M-domain and myosin S2 are required for functional effects of cMyBP-C and to investigate whether cMyBP-C interactions with the thin filament contribute to the regulation of muscle contraction. Recombinant proteins containing N-terminal domains of cMyBP-C were created and in vitro motility and myosin ATPase assays were used to assess effects of cMyBP-C on actomyosin interactions and cosedimentation assays were used to assess binding of cMyBP-C to F-actin. Results showed that myosin S2 is not required for the Cl, M, and C2 domains to activate or inhibit Ca2+ regulated thin filament motility in in vitro motility assays either in the absence and presence of Ca2+, providing indirect evidence that interactions with actin may be important for the regulatory effects of cMyBP-C. Cosedimentation assays showed that the Cl and M domains bound to F-actin with affinity, K d, of ∼10 muM and at a molar binding ratio, Bmax, of 1.0 mol/mol. The binding interaction between the M-domain and F-actin was eliminated when the M-domain was phosphorylated by protein kinase A (PKA). Finally, specific residues in the M-domain were identified that are important for actin binding and actin binding was shown to be correlated with inhibitory effects of cMyBP-C in in vitro motility assays. The results of this thesis form the basis for a novel hypothesis describing the function of cMyBP-C in sarcomeres and suggest that cMyBP-C may interact with the thin filament to regulate actomyosin interactions in vivo.