Cardiac myosin-binding protein C (cMyBP-C) is a regulatory protein expressed in cardiac sarcomeres that is known to interact with myosin, titin, and actin. cMyBP-C modulates actomyosin interactions in a phosphorylation-dependent way, but it is unclear whether interactions with myosin, titin, or actin are required for these effects. Here we show using cosedimentation binding assays, that the 4 N-terminal domains of murine cMyBP-C (i.e. C0-C1-m-C2) bind to F-actin with a dissociation constant (K d ) of ϳ10 M and a molar binding ratio (B max ) near 1.0, indicating 1:1 (mol/mol) binding to actin. Electron microscopy and light scattering analyses show that these domains cross-link F-actin filaments, implying multiple sites of interaction with actin. Phosphorylation of the MyBP-C regulatory motif, or m-domain, reduced binding to actin (reduced B max ) and eliminated actin cross-linking. These results suggest that the N terminus of cMyBP-C interacts with F-actin through multiple distinct binding sites and that binding at one or more sites is reduced by phosphorylation. Reversible interactions with actin could contribute to effects of cMyBP-C to increase crossbridge cycling. Cardiac myosin-binding protein C (cMyBP-C)2 is a thick filament accessory protein that performs both structural and regulatory functions within vertebrate sarcomeres. Both roles are likely to be essential in deciphering how a growing number of mutations found in the cMyBP-C gene, i.e. MYBPC3, lead to cardiomyopathies and heart failure in a substantial number of the world's population (1, 2).Considerable progress has recently been made in determining the regulatory functions of cMyBP-C and it is now apparent that cMyBP-C normally limits cross-bridge cycling kinetics and is critical for cardiac function (3-5). Phosphorylation of cMyBP-C is essential for its regulatory effects because elimination of phosphorylation sites (serine to alanine substitutions) abolishes the ability of protein kinase A (PKA) to accelerate cross-bridge cycling kinetics and blunts cardiac responses to inotropic stimuli (6). The substitutions further impair cardiac function, reduce contractile reserve, and cause cardiac hypertrophy in transgenic mice (6, 7). By contrast, substitution of aspartic acids at these sites to mimic constitutive phosphorylation is benign or cardioprotective (8).Although a role for cMyBP-C in modulating cross-bridge kinetics is supported by several transgenic and knock-out mouse models (6, 7, 9, 10), the precise mechanisms by which cMyBP-C exerts these effects are not completely understood. For instance, the unique regulatory motif or "m-domain" of cMyBP-C binds to the S2 subfragment of myosin in vitro (11) and binding is abolished by PKA-mediated phosphorylation of the m-domain (12). These observations have led to the idea that (un)binding of the m-domain from myosin S2 mediates PKA-induced increases in cross-bridge cycling kinetics. Consistent with this idea, Calaghan and colleagues (13) showed that S2 added to transiently permeabilized myocytes increa...

Myosin binding protein-C (MyBP-C) is a thick-filament protein whose precise function within the sarcomere is not known. However, recent evidence from cMyBP-C knock-out mice that lack MyBP-C in the heart suggest that cMyBP-C normally slows cross-bridge cycling rates and reduces myocyte power output. To investigate possible mechanisms by which cMyBP-C limits cross-bridge cycling kinetics we assessed effects of recombinant N-terminal domains of MyBP-C on the ability of heavy meromyosin (HMM) to support movement of actin filaments using in vitro motility assays. Here we show that N-terminal domains of cMyBP-C containing the MyBP-C "motif," a sequence of ϳ110 amino acids, which is conserved across all MyBP-C isoforms, reduced actin filament velocity under conditions where fila- Myosin binding protein-C (MyBP-C)2 is a sarcomeric protein associated with the thick filaments of vertebrate striated muscle (1). Although the precise function of MyBP-C within the sarcomere is not well understood, evidence from MyBP-C knock-out mice that lack cardiac MyBP-C (2) indicate cMyBP-C slows cross-bridge cycling and rates of force development, especially at submaximal [Ca 2ϩ ] (3-5). The idea that MyBP-C limits cross-bridge kinetics was initially proposed by Hofmann et al. (6) who suggested that MyBP-C acts as an internal load within the sarcomere based on their observations that partial extraction of MyBP-C from skeletal fibers reversibly accelerated a low velocity phase of shortening at submaximal Ca 2ϩ activation (7). Although the exact structural arrangement of MyBP-C within the sarcomere is not known, MyBP-C could contribute to an internal load by tethering myosin heads to the thick filament and thereby limiting the extension of attached myosin heads as shortening proceeds (6). Consistent with this idea, Calaghan et al. (8) proposed that simultaneous binding of MyBP-C to two positions on myosin, i.e. to myosin S2 (near the S1/S2 junction) and to the light meromyosin segment of myosin rod, could restrict the extension of myosin heads away from the thick filament. The net effect might be to limit myosin interactions with actin. However, a recombinant MyBP-C protein containing only the C1C2 domains and thus a single S2 binding site increased Ca 2ϩ sensitivity of force in myocytes from cMyBP-C knock-out mice (9). Because effects of C1C2 did not depend on a second myosin binding site, the results implied that the C1C2 domains could affect actomyosin interactions independent of tethering myosin heads to thick filaments.The current experiments were performed to investigate mechanisms by which N-terminal domains of MyBP-C influence myosin contractile properties and whether these effects depend on organization of myosin into thick filaments. Results from in vitro motility assays demonstrate that organized thick filaments are not required for recombinant proteins containing N-terminal domains of MyBP-C to affect mechanical properties of myosin and further suggest that effects of MyBP-C to slow cross-bridge kinetics may be due to slow...

Cardiac myosin binding protein C (cMyBP-C), a major accessory protein of cardiac thick filaments, is thought to play a key role in the regulation of myocardial contraction. Although current models for the function of the protein focus on its binding to myosin S2, other evidence suggests that it may also bind to F-actin. We have previously shown that the N-terminal fragment C0-C2 of cardiac myosin binding protein-C (cMyBP-C) bundles actin, providing evidence for interaction of cMyBP-C and actin. In this paper we directly examined the interaction between C0-C2 and F-actin at physiological ionic strength and pH by negative staining and electron microscopy. We incubated C0-C2

Troponin I (TnI) and myosin binding protein-C (MyBP-C) are key regulatory proteins of contractile function in vertebrate muscle. TnI modulates the Ca(2+) activation signal, while MyBP-C regulates cross-bridge cycling kinetics. In vertebrates, each protein is distributed as tissue-specific paralogs in fast skeletal (fs), slow skeletal (ss), and cardiac (c) muscles. The purpose of this study is to characterize how TnI and MyBP-C have changed during the evolution of vertebrate striated muscle and how tissue-specific paralogs have adapted to different physiological conditions. To accomplish this we have completed phylogenetic analyses using the amino acid sequences of all known TnI and MyBP-C isoforms. This includes 99 TnI sequences (fs, ss, and c) from 51 different species and 62 MyBP-C sequences from 26 species, with representatives from each vertebrate group. Results indicate that the role of protein kinase A (PKA) and protein kinase C (PKC) in regulating contractile function has changed during the evolution of vertebrate striated muscle. This is reflected in an increased number of phosphorylatable sites in cTnI and cMyBP-C in endothermic vertebrates and the loss of two PKC sites in fsTnI in a common ancestor of mammals, birds, and reptiles. In addition, we find that His(132), Val(134), and Asn(141) in human ssTnI, previously identified as enabling contractile function during cellular acidosis, are present in all vertebrate cTnI isoforms except those from monotremes, marsupials, and eutherian mammals. This suggests that the replacement of these residues with alternative residues coincides with the evolution of endothermy in the mammalian lineage.