Myosin Binding protein-C Isoforms Research Articles

Myosin binding protein h dominates the "c-zone" in ultrafast swimming muscles of developing zebrafish.

The ‘C-zone’ of mature vertebrate skeletal muscle sarcomeres is characterized by the presence of Myosin-Binding Protein C (MyBP-C), which ‘tunes’ contractility via its N-terminal domain interactions with the myosin head region and/or the thin filament. The ultrafast swimming muscles of zebrafish larvae are a perfect model system to define MyBP-C isoform function, due to tractable genetics and easily characterized muscle mechanics. However, liquid chromatography mass spectrometry shows 5-day-old larval muscle expresses MyBP-C at only ∼5% of the ∼1:10 molar ratio with myosin heavy chain (MYH) typically associated with full C-zone occupancy, while a little-studied MyBP-C paralog, Myosin-Binding Protein H (MyBP-H) comprises the remaining ∼95%. MyBP-H is homologous to MyBP-C's C terminus, which binds to the myosin thick filament backbone, but it lacks analagous N-terminal regulatory domains, which are critical to MyBP-C's slowing of muscle shortening. Therefore, the presence of MyBP-H may underlie the extreme contractile speed of larval muscle (>30 lengths/s), and/or indicate its involvement in muscle development. To define MyBP-H's potential roles, we first imaged its sarcomeric localization in larvae expressing transgenic, FLAG-tagged MyBP-H. Immunofluorescence revealed characteristic fluorescence doublets at the sarcomere center, indicating MyBP-H binds throughout the C-zone. Interestingly, MyBP-H-null mutants engineered using CRISPR/Cas9 appear to develop normally, suggesting the developmental impact of MyBP-H may be subtle. Preliminary mechanical studies of MyBP-H-null larval muscles showed no change in twitch kinetics, tetanic force, or the Force:Velocity relationship, which might be expected, given MyBP-C content increases only minimally (From ∼5% to ∼10% of full C-zone occupancy). Although the role of MyBP-H in development remains an enigma, physiologically stressing the larvae may reveal MyBP-H's function. At a minimum, the MyBP-H null larvae provide a clean slate to repopulate the C-zone using transgenically expressed human or disease-causing MyBP-C isoforms.

Skeletal Myosin-Binding Protein C Isoforms Differentially Regulate Fast- and Slow-Twitch Skeletal Muscle Function

Skeletal myosin-binding protein C (MyBP-C) is a myosin thick filament-associated protein that is encoded by two paralogous genes (slow-type (MYBPC1) and fast-type (MYBPC2)) and may regulate skeletal muscle contractility. We aimed to characterize the expression pattern, regulatory function, and phosphorylation-dependent modulation of this protein in in vitro actomyosin systems of rat slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL). By immunofluorescence imaging, slow-type MyBP-C is expressed in both EDL and SOL, while the fast-type MyBP-C is exclusive to EDL. Interestingly, these MyBP-C isoforms differentially localize within a discrete region of the sarcomere (the C-zone) that is muscle type-dependent. Mass spectrometry demonstrated expression of 3 distinct N-terminal splice variants of slow-type MyBP-C, which were present in both muscle types. When tissue-purified native thin filaments slid over tissue-purified native myosin thick filaments (containing the physiological MyBP-C compliment) in a calcium-dependent manner, only in the C-zone were thin filaments sensitized to calcium and their velocity slowed. Upon proteolysis of MyBP-C's N terminus, these modulatory capacities were attenuated, implicating this domain in MyBP-C's regulatory effects, as confirmed by in vitro motility experiments using recombinant N-terminal fragments of the various MyBP-C variant isoforms. These isoform fragments displayed differential impacts on thin filament sensitization and slowing, which were significantly reduced following PKA phosphorylation. Finally, we developed a “Monte Carlo” simulation that recapitulates thin filament trajectories observed over thick filaments by assuming that the various MyBP-C isoforms all exist within a C-zone and contribute their variant-specific modulatory impact. Thus, fast- and slow-twitch skeletal muscle contractility are differentially regulated by MyBP-C isoforms, and that MyBP-C function itself is, in turn, regulated by phosphorylation.

Heterozygous variants in MYBPC1 are associated with an expanded neuromuscular phenotype beyond arthrogryposis.

Encoding the slow skeletal muscle isoform of myosin binding protein-C, MYBPC1 is associated with autosomal dominant and recessive forms of arthrogryposis. The authors describe a novel association for MYBPC1 in four patients from three independent families with skeletal muscle weakness, myogenic tremors, and hypotonia with gradual clinical improvement. The patients carried one of two de novo heterozygous variants in MYBPC1, with the p.Leu263Arg variant seen in three individuals and the p.Leu259Pro variant in one individual. Both variants are absent from controls, well conserved across vertebrate species, predicted to be damaging, and located in the M-motif. Protein modeling studies suggested that the p.Leu263Arg variant affects the stability of the M-motif, whereas the p.Leu259Pro variant alters its structure. In vitro biochemical and kinetic studies demonstrated that the p.Leu263Arg variant results in decreased binding of the M-motif to myosin, which likely impairs the formation of actomyosin cross-bridges during muscle contraction. Collectively, our data substantiate that damaging variants in MYBPC1 are associated with a new form of an early-onset myopathy with tremor, which is a defining and consistent characteristic in all affected individuals, with no contractures. Recognition of this expanded myopathic phenotype can enable identification of individuals with MYBPC1 variants without arthrogryposis.

N-Terminal Domains of Cardiac Myosin Binding Protein C Cooperatively Activate the Thin Filament

Muscle contraction relies on interaction between myosin-based thick filaments and actin-based thin filaments. Myosin binding protein C (MyBP-C) is a key regulator of actomyosin interactions. Recent studies established that the N'-terminal domains (NTDs) of MyBP-C can either activate or inhibit thin filaments, but the mechanism of their collective actionis poorly understood. Cardiac MyBP-C (cMyBP-C) harbors an extra NTD, which is absent in skeletal isoforms of MyBP-C, and its role in regulation of cardiac contraction is unknown. Here we show that the first two domains of human cMyPB-C (i.e., C0 and C1) cooperate to activate the thin filament. We demonstrate that C1 interacts with tropomyosin via a positively charged loop and that this interaction, stabilized by the C0 domain, is required for thin filament activation by cMyBP-C. Our data reveal a mechanism by which cMyBP-C can modulate cardiac contraction and demonstrate a function of the C0 domain.

Skeletal myosin binding protein-C isoforms regulate thin filament activity in a Ca2+-dependent manner

Muscle contraction, which is initiated by Ca2+, results in precise sliding of myosin-based thick and actin-based thin filament contractile proteins. The interactions between myosin and actin are finely tuned by three isoforms of myosin binding protein-C (MyBP-C): slow-skeletal, fast-skeletal, and cardiac (ssMyBP-C, fsMyBP-C and cMyBP-C, respectively), each with distinct N-terminal regulatory regions. The skeletal MyBP-C isoforms are conditionally coexpressed in cardiac muscle, but little is known about their function. Therefore, to characterize the functional differences and regulatory mechanisms among these three isoforms, we expressed recombinant N-terminal fragments and examined their effect on contractile properties in biophysical assays. Addition of the fragments to in vitro motility assays demonstrated that ssMyBP-C and cMyBP-C activate thin filament sliding at low Ca2+. Corresponding 3D electron microscopy reconstructions of native thin filaments suggest that graded shifts of tropomyosin on actin are responsible for this activation (cardiac > slow-skeletal > fast-skeletal). Conversely, at higher Ca2+, addition of fsMyBP-C and cMyBP-C fragments reduced sliding velocities in the in vitro motility assays and increased force production in cardiac muscle fibers. We conclude that due to the high frequency of Ca2+ cycling in cardiac muscle, cardiac MyBP-C may play dual roles at both low and high Ca2+. However, skeletal MyBP-C isoforms may be tuned to meet the needs of specific skeletal muscles.

Calcium-Dependent Interaction Occurs between Slow Skeletal Myosin Binding Protein C and Calmodulin

Myosin binding protein C (MyBP-C) is a multi-domain protein that participates in the regulation of muscle contraction through dynamic interactions with actin and myosin. Three primary isoforms of MyBP-C exist: cardiac (cMyBP-C), fast skeletal (fsMyBP-C), and slow skeletal (ssMyBP-C). The N-terminal region of cMyBP-C contains the M-motif, a three-helix bundle that binds Ca2+-loaded calmodulin (CaM), but less is known about N-terminal ssMyBP-C and fsMyBP-C. Here, we characterized the conformation of a recombinant N-terminal fragment of ssMyBP-C (ssC1C2) using differential scanning fluorimetry, nuclear magnetic resonance, and molecular modeling. Our studies revealed that ssC1C2 has altered thermal stability in the presence and absence of CaM. We observed that site-specific interaction between CaM and the M-motif of ssC1C2 occurs in a Ca2+-dependent manner. Molecular modeling supported that the M-motif of ssC1C2 likely adopts a three-helix bundle fold comparable to cMyBP-C. Our study provides evidence that ssMyBP-C has overlapping structural determinants, in common with the cardiac isoform, which are important in controlling protein–protein interactions. We shed light on the differential molecular regulation of contractility that exists between skeletal and cardiac muscle.

Skeletal Myosin-Binding Protein C Modulates Actomyosin Contractility in an Isoform-Dependent Manner

Myosin-binding protein C (MyBP-C) is a sarcomeric protein localized to discrete regions (C-zones) of myosin thick filaments in all striated muscles. Unlike cardiac muscle with its specific MyBP-C isoform, mass spectrometry shows that rat slow-twitch soleus (SOL) muscle expresses two different slow MyBP-C isoforms (sMyBP-C), having one (sMyBP-C1) or two N-terminal inserts (sMyBP-C2), while fast-twitch extensor digitorum longus (EDL) expresses these same sMyBP-C isoforms and a fast MyBP-C isoform (fMyBP-C). Do these MyBP-C isoforms differentially regulate skeletal actomyosin contractility in a muscle-specific manner? To address this, we characterized Ca2+-regulated motion of SOL and EDL native actin-thin filaments (NTFs) over their corresponding native myosin-thick filaments, having the physiological complement of MyBP-C in the C-zone. At low calcium (pCa7.5) where NTFs should be inhibited, motion occurred within the C-zone of both SOL and EDL native thick filaments. Whereas, at high Ca2+ (pCa5), SOL and EDL NTFs had initial fast velocities followed by ∼30% slower velocities within the C-zone. These data suggest that one or more of these MyBP-C can sensitize NTFs to Ca2+ and modulate contractility at high Ca2+. To determine how each isoform contributes to these modulatory capacities, we bacterially-expressed N-terminal fragments (sMyBP-C1, sMyBP-C2, fMyBP-C) and assessed their individual impact on SOL or EDL NTF motility over their respective monomeric myosins. For the EDL where all three MyBP-Cs are expressed, at pCa7.5 sMyBP-C1 was most effective at activating NTFs, while at pCa5 all three reduced velocity by ∼50%. For SOL, where only sMyBP-C isoforms are expressed, at pCa7.5 sMyBP-C1 activated NTFs while at pCa5 sMyBP-C2 reduced velocity ∼40%. Our data suggest that the distinctive mixture of MyBP-C isoforms expressed in the SOL and EDL is matched to the muscle's physiological demands. Each MyBP-C isoform possesses specific modulatory capacities that may be called upon under different contractile states. Specifically, to fine-tune actomyosin contractility within the C-zone by Ca2+-sensitizing the thin filament and/or modulating velocity at maximal activation.

A Highly Conserved Yet Flexible Linker Is Part of a Polymorphic Protein-Binding Domain in Myosin-Binding Protein C

The nuclear magnetic resonance (NMR) structure of the tri-helix bundle (THB) of the m-domain plus C2 (ΔmC2) of myosin-binding protein C (MyBP-C) has revealed a highly flexible seven-residue linker between the structured THB and C2. Bioinformatics shows significant patterns of conservation across the THB-linker sequence, with the linker containing a strictly conserved serine in all MyBP-C isoforms. Clinically linked mutations further support the functional significance of the THB-linker region. NMR, small-angle X-ray scattering, and binding studies show the THB-linker plus the first ten residues of C2 undergo dramatic changes when ΔmC2 binds Ca2+-calmodulin, with the linker and C2 N-terminal residues contributing significantly to the affinity. Modeling of all available experimental data indicates that the THB tertiary structure must be disrupted to form the complex. These results are discussed in the context of the THB-linker and the N-terminal residues of C2 forming a polymorphic binding domain that could accommodate multiple binding partners in the dynamic sarcomere.

The N-Terminal Domains of MyBPC3 Restrict Actin Dynamics and Increase Resilience in a Phosphorylation-Dependent Manner

We have determined the effects of myosin binding protein-C (MyBP-C) N-terminal domains on the microsecond rotational dynamics of actin, detected by time-resolved phosphorescence anisotropy (TPA). MyBP-C modulates muscle contractility and is capable of interacting with thin filaments. Protein kinase A (PKA) phosphorylates cardiac and slow skeletal MyBP-C, altering myofilament structure and function. We previously used TPA to show that full-length MyBP-C restricts actin torsional dynamics, and effects by cardiac and slow skeletal MyBP-C (cMyBP-C; ssMyBP-C) are relieved by PKA phosphorylation. To determine the effects of MyBP-C N-terminal domains on actin structural dynamics, we labeled actin at C374 with a phosphorescent dye and performed TPA experiments. The interaction of all three MyBP-C isoforms with actin increased the final anisotropy of the TPA decay, indicating restriction of the amplitude of actin flexibility at saturation of the TPA effect. PKA phosphorylation of ssMyBP-C domains C1-C2 and cMyBP-C domains C0-C2 relieved the restriction of torsional amplitude but also increased the rate of torsional motion, thus increasing actin resilience (rate/amplitude; less brittle). Moreover, effects of cardiac C0-C2 on actin resilience were also PKA-dependent, whereas slow skeletal C1-C2 effects which promote actin resilience persisted independently of phosphorylation. In the case of fast skeletal C1-C2, actin resilience was unaffected and its effect to restrict actin dynamics was unchanged by phosphorylation. The N-terminal domains of cMyBP-C (C0-C2) and skeletal MyBP-C's (C1-C2) had effects on actin dynamics similar to those determined for the full-length MyBP-C isoforms. Effects of phosphomimetic mutations were also investigated. These unique isoform-dependent MyBP-C-induced changes in actin dynamics may play a role in the functional effects of MyBP-C on contraction. This work was supported by NIH grants to DDT (R01 AR032961), to SS (R01 AR067279), and to BC (R00 HL122397).

Skeletal Myosin Binding Protein-C Isoforms Modulate Actomyosin Contractility and are Regulated by Phosphorylation

Myosin binding protein C (MyBP-C) is a thick filament-associated protein found in striated muscle and may regulate muscle contractility. Separate genes encode the fast and slow skeletal isoforms, and there are potential PKA phosphorylation sites in their functionally important N-terminal regions. Here, we compare mouse N-terminal fast (fC1C2) and slow (sC1C2) skeletal fragments containing the initial ∼50 aa Pro/Ala-rich domain and the C1 and C2 Ig-domains that are linked by the ∼100 aa M-domain. Of the known slow skeletal splice variants, we chose a highly expressed variant lacking the N-terminal 34-59 residue insert. To define the potential mechanisms by which skeletal MyBP-Cs affect contractility and whether these effects are modulated by PKA phosphorylation, we assessed the Ca2+-dependent motility of rabbit skeletal native thin filaments over a surface of rabbit psoas myosin in the presence of C1C2 fragments. While thin filaments were fully regulated, with no motion observed at pCa > 7 in the absence of fragments, the addition of either 0.50 μM fC1C2 or sC1C2 resulted in significant motility. This suggests that skeletal MyBP-C isoforms effectively sensitize the thin filament. Under fully activating conditions (pCa ≤ 5), sC1C2 had little effect on motility whereas fC1C2 inhibited sliding velocities by nearly 50%. Thus, these fragments differ in their modulatory capacities with fC1C2 sensitizing the thin filament to Ca2+ and inhibiting maximal velocities, while the sC1C2 variant exhibits only a single mode of contractile modulation; i.e. thin filament sensitization. Interestingly, PKA phosphorylation in the Pro/Ala (sC1C2) and M-domains (sC1C2 and fC1C2), as confirmed by mass spectrometry, reduced both fragments’ Ca2+ sensitization of the thin filament. Thus, the function of MyBP-C isoforms may be tuned to match the physiological demands of the muscle in which they are expressed.

Cardiac myosin binding protein-C plays no regulatory role in skeletal muscle structure and function.

Myosin binding protein-C (MyBP-C) exists in three major isoforms: slow skeletal, fast skeletal, and cardiac. While cardiac MyBP-C (cMyBP-C) expression is restricted to the heart in the adult, it is transiently expressed in neonatal stages of some skeletal muscles. However, it is unclear whether this expression is necessary for the proper development and function of skeletal muscle. Our aim was to determine whether the absence of cMyBP-C alters the structure, function, or MyBP-C isoform expression in adult skeletal muscle using a cMyBP-C null mouse model (cMyBP-C(t/t)). Slow MyBP-C was expressed in both slow and fast skeletal muscles, whereas fast MyBP-C was mostly restricted to fast skeletal muscles. Expression of these isoforms was unaffected in skeletal muscle from cMyBP-C(t/t) mice. Slow and fast skeletal muscles in cMyBP-C(t/t) mice showed no histological or ultrastructural changes in comparison to the wild-type control. In addition, slow muscle twitch, tetanus tension, and susceptibility to injury were all similar to the wild-type controls. Interestingly, fMyBP-C expression was significantly increased in the cMyBP-C(t/t) hearts undergoing severe dilated cardiomyopathy, though this does not seem to prevent dysfunction. Additionally, expression of both slow and fast isoforms was increased in myopathic skeletal muscles. Our data demonstrate that i) MyBP-C isoforms are differentially regulated in both cardiac and skeletal muscles, ii) cMyBP-C is dispensable for the development of skeletal muscle with no functional or structural consequences in the adult myocyte, and iii) skeletal isoforms can transcomplement in the heart in the absence of cMyBP-C.

Mechanistic Understanding of the Involvement of MyBP-C Slow in the Development of Distal Arthrogryposis

Myosin Binding Protein-C (MyBP-C), expressed in striated muscles, is a thick filament associated protein that plays important roles in thick filament assembly and stabilization, and the modulation of actomyosin cross-bridges formation. Several studies have implicated MyBP-C in the development of cardiac and recently skeletal myopathies, underlining its importance in normal sarcomeric function. Mutations in the slow skeletal isoform of MyBP-C (sMyBP-C) have been causally linked to severe and lethal forms of arthrogryposis, a family of neuromuscular diseases. Two missense mutations within MYBPC1, the gene that encodes sMyBP-C, located within the NH2-terminal M-motif (W236R) and the COOH-terminal immunoglobulin (Ig) C8 domain (Y856H) have been linked to the development of distal arthrogryposis type-1 (DA-1), a skeletal muscle disorder characterized by contractures of the distal limbs. Here we explore the molecular defects that underlie the development of DA-1. using a combination of in vitro binding and motility assays, we show that the presence of the W236R mutation abolishes the ability of the NH2-terminus to modulate the formation of actomyosin cross-bridges, while the presence of the Y856H mutation inhibits Ig domains C8-C10 from interacting with native myosin filaments. Our findings therefore suggest that distinct mechanisms may underlie the pathogenesis of DA-1 myopathy depending on the location of the individual mutation within sMyBP-C. We are currently examining the effects of overexpressing mutant sMyBP-C proteins carrying either the W236R or the Y856H mutation in a sMyBP-C-null background, via in vivo gene transfer. In summary, our studies are the first to provide a mechanistic interpretation of the pathogenesis of distal arthrogryposis.

Cardiac myosin binding protein-C restricts intrafilament torsional dynamics of actin in a phosphorylation-dependent manner

We have determined the effects of myosin binding protein-C (MyBP-C) and its domains on the microsecond rotational dynamics of actin, detected by time-resolved phosphorescence anisotropy (TPA). MyBP-C is a multidomain modulator of striated muscle contraction, interacting with myosin, titin, and possibly actin. Cardiac and slow skeletal MyBP-C are known substrates for protein kinase-A (PKA), and phosphorylation of the cardiac isoform alters contractile properties and myofilament structure. To determine the effects of MyBP-C on actin structural dynamics, we labeled actin at C374 with a phosphorescent dye and performed TPA experiments. The interaction of all three MyBP-C isoforms with actin increased the final anisotropy of the TPA decay, indicating restriction of the amplitude of actin torsional flexibility by 15-20° at saturation of the TPA effect. PKA phosphorylation of slow skeletal and cardiac MyBP-C relieved the restriction of torsional amplitude but also decreased the rate of torsional motion. In the case of fast skeletal MyBP-C, its effect on actin dynamics was unchanged by phosphorylation. The isolated C-terminal half of cardiac MyBP-C (C5-C10) had effects similar to those of the full-length protein, and it bound actin more tightly than the N-terminal half (C0-C4), which had smaller effects on actin dynamics that were independent of PKA phosphorylation. We propose that these MyBP-C-induced changes in actin dynamics play a role in the functional effects of MyBP-C on the actin-myosin interaction.

Introducing a series of topical special issues of the Journal of Muscle Research and Cell Motility

The Journal of Muscle Research aims to serve its readers not only by publishing original studies in muscle and motility structure and function but also by informing readers of the state of the art in muscle research and cell motility and to promote debate. This issue of the Journal of Muscle Research and Cell Motility is the first of a series of topical special issues that combine original research with review articles related to a specific subject that we plan to publish once or twice a year. Further topical issues are being planned and we would welcome ideas for the themes to be covered in future issues. In this issue, articles are devoted to cardiac myosinbinding protein-C. When C-protein, or myosin-binding protein-C (MyBP-C) was discovered in skeletal muscle in the early 1970s by Gerald Offer (Offer 1972; Offer et al. 1973), MyBP-C was thought to be merely a structural protein component of the thick filament. Interest in the functions of MyBP-C were galvanised when it was found that the cardiac isoform, encoded by the MYBPC3 gene on chromosome 11, was mutated in cases of hereditary hypertrophic cardiomyopathy (HCM) (Bonne et al. 1995; Watkins et al. 1995). By now, mutations in MYBPC3 are the second-most frequently identified mutations that cause HCM (Schlossarek et al. 2011), with carriers of a specific pathogenic mutation amounting up to 4 % of the population of Indian descent (Dhandapany et al. 2009). It has become clear that MyBP-C must play important roles in both the regulation of contractility and as the assembly of muscle myofibrils. MyBP-C is transcribed from three related genes (MYBPC1-3), which encode the slow, fast and cardiac isoforms. Like many other myofibrilassociated structural proteins, MyBP-C is composed of a series of hundred-residue domains of the intracellular immunoglobulin and fibronectin families—10–11 in the case of MyBP-C (Furst and Gautel 1995; Otey et al. 2009). This molecular architecture is shared with the small MyBPC homologue, myosin-binding protein-H (Vaughan et al. 1993) and other myofibrillar proteins like myopalladin, myotilin, the myomesin isogenes and the giant scaffold and signalling proteins titin and obscurin (KontrogianniKonstantopoulos et al. 2009). Earlier work by the laboratories of Gerald Offer, Carl Moos, Roger Starr, Rick Moss, Polly Hofmann, Criss Hartzell and others had implicated MyBP-C in contraction regulation by interactions with actin or myosin filament components, possibly dynamically modulated by phosphorylation by cAMP and calcium/calmodulin dependent protein kinases in the case of the cardiac isoform (see articles in this issue for a review of the literature). The elucidation of the primary structure of both skeletal and cardiac MyBP-C isoforms pinpointed these phosphorylation sites to the N-terminal region within a sequence distinct from the immunoglobulin or fibronectin-like domains that form the remainder of the protein (Einheber and Fischman 1990; Furst et al. 1992; Gautel et al. 1995). This MyBP-C motif has since been implicated as the major domain involved in contraction regulation by interactions that have been localised to both the actin and myosin S. B. Marston NHLI, Myocardial Function, Imperial College London, Imperial Centre for Translational and Experimental Medicine, London W12 0NN, UK e-mail: S.marston@imperial.ac.uk

Effects of Myosin Binding Protein-C Isoform, Phosphorylation, and Domains on the Rotational Dynamics of Actin Filaments

We have determined the effects of Myosin binding protein-C (MyBP-C) and its domains on the microsecond time-scale rotational dynamics of actin, using time-resolved phosphorescence anisotropy (TPA). MyBP-C is a multi-domain thick filament-associated modulator of striated muscle contraction, spanning the interfilament spacing to contact both myosin and actin. Cardiac (c-) and slow skeletal (ss-) MyBP-C are known substrates for Protein kinase-A (PKA), and phosphorylation of cMyBP-C alters contractile properties and myofilament structure. To determine the effects of MyBP-C on actin's microsecond structural dynamics, we labeled actin at C374 with erythrosine iodoacetamide and performed TPA experiments. The interaction of all three MyBP-C isoforms with actin increased the final anisotropy (r∞) of the TPA decay in a concentration-dependent manner, indicating restriction of the rotational amplitude of actin dynamics. While PKA phosphorylation had little effect on fast skeletal (fs-) MyBP-C, phosphorylation of cMyBP-C and ssMyBP-C nearly eliminated the effects of these proteins to restrict actin dynamics, despite no change in binding affinity with phosphorylation. Skeletal MyBP-C (C1-C10) affected actin anisotropy at lower concentrations than cMyBP-C (C0-C10), suggesting that skeletal and cardiac N-terminal MyBP-C interactions with actin have distinct properties. The effects of truncated cMyBP-C on actin anisotropy determined that C-terminal domains are important for restricting rotational dynamics, whereas N-terminal domains are important for regulating this effect. These MyBP-C-induced changes in actin dynamics may play a role in the known effects of MyBP-C on the functional actin-myosin interaction. This work was funded by grants from NIH (F32 HL107039-01 to BAC and T32 AR007612 to DDT).

Myosin Binding Protein-C Slow: An Intricate Subfamily of Phosphoproteins

Myosin Binding Protein-C (MyBP-C), a family of accessory proteins of striated muscles, contributes to the assembly and stabilization of thick filaments, and regulates the formation of actomyosin cross-bridges. Three distinct MyBP-C isoforms have been characterized; cardiac, slow skeletal and fast skeletal. The slow isoform, sMyBP-C, consists of four alternatively spliced forms, variants 1-4 (v1-4) that share common structures and sequences. Variants 1-4 differ, however, in three regions; v1 and v2 contain a 25-residue long insertion within the NH2-terminus, v3 carries an 18-amino acid long segment within immunoglobulin domain C7 and v1 contains a unique COOH-terminus consisting of 26 amino acids, while v4 does not possess any of these insertions.

Structure and Interactions of Myosin-binding Protein C Domain C0: CARDIAC-SPECIFIC REGULATION OF MYOSIN AT ITS NECK?

Myosin-binding protein C (MyBP-C) is a multidomain protein present in the thick filaments of striated muscles and is involved in both sarcomere formation and contraction regulation. The latter function is believed to be located at the N terminus, which is close to the motor domain of myosin. The cardiac isoform of MyBP-C is linked to hypertrophic cardiomyopathy. Here, we use NMR spectroscopy and biophysical and biochemical assays to study the three-dimensional structure and interactions of the cardiac-specific Ig-like domain C0, a part of cardiac MyBP-C of which little is known. The structure confirmed that C0 is a member of the IgI class of proteins, showing many of the characteristic features of this fold. Moreover, we identify a novel interaction between C0 and the regulatory light chain of myosin, thus placing the N terminus of the protein in proximity to the motor domain of myosin. This novel interaction is disrupted by several cardiomyopathy-linked mutations in the MYBPC3 gene. These results provide new insights into how cardiac MyBP-C incorporates in the sarcomere and how it can contribute to the regulation of muscle contraction.

Myosin Binding Protein‐C: A Regulator of Actomyosin Interaction in Striated Muscle

Myosin-Binding protein-C (MyBP-C) is a family of accessory proteins of striated muscles that contributes to the assembly and stabilization of thick filaments, and regulates the formation of actomyosin cross-bridges, via direct interactions with both thick myosin and thin actin filaments. Three distinct MyBP-C isoforms have been characterized; cardiac, slow skeletal, and fast skeletal. Numerous mutations in the gene for cardiac MyBP-C (cMyBP-C) have been associated with familial hypertrophic cardiomyopathy (FHC) and have led to increased interest in the regulation and roles of the cardiac isoform. This review will summarize our current knowledge on MyBP-C and its role in modulating contractility, focusing on its interactions with both myosin and actin filaments in cardiac and skeletal muscles.

Evolution of the regulatory control of vertebrate striated muscle: the roles of troponin I and myosin binding protein-C

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.

Differential Expression of Two Cardiac Myosin-Binding Protein-C Isoforms in Developing Chicken Cardiac and Skeletal Muscle Cells

Myosin-binding protein-C (MyBP-C), also known as C-protein, is a major myosin-binding protein characteristic of striated muscle, and plays a critical role in myofibril organization, especially in registration of thick filaments in the sarcomeres during myofibrillogenesis. We previously demonstrated that cardiac-type MyBP-C is involved early in the process of myofibrillogenesis in both cardiac and skeletal muscle during chicken muscle development. Two variants (type I and type II) have been detected in chicken cardiac MyBP-C; they differ only in the presence or absence of a sequence of 15 amino acid residues (termed P-seq) that includes a phosphorylation site for cyclic AMP-dependent kinase in the cardiac MyBP-C motif ( Yasuda et al, 1995 ). Therefore, types I and II are regarded as phosphorylatable and non-phosphorylatable isoforms, respectively. In this study, an antibody specific for P-seq was prepared. With this and other monoclonal antibodies to cardiac MyBP-C (C-315), expression and localization of the two MyBP-C isoforms in developing chicken cardiac and skeletal muscle were examined by immunocytochemistry and immunoblotting. The results showed that type I is predominantly expressed in the heart and is localized in myofibrils of both atrial and ventricular muscles through development. In contrast, type II is mainly expressed in embryonic skeletal muscle, although type I is faintly expressed in cultured skeletal muscle. These observations were confirmed by RT-PCR.