Dilated cardiomyopathy in homozygous myosin-binding protein-C mutant mice.

To elucidate the role of cardiac myosin-binding protein-C (MyBP-C) in myocardial structure and function, we have produced mice expressing altered forms of this sarcomere protein. The engineered mutations encode truncated forms of MyBP-C in which the cardiac myosin heavy chain-binding and titin-binding domain has been replaced with novel amino acid residues. Analogous heterozygous defects in humans cause hypertrophic cardiomyopathy. Mice that are homozygous for the mutated MyBP-C alleles express less than 10% of truncated protein in M-bands of otherwise normal sarcomeres. Homozygous mice bearing mutated MyBP-C alleles are viable but exhibit neonatal onset of a progressive dilated cardiomyopathy with prominent histopathology of myocyte hypertrophy, myofibrillar disarray, fibrosis, and dystrophic calcification. Echocardiography of homozygous mutant mice showed left ventricular dilation and reduced contractile function at birth; myocardial hypertrophy increased as the animals matured. Left-ventricular pressure-volume analyses in adult homozygous mutant mice demonstrated depressed systolic contractility with diastolic dysfunction. These data revise our understanding of the role that MyBP-C plays in myofibrillogenesis during cardiac development and indicate the importance of this protein for long-term sarcomere function and normal cardiac morphology. We also propose that mice bearing homozygous familial hypertrophic cardiomyopathy-causing mutations may provide useful tools for predicting the severity of disease that these mutations will cause in humans.

Slow tonic muscle fibers in the thyroarytenoid muscles of human vocal folds; a possible specialization for speech.

Most of the sounds of human speech are produced by vibration of the vocal folds, yet the biomechanics and control of these vibrations are poorly understood. In this study the muscle within the vocal fold, the thyroarytenoid muscle (TA), was examined for the presence and distribution of slow tonic muscle fibers (STF), a rare muscle fiber type with unique contraction properties. Nine human TAs were frozen and serially sectioned in the frontal plane. The presence and distribution pattern of STF in each TA were examined by immunofluorescence microscopy using the monoclonal antibodies (mAb) ALD-19 and ALD-58 which react with the slow tonic myosin heavy chain (MyHC) isoform. In addition, TA muscle samples from adjacent frozen sections were also examined for slow tonic MyHC isoform by electrophoretic immunoblotting. STF were detected in all nine TAs and the presence of slow tonic MyHC isoform was confirmed in the immunoblots. The STF were distributed predominantly in the medial aspect of the TA, a distinct muscle compartment called the vocalis which is the vibrating part of the vocal fold. STF do not contract with a twitch like most muscle fibers, instead, their contractions are prolonged, stable, precisely controlled, and fatigue resistant. The human voice is characterized by a stable sound with a wide frequency spectrum that can be precisely modulated and the STF may contribute to this ability. At present, the evidence suggests that STF are not presented in the vocal folds of other mammals (including other primates), therefore STF may be a unique human specialization for speech.

Identification of the A-band localization domain of myosin binding proteins C and H (MyBP-C, MyBP-H) in skeletal muscle.

Although major constituents of the thick filaments of vertebrate striated muscles, the myosin binding proteins (MyBP-C and MyBP-H) are still of uncertain function. Distributed in the cross-bridge bearing zone of the A-bands of myofibrils, in a series of transverse 43 nm stripes, the proteins are constructed of a tandem series of small globular domains, each composed of approximately 90-100 amino acids, which have sequence similarities to either the C2-set of immunoglobulins (IgC2) and the fibronectin type III (FnIII) motifs. MyBP-C is composed of ten globular domains ( approximately 130 kDa) whereas MyBP-H is smaller ( approximately 58 kDa) and consists of a unique N-terminal segment followed by four globular domains, the order of which is identical to that of MyBP-C (FnIII-IgC2-FnIII-IgC2). To improve our understanding of this protein family we have characterized the domains in each of these two proteins which are required for targeting the proteins to their native site(s) in the sarcomere during myogenesis. Cultures of skeletal muscle myoblasts were transfected with expression plasmids encoding mutant constructs of the MyBPs bearing an N-terminal myc epitope, and their localization to the A-band examined by immunofluorescence microscopy. Based on the clarity and intensity of the myc A-band signals we concluded that constructs encoding the four C-terminal motifs of MyBP-C and MyBP-H ( approximately 360 amino acids) were all that was necessary to efficiently localize each of these peptides to the A-band. Truncation mutants lacking one of these 4 domains were less efficiently targeted to the C-zone of the sarcomere. Deletion of the last C-terminal motif of MyBP-H, its myosin binding domain, abolished all localization to the A-band. A chimeric construct, HU-3C10, in which the C-terminal motif of MyBP-H was replaced by the myosin binding domain of MyBP-C, efficiently localized to the A-band. Taken together, these observations indicate that MyBP-C and MyBP-H are localized to the A-band by the same C-terminal domain, composed of two IgC2 and two FnIII motifs. A model has been proposed for the interaction and positioning of the MyBPs in the thick filament through a ternary complex of the four C-terminal motifs with the myosin rods and titin.

The interface between MyBP-C and myosin: site-directed mutagenesis of the CX myosin-binding domain of MyBP-C.

Myosin-binding protein-C (MyBP-C or C-protein) is a ca. 130 kDa protein present in the thick filaments of all vertebrate striated muscle. The protein contains ten domains, each of ca. 90-100 amino acids; seven are members of the IgI family of proteins, three of the fibronectin type III family. The motifs are arranged in the following order (from N- to C-terminus): Ig-Ig-Ig-Ig-Ig-Fn-Fn-Ig-Fn-Ig. The C-terminal Ig motif (domain X or CX) contains its light meromyosin-binding site. A recombinant form of CX, beginning at Met-1027, exhibits saturable binding to myosin with an affinity comparable to the C-terminal 13 kDa chymotryptic fragment of native MyBP-C. To identify the surface in CX involved in its interaction with myosin, nine site-directed mutants (R37E, K43E, N49D, E52R, D56K, R73E, R74E, G80D and R103E) were constructed. Using a new assay for assessing the binding of CX with the light meromyosin (LMM) portion of myosin, we demonstrate that recombinant CX, just as the full-length protein, is able to facilitate LMM polymerization. Moreover, we show that residues Arg-37, Glu-52, Asp-56, Arg-73, and Arg-74 are involved in this interaction with the myosin rod. All of these amino acids interact with negatively charged residues of LMM, since the mutants R37E, R73E and R74E are unable to bind myosin, whereas E52R and D56K bind myosin with higher affinity than wild-type CX. Residues Lys-43 and Arg-103 show a small but significant influence on the binding reaction; residues Asn-49 and Gly-80 seem not to be involved in this interaction. Based on these data, a model is proposed for the interaction between MyBP-C CX and myosin filaments. In this model, CX interacts with four molecules of LMM at four different sites of the binding protein, thus explaining the effects of MyBP-C on the critical concentration of myosin polymerization.

Isoform-specific interaction of the myosin-binding proteins (MyBPs) with skeletal and cardiac myosin is a property of the C-terminal immunoglobulin domain.

Full-length cDNAs encoding chicken and human skeletal MyBP-H and MyBP-C have been isolated and sequenced (1-5). All are members of a protein family with repetitive immunoglobulin C2 and fibronectin type III motifs. The myosin binding domain was mapped to a single immunoglobulin motif in cardiac MyBP-C and skeletal MyBP-H. Limited alpha-chymotryptic digestion of cardiac MyBP-C generated three peptides, similar in relative mobility to those of skeletal MyBP-C: approximately 100, 40, and 15 kDa. Tryptic digestion of MyBP-H yielded two peptides: approximately 50 and 14 kDa. Partial amino acid sequences proved that the 15- and 14-kDa fragments are located at the C termini of cardiac MyBP-C and skeletal MyBP-H, respectively. Only the 14- and 15-kDa peptides bound to myosin. Thus, the myosin binding site in all three proteins resides within an homologous, C-terminal immunoglobulin domain. Binding reactions (2) between the skeletal and cardiac MyBPs and corresponding myosin isoforms demonstrated saturable binding of the MyBP proteins and their C-terminal peptides to myosin, but there are higher limiting stoichiometries with the homologous isoform partners. Evidence is presented indicating that MyBP-H and -C compete for binding to a discrete number of sites in myosin filaments.

Skeletal muscle-specific myosin binding protein-H is expressed in Purkinje fibers of the cardiac conduction system.

Heart contraction is coordinated by conduction of electrical excitation through specialized tissues of the cardiac conduction system. By retroviral single-cell tagging and lineage analyses in the embryonic chicken heart, we have recently demonstrated that a subset of cardiac muscle cells terminally differentiates as cells of the peripheral conduction system (Purkinje fibers) and that this occurs invariably in perivascular regions of developing coronary arteries. Cis regulatory elements that function in transcriptional regulation of cells in the conducting system have been distinguished from those in contractile cardiac muscle cells; eg, 5' regulatory sequences of the desmin gene act as enhancer elements in skeletal muscle and in the conduction system but not in cardiac muscle. We hypothesize that Purkinje fiber differentiation involves a switch of the gene expression program from that characteristic of cardiac muscle to one typical of skeletal muscle. To test this hypothesis, we examined the expression of myosin binding protein-H (MyBP-H) in Purkinje fibers of chicken hearts. This unique myosin binding protein is present in skeletal but not cardiac myocytes. A site-directed polyclonal antibody (AB105) was generated against MyBP-H. Immunohistological analysis of the myocardium mapped the AB105 antigen predominantly to A bands of myofibrils within Purkinje fibers. Western blot analysis of whole extracts from the ventricular wall of adult chicken hearts revealed that the AB105 epitope was restricted to a single protein of approximately 86 kD, the same size as MyBP-H in skeletal muscle. Biochemical properties of the Purkinje fiber 86-kD protein and RNase protection analyses of its mRNA indicate that Purkinje fiber 86-kD protein is indistinguishable from skeletal muscle MyBP-H. The results provide evidence that skeletal muscle MyBP-H is expressed in a subset of cardiac muscle cells that differentiate into Purkinje fibers of the heart.

The carboxyl terminus of myosin binding protein C (MyBP-C, C-protein) specifies incorporation into the A-band of striated muscle.

Myosin binding protein-C (MyBP-C), also known as C-protein, is a major constituent of the thick filaments of vertebrate striated muscles. The protein, approximately 130 kDa, consists of a series of 10 globular motifs (numbered I to X) each of approximately 90-100 amino acids, bearing resemblance to the C2-set of immunoglobins (Ig C2) and to the fibronectin type III (FnIII) motifs. Using pure preparations of myosin and MyBP-C, it has been demonstrated that the major myosin binding domain of MyBP-C resides within the C-terminal Ig C2 motif (motif X). However, in the context of the in vivo thick filament, it is uncertain if the latter domain is sufficient to target MyBP-C correctly to the A-band or if other regions of the molecule are required for this process. To answer this question, cultures of skeletal muscle myoblasts were transfected with expression plasmids encoding seven truncation mutants of MyBP-C, and their targeting to the A-band investigated by immunofluorescence microscopy. To distinguish the recombinant proteins from endogenous MyBP-C, a myc epitope was inserted at each amino terminus. Recombinant MyBP-C exhibited an identical distribution in the sarcomere to that of native MyBP-C; i.e. it was found exclusively in the C-zone of the A-band. A mutant encoding the C-terminal 372 amino acids, but lacking motifs I-VI (termed delta 1-6), also targeted correctly to the A-band. This fragment, which is composed of two Ig C2 and two FnIII motifs, was the minimal protein fragment required for correct A-band incorporation. Larger amino-terminal deletions or deletion of motif X, the myosin binding domain, abolished all localization to the A-band. One construct (delta 10) lacking only motif X strongly inhibited myofibril assembly. We conclude that the myosin binding domain of MyBP-C, although essential, is not sufficient for correct incorporation into the A-band and that motifs VII to IX are required for this process. The data suggest a topological model in which MyBP-C is associated with the thick filament through its C terminus.

The polyclonal origin of myocyte lineages.

The heart beat is coordinated by the integrated activities of three myocyte subpopulations: atrial myocytes, ventricular myocytes, and cells of the cardiac conduction system. In this review we discuss the classic fate map and recent retroviral cell lineage studies to better understand the origin, timing, and mechanisms regulating (a) the formation of these three myocyte lineages and (b) the morphogenetic plan underlying formation of the myocardial walls and the conduction system.

Modulation of myosin filament organization by C-protein family members.

We have analyzed the interactions between two types of sarcomeric proteins: myosin heavy chain (MyHC) and members of an abundant thick filament-associated protein family (myosin-binding protein; MyBP). Previous work has demonstrated that when MyHC is transiently transfected into mammalian nonmuscle COS cells, the expressed protein forms spindle-shaped structures consisting of bundles of myosin thick filaments. Co-expression of MyHC and MyBP-C or -H modulates the MyHC structures, resulting in dramatically longer cables consisting of myosin and MyBP encircling the nucleus. Immunoelectron microscopy indicates that these cable structures are more uniform in diameter than the spindle structures consisting solely of MyHC, and that the myosin filaments are compacted in the presence of MyBP. Deletion analysis of MyBP-H indicates that cable formation is dependent on the carboxy terminal 24 amino acids. Neither the MyHC spindles nor the MyHC/MyBP cables associate with the endogenous actin cytoskeleton of the COS cell. While there is no apparent co-localization between these structures and the microtubule network, colchicine treatment of the cells promotes the formation of longer assemblages, suggesting that cytoskeletal architecture may physically impede or regulate polymer formation/extension. The data presented here contribute to a greater understanding of the interactions between the MyBP family and MyHC, and provide additional evidence for functional homology between MyBP-C and MyBP-H.

Immunohistochemical analysis of C-protein isoforms in cardiac and skeletal muscle of the axolotl, Ambystoma mexicanum.

Of the several proteins located within sarcomeric A-bands, C-protein, a myosin binding protein (MyBP) is thought to regulate and stabilize thick filaments during assembly. This paper reports the characterization of C-protein isoforms in juvenile and adult axolotls, Ambystoma mexicanum, by means of immunofluorescent microscopy and Western blot analyses. C-protein and myosin are found specifically within the A-bands, whereas tropomyosin and alpha-actin are detected in the I-bands of axolotl myofibrils. The MF1 antibody prepared against the fast skeletal muscle isoform of chicken C-protein specifically recognizes a cardiac isoform (Axcard1) in juvenile and adult axolotls but does not label axolotl skeletal muscle. The ALD66 antibody, which reacts with the C-protein slow isoform in chicken, local- izes only in skeletal muscle of the axolotl. This slow axolotl isoform (Axslow) displays a heterogeneous distribution in fibers of dorsalis trunci skeletal muscle. The C315 antibody against the chicken C-protein cardiac isoform identifies a second axolotl cardiac isoform (Axcard2), which is present also in axolotl skeletal muscle. No C-protein was detected in smooth muscle of the juvenile and adult axolotl with these antibodies.

Antisense suppression of skeletal muscle myosin light chain-1 biosynthesis impairs myofibrillogenesis in cultured myotubes.

Although the alkali or essential light chains of skeletal muscle myosin are not required for actin-activated myosin ATPase activity, these myosin subunits are necessary for force transmission with in vitro actin motility assays and are believed to stabilize the alpha-helical neck region of myosin subfragment-1. To probe the functions of the essential light chains during myofibril assembly, we used recombinant DNA procedures to deplete this light chain in cultured muscle. Retroviral expression vectors were constructed which encoded the exon-1 sequence of the myosin light chain-1 gene in antisense orientation. These vectors were applied to myogenic cells from embryonic chick and quail pectoralis muscle. Colonies expressing antisense RNA were selected in growth medium containing the neomycin analogue G-418, plus 5-bromo-2'-deoxyuridine (BrdU) and triggered to differentiate by removal of the latter. Expression of antisense myosin light chain-1 mRNA impaired muscle development. In the antisense cultures there were more mononucleated cells, fewer and smaller myotubes which had poorly developed myofibrils and high levels of diffusely staining myosin heavy chain, not apparent in control myotubes. Protein synthesis in the myotube cultures was analyzed by 35S-methionine labelling and two-dimensional gel electrophoresis. Except for a suppression of approximately 80% of myosin light chain-1f synthesis, the overall pattern of protein synthesis was not altered significantly. These studies suggest that retardation of myosin light chain-1f accumulation inhibits or delays myofibrillogenesis.

Fibroblast growth factor receptor is required for in vivo cardiac myocyte proliferation at early embryonic stages of heart development.

In birds and mammals, cardiac myocytes terminate mitotic activity in the neonatal period and regeneration of cardiac muscle does not occur after myocardial injury in adult hearts. Even embryonic myocytes, which actively proliferate in vivo, quickly lose mitotic activity when placed in cell culture. Several growth factors, including fibroblast growth factor (FGF), have been documented in embryonic hearts and some have been shown to influence myocyte terminal differentiation in culture. However, none of these growth factors have been shown to reactivate cell division in postmitotic myocytes nor have their in vivo functions been defined satisfactorily. To clarify the role of FGF signaling in heart growth, we prepared two retroviral vectors capable of suppressing (i) functions of FGF receptors (FGFRs) with a dominant-negative mutant of receptor type 1 (FGFR1) or (ii) the translation of endogenous FGFR1 by transcribing its antisense RNA. Both vectors inhibited myocyte proliferation and/or survival during the first week of chicken embryonic development but had much less effect after the second week. No apparent alteration of myocyte growth was observed after overexpression of full-length FGFR1. These results suggest that receptor-coupled FGF signaling regulates cardiac myocyte growth during tubular stages of cardiogenesis but that myocyte growth becomes FGF-independent after the second week of embryogenesis.

Sequential appearance of muscle-specific proteins in myoblasts as a function of time after cell division: evidence for a conserved myoblast differentiation program in skeletal muscle.

Based on the assumption that a conserved differentiation program governs the assembly of sarcomeres in skeletal muscle in a manner analogous to programs for viral capsid assembly, we have defined the temporal and spatial distribution of 10 muscle-specific proteins in mononucleated myoblasts as a function of the time after terminal cell division. Single cells in mitosis were identified in monolayer cultures of embryonic chicken pectoralis, followed for selected time points (0-24 h postmitosis) by video time-lapse microscopy, and then fixed for immunofluorescence staining. For convenience, the myoblasts were termed x-h-old to define their age relative to their mitotic "birthdate." All 6 h myoblasts that emerged in a mitogen-rich medium were desmin+ but only 50% were positive for a alpha-actin, troponin-I, alpha-actinin, MyHC, zeugmatin, titin, or nebulin. By 15 h postmitosis, approximately 80% were positive for all of the above proteins. The up-regulation of these 7 myofibrillar proteins appears to be stochastic, in that many myoblasts were alpha-actinin+ or zeugmatin+ but MyHC- or titin- whereas others were troponin-I+ or MyHC+ but alpha-actinin- or alpha-actin-. In 15-h-old myoblasts, these contractile proteins were organized into nonstriated myofibrils (NSMFs). In contrast to striated myofibrils (SMFs), the NSMFs exhibited variable stoichiometries of the sarcomeric proteins and these were not organized into any consistent pattern. In this phase of maturation, two other changes occurred: (1) the microtubule network was reorganized into parallel bundles, driving the myoblasts into polarized, needle-shaped cells; and (2) the sarcolemma became fusion-competent. A transition from NSMFs to SMFs took place between 15 and 24 h (or later) postmitosis and was correlated with the late appearance of myomesin, and particularly, MyBP-C (C protein). The emergence of one, or a string of approximately 2 mu long sarcomeres, was invariably characterized by the localization of myomesin and MyBP-C to their mature positions in the developing A-bands. The latter group of A-band proteins may be rate-limiting in the assembly program. The great majority of myoblasts stained positively for desmin and myofibrillar proteins prior to, rather than after, fusing to form myotubes. This sequential appearance of muscle-specific proteins in vitro fully recapitulates myofibrillar assembly steps in myoblasts of the myotome and limb bud in vivo, as well as in nonmuscle cells converted to myoblasts by MyoD. We suggest that this cell-autonomous myoblast differentiation program may be blocked at different control points in immortalized myogenic cell lines.

The major myosin-binding domain of skeletal muscle MyBP-C (C protein) resides in the COOH-terminal, immunoglobulin C2 motif.

A common feature shared by myosin-binding proteins from a wide variety of species is the presence of a variable number of related internal motifs homologous to either the Ig C2 or the fibronectin (Fn) type III repeats. Despite interest in the potential function of these motifs, no group has clearly demonstrated a function for these sequences in muscle, either intra- or extracellularly. We have completed the nucleotide sequence of the fast type isoform of MyBP-C (C protein) from chicken skeletal muscle. The deduced amino acid sequence reveals seven Ig C2 sets and three Fn type III motifs in MyBP-C. alpha-chymotryptic digestion of purified MyBP-C gives rise to four peptides. NH2-terminal sequencing of these peptides allowed us to map the position of each along the primary structure of the protein. The 28-kD peptide contains the NH2-terminal sequence of MyBP-C, including the first C2 repeat. It is followed by two internal peptides, one of 5 kD containing exclusively spacer sequences between the first and second C2 motifs, and a 95-kD fragment containing five C2 domains and three fibronectin type III motifs. The C-terminal sequence of MyBP-C is present in a 14-kD peptide which contains only the last C2 repeat. We examined the binding properties of these fragments to reconstituted (synthetic) myosin filaments. Only the COOH-terminal 14-kD peptide is capable of binding myosin with high affinity. The NH2-terminal 28-kD fragment has no myosin-binding, while the long internal 100-kD peptide shows very weak binding to myosin. We have expressed and purified the 14-kD peptide in Escherichia coli. The recombinant protein exhibits saturable binding to myosin with an affinity comparable to that of the 14-kD fragment obtained by proteolytic digestion (1/2 max binding at approximately 0.5 microM). These results indicate that the binding to myosin filaments is mainly restricted to the last 102 amino acids of MyBP-C. The remainder of the molecule (1,032 amino acids) could interact with titin, MyBP-H (H protein) or thin filament components. A comparison of the highly conserved Ig C2 domains present at the COOH-terminus of five MyBPs thus far sequenced (human slow and fast MyBP-C, human and chicken MyBP-H, and chicken MyBP-C) was used to identify residues unique to these myosin-binding Ig C2 repeats.

Complete sequence of human fast-type and slow-type muscle myosin-binding-protein C (MyBP-C). Differential expression, conserved domain structure and chromosome assignment.

Myosin-binding-protein C (MyBP-C) is a myosin-associated protein of unknown function found in the cross-bridge-bearing zone (C region) of A bands in striated muscle. Using a cDNA clone encoding the fast-type isoform of chicken MyBP-C, we screened a human fetal muscle cDNA library and isolated clones encoding the full-length human fast-type isoform of MyBP-C. cDNA clones encoding the slow-type isoform of human MyBP-C, were also isolated and fully sequenced. Northern-blot analysis demonstrated skeletal muscle-specific expression of these gene products. Using human/hamster somatic-cell hybrids, we were able to map the slow-type MyBP-C to human chromosome 12, and the fast-type MyBP-C to chromosome 19. The cDNA for human fast-type MyBP-C encodes a polypeptide of 1142 amino acids with an expected molecular mass of 128.1 kDa. Comparison of this cDNA with other members of the MyBP family reveals extensive primary-sequence conservation. Each MyBP-C contains seven immunoglobulin C2 motifs and three fibronectin type-III repeats in the arrangement C2-C2-C2-C2-C2-III-III-C2-III-C2. Regions of high identity shared by the chicken and the two human proteins are not restricted to the immunoglobulin and fibronectin motifs. Sequence comparison of all three proteins has allowed us to map a highly conserved region between the first and second C2 motifs, the only large spacer sequence present between motifs in these proteins.

Human myosin-binding protein H (MyBP-H): complete primary sequence, genomic organization, and chromosomal localization.

Vertebrate striated muscle contains a set of myosin-associated proteins with discrete distributions in the A-band. Some of these proteins, including MyBP-H and MyBP-C, are characterized by a series of internal repeats (motifs) with homology to either the C2-set of the immunoglobulin superfamily or the fibronectin type III repeat. These repeats are predicted to be involved in protein-protein interactions within the myofibril. The cDNA sequence, the genomic organization, and the chromosomal localization of the human homologue of MyBP-H are presented. The 1.8-kb cDNA encodes a 52-kDa polypeptide containing two Ig-C2 and two type III repeats. The mRNA is expressed in a skeletal muscle-specific pattern. A 28-kb region of genomic sequence has been isolated that encompasses the 5' and 3' ends of the cDNA. This region includes 4.2 kb of upstream sequence with a potential promoter and 14 kb of downstream sequence containing the polyadenylation site. The chromosomal assignment was made by high resolution chromosomal in situ hybridization. This method maps the gene to chromosome 1q32.1. The repeat structure described previously in chicken MyBP-H and MyBP-C was also detected in human MyBP-H. The primary sequence of the C-terminal Ig-C2 motif and its predicted secondary structure have been extensively conserved in MyBP-H homologues and other members of the MyBP family. This Ig-C2 motif has been implicated in myosin binding.