Mutations In Myosin Binding protein-C Research Articles

Cryo-electron tomography of intact cardiac muscle reveals myosin binding protein-C linking myosin and actin filaments.

Myosin binding protein C (MyBP-C) is an accessory protein of the thick filament in vertebrate cardiac muscle arranged over 9 stripes of intervals of 430Å in each half of the A-band in the region called the C-zone. Mutations in cardiac MyBP-C are a leading cause of hypertrophic cardiomyopathy the mechanism of which is unknown. It is a rod-shaped protein composed of 10 or 11 immunoglobulin- or fibronectin-like domains labelled C0 to C10 which binds to the thick filament via its C-terminal region. MyBP-C regulates contraction in a phosphorylation dependent fashion that may be through binding of its N-terminal domains with myosin or actin. Understanding the 3D organisation of MyBP-C in the sarcomere environment may provide new light on its function. We report here the fine structure of MyBP-C in relaxed rat cardiac muscle by cryo-electron tomography and subtomogram averaging of refrozen Tokuyasu cryosections. We find that on average MyBP-C connects via its distal end to actin across a disc perpendicular to the thick filament. The path of MyBP-C suggests that the central domains may interact with myosin heads. Surprisingly MyBP-C at Stripe 4 is different; it has weaker density than the other stripes which could result from a mainly axial or wavy path. Given that the same feature at Stripe 4 can also be found in several mammalian cardiac muscles and in some skeletal muscles, our finding may have broader implication and significance. In the D-zone, we show the first demonstration of myosin crowns arranged on a uniform 143Å repeat.

Impaired contractile function due to decreased cardiac myosin binding protein C content in the sarcomere

Mutations in cardiac myosin binding protein C (MyBP-C) are a common cause of familial hypertrophic cardiomyopathy (FHC). The majority of MyBP-C mutations are expected to reduce MyBP-C expression; however, the consequences of MyBP-C deficiency on the regulation of myofilament function, Ca²⁺ homeostasis, and in vivo cardiac function are unknown. To elucidate the effects of decreased MyBP-C expression on cardiac function, we employed MyBP-C heterozygous null (MyBP-C+/-) mice presenting decreases in MyBP-C expression (32%) similar to those of FHC patients carrying MyBP-C mutations. The levels of MyBP-C phosphorylation were reduced 53% in MyBP-C+/- hearts compared with wild-type hearts. Skinned myocardium isolated from MyBP-C+/- hearts displayed decreased cross-bridge stiffness at half-maximal Ca²⁺ activations, increased steady-state force generation, and accelerated rates of cross-bridge recruitment at low Ca²⁺ activations (<15% and <25% of maximum, respectively). Protein kinase A treatment abolished basal differences in rates of cross-bridge recruitment between MyBP-C+/- and wild-type myocardium. Intact ventricular myocytes from MyBP-C+/- hearts displayed abnormal sarcomere shortening but unchanged Ca²⁺ transient kinetics. Despite a lack of left ventricular hypertrophy, MyBP-C+/- hearts exhibited elevated end-diastolic pressure and decreased peak rate of LV pressure rise, which was normalized following dobutamine infusion. Furthermore, electrocardiogram recordings in conscious MyBP-C+/- mice revealed prolonged QRS and QT intervals, which are known risk factors for cardiac arrhythmia. Collectively, our data show that reduced MyBP-C expression and phosphorylation in the sarcomere result in myofilament dysfunction, contributing to contractile dysfunction that precedes compensatory adaptations in Ca²⁺ handling, and chamber remodeling. Perturbations in mechanical and electrical activity in MyBP-C+/- mice could increase their susceptibility to cardiac dysfunction and arrhythmia.

Determination of MyBP-C Orientation in the Cardiac Sarcomere by Immuno-EM

Myosin binding protein-C (MyBP-C) is a thick filament accessory protein of vertebrate striated muscle. It is essential for normal cardiac function, and mutations in MyBP-C cause cardiac and skeletal muscle disease. The 40 nm long molecule is composed of 10 or 11 immunoglobulin- or fibronectin-like domains and is located in up to 9 axial stripes 43 nm apart in each half of the A-band. To understand MyBP-C function it is important to know its structural organization in the sarcomere. Several models have been proposed, in which MyBP-C either wraps around the thick filament, or extends radially from or longitudinally along the filament (or some combination of these). To distinguish between these models, we have used immuno-EM of mouse cardiac myofibrils labeled with antibodies to different domains of MyBP-C to determine the relative axial positions of the domains. Myofibrils were obtained from mouse cardiac muscle by homogenizing chemically skinned hearts under rigor conditions. They were labeled with antibodies specific for the N-terminal domain, the middle of the molecule, and the C-terminal domain, and observed by negative stain EM. Labeled myofibrils showed nine stain-excluding stripes in each half of the A-band. The average distance of each stripe from the middle of the M-line was measured. All antibodies labeled axially within 10 nm of each other, at the same axial positions as the unlabeled stripes, suggesting that the bulk of MyBP-C runs radially or circumferentially. A small axial difference between the C-terminal and the central and N-terminal antigenic sites suggests that a short portion of the C-terminus runs longitudinally. Electron tomography of muscle sections (Luther et al., PNAS 2011) and 3D reconstruction of isolated thick filaments (Zoghbi et al., PNAS 2008) support the radial arrangement.

Activation and Inhibition of F-Actin and Cardiac Thin Filaments by the N-Terminal Domains of Cardiac Myosin Binding Protein C

Myosin binding protein-C (MyBPC) has been known for over thirty years to inhibit actomyosin ATP hydrolysis and more recently has been shown to affect the calcium sensitivity of force in muscle fibers. The various domains of MyBPC have previously been shown to have complex interactions with other myofibrillar proteins. The C-terminal domains have been shown to bind to the thick filament and the N-terminal domains interact with the S2 region of myosin and with f-actin. The number of mutations in cardiac MyBPC (cMyBPC) are the second most prevalent of sarcomeric proteins in producing cardiomyopathies. We have used steady state kinetics to study the molecular mechanism by which the soluble N-terminal C0C1, C1C2 and C0C2 domains of mouse and human cMyBPC affect the activation of myosin ATP hydrolysis by f-actin and native porcine thin filaments.The N-terminal domains of cMyBPC inhibit the activation of the steady state ATPase myosin by f-actin. However, in the presence of native cardiac thin filaments.the N-terminal domains C1C2 and C0C2 of mouse and human cMyBPC produce biphasic effects on the steady state ATP hydrolysis of cardiac myosin-S1. That is, ATPase is activated at low ratios of cMyBPC-N-terminal domain to thin filament and is inhibited by higher ratios similar to the effects observed with f-actin. These data suggest that low ratios of cMyBPC N-terminal domains activate thin filaments in a mechanism similar to that of rigor myosin-S1 but higher ratios inhibit the ATPase rate by competing with myosin-S1-ADP-Pi binding to actin and thin filaments. Effects also appear species-dependent since the C0C1 domains of human cMyBPC produce similar effects as C1C2 and C0C2 on ATPase in the presence of thin filaments, but mouse C0C1 does not produce significant activation.

Axial distribution of myosin binding protein-C is unaffected by mutations in human cardiac and skeletal muscle

Myosin binding protein-C (MyBP-C), a major thick filament associated sarcomeric protein, plays an important functional and structural role in regulating sarcomere assembly and crossbridge formation. Missing or aberrant MyBP-C proteins (both cardiac and skeletal) have been shown to cause both cardiac and skeletal myopathies, thereby emphasising its importance for the normal functioning of the sarcomere. Mutations in cardiac MyBP-C are a major cause of hypertrophic cardiomyopathy (HCM), while mutations in skeletal MyBP-C have been implicated in a disease of skeletal muscle—distal arthrogryposis type 1 (DA-1). Here we report the first detailed electron microscopy studies on human cardiac and skeletal tissues carrying MyBP-C gene mutations, using samples obtained from HCM and DA-1 patients. We have used established image averaging methods to identify and study the axial distribution of MyBP-C on the thick filament by averaging profile plots of the A-band of the sarcomere from electron micrographs of human cardiac and skeletal myopathy specimens. Due to the difficulty of obtaining normal human tissue, we compared the distribution to the A-band structure in normal frog skeletal, rat cardiac muscle and in cardiac muscle of MyBP-C-deficient mice. Very similar overall profile averages were obtained from the C-zones in cardiac HCM samples and skeletal DA-1 samples with MyBP-C gene mutations, suggesting that mutations in MyBP-C do not alter its mean axial distribution along the thick filament.

Modulating Contraction by Binding of MyBP-C to Actin

Myosin-binding protein C (MyBP-C) is a ∼130 kDa protein of the thick filaments of vertebrate skeletal and cardiac muscle. It consists of a linear array of ten or eleven globular, 10-kDa domains from the immunoglobulin (Ig) and fibronectin type III families, and an additional, MyBP-C-specific motif. The cardiac isoform, cMyBP-C, plays a key role in modulating cardiac function, and mutations in MyBP-C cause heart disease. Despite its discovery 40 years ago, the mechanism of MyBP-C function remains poorly understood. In vitro studies suggest that it could modulate contraction by binding to thin filaments, but there has been no evidence for this in situ. We used electron tomography of exceptionally well-preserved skeletal muscle to study the 3D organization of MyBP-C in the intact sarcomere. The tomogram shows that MyBP-C projects perpendicular to the thick filament surface and reaches neighboring thin filaments. This thick-thin filament bridge suggests a possible physical basis for modulating filament sliding and thus contraction. In vitro, binding to actin has been shown to occur via MyBP-C's N-terminal end. To understand the structural basis of this binding, we used negative stain electron microscopy and 3D reconstruction to study F-actin decorated with bacterially expressed N-terminal cMyBP-C fragments. Clear decoration was obtained under a variety of salt conditions. 3D reconstructions showed MyBP-C density starting over subdomain 1 of actin and extending tangentially towards actin's pointed end. Molecular fitting with an atomic structure of a MyBP-C Ig domain suggested that most of the N-terminal domains may be well ordered on actin. The location of binding was such that it appeared to overlap the relaxed (low Ca2+) position of tropomyosin but not the activated position. This suggests that MyBP-C might help determine the state of thin filament activity by modulating tropomyosin position on actin.

Cardiomyocyte Hypertrophy and Reduced Myofibril Density Underlie Decreased Maximal Force Generating Capacity in Familial Hypertrophic Cardiomyopathy

Background: Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in genes encoding sarcomeric proteins. Previously, we observed a reduced maximal force generating capacity (Fmax) of cardiomyocytes from HCM patients with mutations in myosin-binding protein-C (MYBPC3). In this study we investigated the contribution of myofibril density (MyoD) and cross-sectional area (CSA) to the reduction in Fmax.Methods: We measured Fmax and CSA of single permeabilized ventricular cardiomyocytes of 27 HCM patients with mutations in MYBPC3, myosin heavy chain: MYH7, troponin T: TNNT2 and troponin I: TNNI3 and 10 non-failing donor hearts. MyoD was calculated as the sum of myofibril area related to cell area assessed by electron microscopy.Results: Average Fmax was significantly lower in HCM groups compared to donor (Figure A). Figure B shows that MyoD of HCM hearts was reduced compared to donors. CSA of HCM hearts (529±48μm2) was significantly higher compared to donors (342±12μm2). Linear regression analysis revealed a significant negative linear relationship between CSA and Fmax (R2= 0.50, β=-0.023, p=0.014).Conclusion: Cardiomyocyte hypertrophy and reduced myofibrillar density underlie depressed Fmax in HCM with mutations in thick and thin sarcomeric proteins.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Direct visualization of myosin-binding protein C bridging myosin and actin filaments in intact muscle

Myosin-binding protein C (MyBP-C) is a thick filament protein playing an essential role in muscle contraction, and MyBP-C mutations cause heart and skeletal muscle disease in millions worldwide. Despite its discovery 40 y ago, the mechanism of MyBP-C function remains unknown. In vitro studies suggest that MyBP-C could regulate contraction in a unique way--by bridging thick and thin filaments--but there has been no evidence for this in vivo. Here we use electron tomography of exceptionally well preserved muscle to demonstrate that MyBP-C does indeed bind to actin in intact muscle. This binding implies a physical mechanism for communicating the relative sliding between thick and thin filaments that does not involve myosin and which could modulate the contractile process.

Electron Microscopy and 3D Reconstruction of F-Actin Decorated with Cardiac Myosin-Binding Protein C (cMyBP-C)

Myosin-binding protein C (MyBP-C) is an ∼ 130-kDa rod-shaped protein of the thick (myosin containing) filaments of vertebrate striated muscle. It is composed of 10 or 11 globular 10-kDa domains from the immunoglobulin and fibronectin type III families and an additional MyBP-C-specific motif. The cardiac isoform cMyBP-C plays a key role in the phosphorylation-dependent enhancement of cardiac function that occurs upon β-adrenergic stimulation, and mutations in MyBP-C cause skeletal muscle and heart diseases. In addition to binding to myosin, MyBP-C can also bind to actin via its N-terminal end, potentially modulating contraction in a novel way via this thick–thin filament bridge. To understand the structural basis of actin binding, we have used negative stain electron microscopy and three-dimensional reconstruction to study the structure of F-actin decorated with bacterially expressed N-terminal cMyBP-C fragments. Clear decoration was obtained under a variety of salt conditions varying from 25 to 180 mM KCl concentration. Three-dimensional helical reconstructions, carried out at the 180-mM KCl level to minimize nonspecific binding, showed MyBP-C density over a broad portion of the periphery of subdomain 1 of actin and extending tangentially from its surface in the direction of actin's pointed end. Molecular fitting with an atomic structure of a MyBP-C Ig domain suggested that most of the N-terminal domains may be well ordered on actin. The location of binding was such that it could modulate tropomyosin position and would interfere with myosin head binding to actin.

Electron tomography reveals the structure of the C-zone in striated muscle

The C-zone in vertebrate striated muscle is the region in each half A-band where myosin binding protein C (MyBPC) forms 7 bands of 43 nm spacing. Mutations in MyBPC are a major cause of familial hypertrophic cardiomyopathy; hence there is great interest in understanding its role in the sarcomere. We have found that the organisation of the C-zone is very similar in cardiac and skeletal muscle. Here we report on the 3D organisation of the C-zone by electron tomography of an exceptionally well-preserved frog skeletal muscle.

Myosin binding protein—C: Enigmatic regulator of cardiac contraction

Myosin binding protein C (MyBPC) is a sarcomeric protein whose role in sarcomere structure and regulation of contraction is currently under investigation. It is a member of the immunoglobulin superfamily and is found in the C-zone of the A-band of the sarcomere. The elongated structure of MyBPC is composed of a series of immunoglobulin and fibronectin domains, with the C-terminal domains binding to the myosin thick filament and the N-terminal domains interacting with the myosin subfragment-2 (S2) neck region and possibly the actin thin filament. The functions of MyBPC are to stabilise the sarcomere structure and to regulate contraction. When phosphorylated near its N-terminus, MyBPC no longer binds myosin-S2, causing an increase in the ordering of the myosin heads, ATPase activity, F max and Ca 2+ sensitivity of contraction. Mutations in MyBPC have been found to cause familial hypertrophic cardiomyopathy (FHC) and changes in MyBPC phosphorylation have been linked to cardiac ischaemia-reperfusion injury.

Four and a Half LIM Protein 1 Binds Myosin-binding Protein C and Regulates Myosin Filament Formation and Sarcomere Assembly

Four and a half LIM protein 1 (FHL1/SLIM1) is highly expressed in skeletal and cardiac muscle; however, the function of FHL1 remains unknown. Yeast two-hybrid screening identified slow type skeletal myosin-binding protein C as an FHL1 binding partner. Myosin-binding protein C is the major myosin-associated protein in striated muscle that enhances the lateral association and stabilization of myosin thick filaments and regulates actomyosin interactions. The interaction between FHL1 and myosin-binding protein C was confirmed using co-immunoprecipitation of recombinant and endogenous proteins. Recombinant FHL2 and FHL3 also bound myosin-binding protein C. FHL1 impaired co-sedimentation of myosin-binding protein C with reconstituted myosin filaments, suggesting FHL1 may compete with myosin for binding to myosin-binding protein C. In intact skeletal muscle and isolated myofibrils, FHL1 localized to the I-band, M-line, and sarcolemma, co-localizing with myosin-binding protein C at the sarcolemma in intact skeletal muscle. Furthermore, in isolated myofibrils FHL1 staining at the M-line appeared to extend partially into the C-zone of the A-band, where it co-localized with myosin-binding protein C. Overexpression of FHL1 in differentiating C2C12 cells induced "sac-like" myotube formation (myosac), associated with impaired Z-line and myosin thick filament assembly. This phenotype was rescued by co-expression of myosin-binding protein C. FHL1 knockdown using RNAi resulted in impaired myosin thick filament formation associated with reduced incorporation of myosin-binding protein C into the sarcomere. This study identified FHL1 as a novel regulator of myosin-binding protein C activity and indicates a role for FHL1 in sarcomere assembly.

Morphologic Characteristics of Hypertrophic Cardiomyopathy of the Elderly With Cardiac Myosin-Binding Protein C Gene Mutations

Several morphologic distinctions between elderly and young patients with hypertrophic cardiomyopathy (HCM) have been reported. In particular, a crescent-shaped left ventricular (LV) cavity with reversed septal curvature, which is often seen in young patients, is rare in elderly patients. However, those studies were carried out before gene testing became available and heterogeneous causes or age-related changes may have been included. The purpose of this study was to determine the morphologic characteristics of elderly patients with HCM definitely caused by a mutation in the cardiac myosin-binding protein C (MyBPC). Twenty-seven patients with HCM caused by MyBPC gene abnormality were evaluated. Patients were divided into an elderly group (> or = 65 years of age, n = 8) and a young group (< 65 years of age, n = 19). LV hypertrophy was milder in the elderly than in the young for maximum LV wall thickness (18 +/- 5 mm vs 24 +/- 6 mm, p = 0.008) and Wigle score (5.7 +/- 1.5 vs 7.6 +/- 1.6, p < 0.005). However, an abnormal crescent-shaped LV was similarly prominent in both groups (75% in the elderly vs 95% in the young, p = NS). None of the elderly patients showed a proximal septal bulge. An abnormal crescent-shaped LV cavity was frequently present in the elderly as in the young when there are MyBPC mutations. It is possible that this morphologic feature could become useful for determining the etiology of HCM in elderly patients.

A novel mutation in the cardiac myosin-binding protein C gene is responsible for hypertrophic cardiomyopathy with severe ventricular hypertrophy and sudden death

It has been demonstrated previously that clinical phenotypes of HCM (hypertrophic cardiomyopathy) caused by mutations in the cardiac MyBP-C (myosin-binding protein C) gene show late onset, low penetrance and favourable clinical course. However, we have encountered severe phenotypes in several carriers of the MyBP-C gene mutations. The aim of the present study was to screen novel MyBP-C gene mutations in patients with HCM and to investigate the genetic differences in affected subjects with severe phenotypes. The MyBP-C gene was screened in 292 Japanese probands with HCM, and a novel c.2067+1G-->A mutation was present in 15 subjects in five families. Clinical phenotypes of carriers of the c.2067+1G-->A mutation were compared with those of a previously identified Arg820Gln (Arg820-->Gln) mutation in the MyBP-C gene. The disease penetrance in subjects aged > or =30 years was 90% in carriers of the c.2067+1G-->A mutation and 61% in carriers of the Arg820Gln mutation. Sudden death occurred in four subjects from three families with the c.2067+1G-->A mutation and in two subjects from one family with the Arg820Gln mutation. Two carriers of the c.2067+1G-->A mutation had substantial hypertrophy (maximal wall thickness > or =30 mm). In contrast, two carriers of the Arg820Gln mutation had end-stage HCM. In conclusion, the c.2067+1G-->A mutation is associated with HCM with substantial hypertrophy and moderate incidence of sudden death, whereas the Arg820Gln mutation is associated with end-stage HCM. These observations may provide important prognostic information regarding the clinical practice of HCM.

Myosin binding protein C: structural abnormalities in familial hypertrophic cardiomyopathy.

The muscle protein myosin binding protein C (MyBPC) is a large multi-domain protein whose role in the sarcomere is complex and not yet fully understood. Mutations in MyBPC are strongly associated with the heart disease familial hypertrophic cardiomyopathy (FHC) and these experiments of nature have provided some insight into the intricate workings of this protein in the heart. While some regions of the MyBPC molecule have been assigned a function in the regulation of muscle contraction, the interaction of other regions with various parts of the myosin molecule and the sarcomeric proteins, actin and titin, remain obscure. In addition, several intra-domain interactions between adjacent MyBPC molecules have been identified. Although the basic structure of the molecule (a series of immunoglobulin and fibronectin domains) has been elucidated, the assembly of MyBPC in the sarcomere is a topic for debate. By analysing the MyBPC sequence with respect to FHC-causing mutations it is possible to identify individual residues or regions of each domain that may be important either for binding or regulation. This review looks at the current literature, in concert with alignments and the structural models of MyBPC, in an attempt to understand how FHC mutations may lead to the disease state.

Hypertrophic cardiomyopathy in cardiac myosin binding protein-C knockout mice.

Familial hypertrophic cardiomyopathy (FHC) is an inherited autosomal dominant disease caused by mutations in sarcomeric proteins. Among these, mutations that affect myosin binding protein-C (MyBP-C), an abundant component of the thick filaments, account for 20% to 30% of all mutations linked to FHC. However, the mechanisms by which MyBP-C mutations cause disease and the function of MyBP-C are not well understood. Therefore, to assess deficits due to elimination of MyBP-C, we used gene targeting to produce a knockout mouse that lacks MyBP-C in the heart. Knockout mice were produced by deletion of exons 3 to 10 from the endogenous cardiac (c) MyBP-C gene in murine embryonic stem (ES) cells and subsequent breeding of chimeric founder mice to obtain mice heterozygous (+/-) and homozygous (-/-) for the knockout allele. Wild-type (+/+), cMyBP-C(+/-), and cMyBP-C(-/-) mice were born in accordance with Mendelian inheritance ratios, survived into adulthood, and were fertile. Western blot analyses confirmed that cMyBP-C was absent in hearts of homozygous knockout mice. Whereas cMyBP-C(+/-) mice were indistinguishable from wild-type littermates, cMyBP-C(-/-) mice exhibited significant cardiac hypertrophy. Cardiac function, assessed using 2-dimensionally guided M-mode echocardiography, showed significantly depressed indices of diastolic and systolic function only in cMyBP-C(-/-) mice. Ca2+ sensitivity of tension, measured in single skinned myocytes, was reduced in cMyBP-C(-/-) but not cMyBP-C(+/-) mice. These results establish that cMyBP-C is not essential for cardiac development but that the absence of cMyBP-C results in profound cardiac hypertrophy and impaired contractile function.

Development of left ventricular hypertrophy in adults with hypertrophic cardiomyopathy caused by cardiac myosin-binding protein C gene mutations

OBJECTIVESWe sought to determine whether the development of left ventricular hypertrophy (LVH) can be demonstrated during adulthood in genetically affected relatives with hypertrophic cardiomyopathy (HCM).BACKGROUNDHypertrophic cardiomyopathy is a heterogeneous cardiac disease caused by mutations in nine genes that encode proteins of the sarcomere. Mutations in cardiac myosin-binding protein C (MyBPC) gene have been associated with age-related penetrance.METHODSTo further analyze dormancy of LVH in patients with HCM, we studied, using echocardiography and 12-lead electrocardiography, the phenotypic expression caused by MyBPC mutations in seven genotyped pedigrees.RESULTSOf 119 family members studied, 61 were identified with a MyBPC mutation, including 21 genetically affected relatives (34%) who did not express the HCM morphologic phenotype (by virtue of showing normal left ventricular wall thickness). Of these 21 phenotype-negative individuals, 9 were children, presumably in the prehypertrophic phase, and 12 were adults. Of the 12 adults with normal wall thickness ≤12 mm (7 also with normal electrocardiograms), 5 subsequently underwent serial echocardiography prospectively over four to six years. Of note, three of these five adults showed development of LVH in mid-life, appearing for the first time at 33, 34 and 42 years of age, respectively, not associated with outflow obstruction or significant symptoms.CONCLUSIONSIn adults with HCM, disease-causing MyBPC mutations are not uncommonly associated with absence of LVH on echocardiogram. Delayed remodeling with the development of LVH appearing de novo in adulthood, demonstrated here for the first time in individual patients with prospectively obtained serial echocardiograms, substantiates the principle of age-related penetrance for MyBPC mutations in HCM. These observations alter prevailing perceptions regarding the HCM clinical spectrum and family screening strategies and further characterize the evolution of LVH in this disease.

Myosin binding protein C, a phosphorylation-dependent force regulator in muscle that controls the attachment of myosin heads by its interaction with myosin S2.

Myosin binding protein C (MyBP-C) is one of the major sarcomeric proteins involved in the pathophysiology of familial hypertrophic cardiomyopathy (FHC). The cardiac isoform is tris-phosphorylated by cAMP-dependent protein kinase (cAPK) on beta-adrenergic stimulation at a conserved N-terminal domain (MyBP-C motif), suggesting a role in regulating positive inotropy mediated by cAPK. Recent data show that the MyBP-C motif binds to a conserved segment of sarcomeric myosin S2 in a phosphorylation-regulated way. Given that most MyBP-C mutations that cause FHC are predicted to result in N-terminal fragments of the protein, we investigated the specific effects of the MyBP-C motif on contractility and its modulation by cAPK phosphorylation. The diffusion of proteins into skinned fibers allows the investigation of effects of defined molecular regions of MyBP-C, because the endogenous MyBP-C is associated with few myosin heads. Furthermore, the effect of phosphorylation of cardiac MyBP-C can be studied in a defined unphosphorylated background in skeletal muscle fibers only. Triton skinned fibers were tested for maximal isometric force, Ca(2+)/force relation, rigor force, and stiffness in the absence and presence of the recombinant cardiac MyBP-C motif. The presence of unphosphorylated MyBP-C motif resulted in a significant (1) depression of Ca(2+)-activated maximal force with no effect on dynamic stiffness, (2) increase of the Ca(2+) sensitivity of active force (leftward shift of the Ca(2+)/force relation), (3) increase of maximal rigor force, and (4) an acceleration of rigor force and rigor stiffness development. Tris-phosphorylation of the MyBP-C motif by cAPK abolished these effects. This is the first demonstration that the S2 binding domain of MyBP-C is a modulator of contractility. The anchorage of the MyBP-C motif to the myosin filament is not needed for the observed effects, arguing that the mechanism of MyBP-C regulation is at least partly independent of a "tether," in agreement with a modulation of the head-tail mobility. Soluble fragments occurring in FHC, lacking the spatial specificity, might therefore lead to altered contraction regulation without affecting sarcomere structure directly.

Molecular Genetic Basis of Hypertrophic Cardiomyopathy:

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease caused by mutations in sarcomeric proteins. The disease is characterized by left ventricular hypertrophy in the absence of an increased external load, and myofibrillar disarray. A large number of mutations in genes coding for the beta-myosin heavy chain (beta-MyHC), cardiac troponin T (cTnT), cardiac troponin I, alpha-tropomyosin, myosin binding protein C (MyBP-C), and myosin light chain 1 and 2 in patients with HCM have been identified. Genotype-phenotype correlation studies have shown that mutations carry prognostic significance. The Gly256Glu, Val606Met, and Leu908Val mutations in the beta-MyHC are associated with a benign prognosis. In contrast, Arg403Gln, Arg719Trp, and Arg453Cys mutations are associated with a high incidence of sudden cardiac death (SCD). Mutations in cTnT are associated with a mild degree of hypertrophy, but a high incidence of SCD. Mutations in MyBP-C are associated with mild hypertrophy and a benign prognosis. However, it has become evident that factors other than the underlying mutations, such as genetic background and possibly environmental factors, also modulate phenotypic expression of HCM.