Cardiac Myosin Binding protein-C Research Articles

069 Cardiac myosin binding protein C mutations in families with hypertrophic cardiomyopathy: disease expression in relation to age, gender, and long term outcome

<h3>Introduction</h3> Mutations in cardiac myosin binding protein-C (MYBPC3), the most common genetic cause of hypertrophic cardiomyopathy (HCM), have been reported to cause a comparatively benign and late-onset form of the disease with incomplete penetrance. Based upon selected families with small numbers of mutations, these early reports may be misleading however. This study aimed to redefine the clinical characteristics of HCM related to MYBPC3 by evaluating the largest cohort to date of unselected patients and their families, in whom an MYBPC3 mutation had been identified. <h3>Methods</h3> Index cases and their families underwent history, physical examination, electrocardiogram (ECG), transthoracic echocardiography, ambulatory ECG monitoring, metabolic exercise testing and mutation analysis. Long-term follow up data were collected where available. <h3>Results</h3> Forty-four MYBPC3 mutations were identified in 59 index cases. 26 of 44 (44%) were missense with 11 (19%) insertions/deletions, 11 (19%) intronic, and 5 (8%) nonsense mutations. A further 6 (10%) had complex genetic status with two different sequence variations identified. Nine families shared the R502W missense mutation and haplotype analysis confirmed a common founder, the first to be described in a UK cohort. A further 111 mutation carriers were identified, of which 39 were clinically affected – disease penetrance was therefore incomplete (58%) and related to age and gender but not mutation type. Mean age at diagnosis was 40.1+/−15.9 years with a wide range (7–76); 91.8% of affected mutation carriers were diagnosed over the age of 20 years. Most had asymmetric septal hypertrophy (86.4%) and mean maximal wall thickness was 20+/−5.8 mm. Families sharing identical mutations showed significant variability in disease penetrance, age at diagnosis and risk of sudden death, suggesting that modifying factors play a significant role in disease development. During long term follow up (mean 7.9+/−4.5 years) one individual developed hypertrophy as an adult, five individuals died (three suddenly) and overall survival was 94%. <h3>Discussion</h3> The broad spectrum of mutations, disease severity and natural history of disease suggests that earlier reports of late-onset, benign disease related to MYBPC3 mutations were premature. In this study disease expression is broadly similar to non-genotyped HCM cohorts with disease severity ranging from mild to severe, risk of sudden death ranging from low to high and clinical disease being diagnosed in all decades of life. Such variance is not adequately explained by the sarcomeric protein gene or specific mutation per se and other genetic and environmental factors influence disease penetrance, severity and prognosis.

Can Genetic Testing Improve Our Aim in Hypertrophic Cardiomyopathy?

See related article, pages 1549–1552 Hypertrophic cardiomyopathy (HCM) is a common inherited disorder with an estimated prevalence of ≥1 in 500 worldwide. The disease is inherited in families in an autosomal dominant fashion and is usually caused by mutations in genes encoding contractile proteins such as cardiac β-myosin heavy chain ( MYH7 ), cardiac troponin T ( TNNT2 ), cardiac myosin binding protein-C ( MYBPC3 ), cardiac troponin I, α-myosin heavy chain, cardiac α-actin, α-tropomyosin, titin, myosin regulatory light chain, and myosin essential light chain. Several HCM disease genes remain to be identified. The most obvious clinical manifestation of HCM is left ventricular hypertrophy. Although such hypertrophy is classically asymmetrical with prominent involvement of the interventricular septum, both concentric and apical hypertrophy can also occur. In fact, affected individuals may not exhibit any hypertrophy. Other clinical features are similarly variable in their expression and include sudden cardiac death, heart failure, arrhythmias, stroke, heart block, and infective endocarditis. Some patients remain asymptomatic throughout their lifetime. Many HCM patients have no or only minor symptoms, and asymptomatic affected children and adolescents are often diagnosed during family screening after another family member comes to medical attention.1 The average annual risk of sudden cardiac death in a HCM patient is 1%,2 and in high risk patients, prophylactic defibrillator implantation can be lifesaving. Given marked variation in penetrance and expressivity, early diagnosis and reliable prognostic tools are crucial for primary prevention and proper followup of affected individuals and their family members. Current guidelines recommend that all first-degree relatives of an individual affected by HCM should be clinically evaluated by history, physical examination, electrocardiography, and echocardiography. With increased understanding of the molecular genetic causes of HCM and advances in modern laboratory technology, clinical genetic testing for HCM has become increasingly feasible.3 The GeneTests database (http://www.ncbi.nlm.nih.gov/sites/GeneTests) currently lists 5 …

Identification of Novel Protein Kinase A Phosphorylation Sites in the M-domain of Human and Murine Cardiac Myosin Binding Protein-C Using Mass Spectrometry Analysis

Cardiac myosin binding protein-C (cMyBP-C) is a large multidomain accessory protein bound to myosin thick filaments in striated muscle sarcomeres. It plays an important role in the regulation of muscle contraction, and mutations in the gene encoding cMyBP-C are a common cause of familial hypertrophic cardiomyopathy, the leading cause of sudden cardiac death in young people. (1) The N-terminal domains including the C0, C1, cMyBP-C motif, and C2 domains play a crucial role in maintaining and modulating actomyosin interactions (keeping normal cardiac function) in a phosphorylation-dependent manner. The cMyBP-C motif or "M-domain" is a highly conserved linker domain in the N-terminus of cMyBP-C that contains three to five protein kinase A (PKA) phosphorylation sites, depending on species. For the human isoform, three PKA sites were previously identified (Ser(275), Ser(284), and Ser(304)), while three homologous sites exist in the murine isoform (Ser(273), Ser(282), and Ser(302)). The murine cMyBP-C isoform contains an additional conserved consensus site, Ser(307) that is not present in the human isoform. In this study, we investigated sites of PKA phosphorylation of murine and human cMyBP-C by treating the recombinant protein C0C2 ( approximately 50 KDa, which contains the N-terminal C0, C1, M, and C2 domains) and C1C2 (approximately 35 KDa, contains C1, M, and C2 domains) with PKA and assessing the phosphorylation states using SDS-PAGE with ProQ Diamond staining, and powerful hybrid mass spectrometric analyses. Both high-accuracy bottom-up and measurements of intact proteins mass spectrometric approaches were used to determine the phosphorylation states of C0C2 and C1C2 proteins with or without PKA treatment. Herein, we report for the first time that there are four PKA phosphorylation sites in both murine and human M-domains; both murine Ser(307) and a novel human Ser(311) can be phosphorylated in vitro by PKA. Future studies are needed to investigate the phosphorylation state of murine and human cMyBP-C in vivo.

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.

Force, Ca-Sensitivity and Contractile Efficiency in Human Myocardium Expressing a Truncated Cardiac Myosin Binding Protein-C

We have investigated contractile parameters of ventricular myocardium in samples from a patient diagnosed with hypertrophic cardiomyopathy (HCM) caused by a truncation mutation in MYBPC3, the gene encoding cardiac myosin binding protein C (cMyBP-C). The mutation truncates the protein in the C7 domain resulting in the loss of 408 residues. Our earlier work has shown that the truncated protein is not stably expressed and the disease is likely to be mediated by cMyBP-C haploinsufficiency.

Mechanical Unfolding of Cardiac Myosin Binding Protein-C by Atomic Force Microscopy

Cardiac myosin binding protein-C (cMyBP-C) is a member of the immunoglobulin (Ig) superfamily of proteins and consists of 8 Ig- and 3 fibronectin (Fn)-like domains along with a unique regulatory sequence referred to as the M-domain. Domains near the C-terminus bind tightly to myosin and mediate the association of MyBP-C with thick (myosin-containing) filaments, whereas N-terminal domains of MyBP-C, including the M-domain, bind reversibly to myosin S2 and/or actin. The ability of MyBP-C to bind to both myosin and actin raises the possibility that MyBP-C cross links thick and thin (actin-containing) filaments and thereby imposes a drag that regulates shortening velocity during contraction. To investigate the mechanical properties of the proposed thick-thin filament linkage, we used atomic force microscopy (AFM) and electron microscopy (EM) to assess the single molecule elasticity and mechanical stability of full-length mouse cardiac (c) MyBP-C expressed in sF9 cells. Force-extension curves showed that cMyBP-C is extensible via unfolding of individual domains evident as "saw tooth" peaks in force spectra. Spectra with up to 12 peaks were obtained. The force required to unfold the domains varied, with the least and most stable domains unfolding at forces <50 pN and >100 pN, respectively, suggesting that a mechanical hierarchy exists along cMyBP-C. EM images of purified, rotary shadowed cMyBP-C showed that molecules were frequently V- or U-shaped with lengths ∼44 nm. These data indicate that cMyBP-C is extensible and contains regions with variable resistances that could slow sarcomere shortening or limit lattice expansion. Supported by NIH HL080367.

N-Terminal Fragments of Cardiac Myosin Binding Protein-C Inhibit Actomyosin Motility by Tethering Actin

Cardiac myosin binding protein-C (cMyBP-C) mutations are a leading cause of hypertrophic cardiomyopathy. cMyBP-C has 11 domains, C0 through C10, that bind sarcomeric proteins, including myosin and actin. A 29 kD N-terminal fragment (C0C1f) of cMyBP-C containing the first two domains C0 and C1 and the first 15 residues of the conserved MyBP-C motif is cleaved from cMyBP-C following ischemic-reperfusion injury (Sadayappan et al., JMCC 44:S44, 2008). Expressed C0C1f fragments inhibit actin velocities in the motility assay at a 2:1 molar ratio to myosin, similar to other N-terminal fragments: C0C3, C0C2, and C1C2. Interestingly, fragments containing only the C0C1 domains do not alter velocity, suggesting the additional 15 residues in C0C1f are necessary for inhibition. Adding C0C1 to the motility assay can partially reverse the C0C3-mediated inhibition of velocity, suggesting C0C1 may compete with C0C3 for actin binding. cMyBP-C fragments may affect motility by creating a tether between actin and the flowcell surface. To test this, motility experiments were performed under high ionic strength, saturating MgATP, and in the absence of methylcellulose, conditions in which most actin filaments diffuse away from the surface due to weak interactions with myosin. In the presence of C0C2, many actin filaments bound and translocated on the surface, confirming this fragment's tethering capacity. Additionally, in the laser trap we adhered C0C3 fragments to a bead in the absence of myosin and observed C0C3 transiently binding to a single actin filament with an ∼100 ms attached lifetime. We also saw evidence that C0C3 may partially unfold under load. These experiments strongly suggest that N-terminal domains of cMyBP-C containing the MyBP-C motif tether actin filaments and provide one mechanism for modulating actomyosin motion generation, i.e. by imposing an effective viscous load within the sarcomere.

Comparative Effects of the Proline-Alanine Rich Regions of Human and Murine Cardiac Myosin Binding Protein-C

The N-terminus of cMyBP-C can activate actomyosin interactions in the absence of Ca2+, but it is unclear which sequences mediate the activating effects. Herron et al. (Circ Res, 98:1290-8, 2006) found that the Pro-Ala rich region (P-A) of human cMyBP-C could activate tension in the absence of Ca2+, whereas Razumova et al. (J Gen Physiol, 132:575-85, 2008) found that murine C1 and M domains activated tension. The different results might be explained by isoform differences, especially in P-A which is only 46% identical between mouse and human cMyBP-C. The goal of this study was to determine if species-specific differences in P-A account for the different activating effects of murine and human cMyBP-C. Recombinant chimeric proteins containing the C0, P-A, and C1 domains (C0C1) from either human or murine cMyBP-C were engineered and their activating effects assessed using in vitro motility and ATPase assays. Consistent with previous observations, human C0C1 activated actomyosin interactions in the absence of Ca2+, whereas murine C0C1 did not. However, substituting human P-A for murine P-A conferred activating properties to murine C0C1, whereas substituting murine P-A for human P-A depressed the activating effects of human C0C1. Activating effects of the chimera proteins were intermediate between those of murine and human C0C1, suggesting that C0 or C1 also contribute to activation properties. Further chimeric substitutions of C0 and C1 demonstrated that the human C1 domain also contributed to activation, whereas the C0 domain did not. These results suggest that the human P-A and C1 domains are sufficient to activate actomyosin interactions in the absence of Ca2+, and that species-specific differences are likely to contribute to functional differences of cMyBP-C. Supported by NIH HL080367 to SPH and a NSF Graduate Research Fellowship to JFS.

Identification of Amino Acid Residues in the Cardiac Myosin Binding Protein-C Motif Important for Actin Binding

N-terminal domains of cardiac myosin binding protein-C (cMyBP-C) can activate actomyosin interactions in the absence of Ca2+ and bind to actin in a phosphorylation dependent manner. We have previously shown that two N-terminal domains, C1 and the MyBP-C motif (“M”) domain, bind specifically to actin and to thin filaments; however, the sequences or residues that mediate actin binding have not been identified. The goal of this study was to identify residues in the M-domain that contribute to actin binding and to investigate whether interactions between the M-domain and actin mediate the activating properties of cMyBP-C. We therefore used alanine-scanning mutagenesis to target candidate actin binding sites in the M-domain that bear homology to the actin binding motifs in other known actin binding proteins and to assess the effects of mutations on the ability of recombinant proteins to bind actin and activate actomyosin interactions in motility assays. Results demonstrate that mutation of select positively-charged amino acids in the M-domain that are homologous to binding motifs in known actin binding proteins reduced binding of cMyBP-C to actin. The mutations also reduced or eliminated the activating properties of recombinant cMyBP-C in in vitro motility assays. However, mutation of other positively-charged amino acids did not affect actin binding or protein functional properties. These results indicate that specific residues within the M-domain confer actin binding and that interactions with actin contribute to the functional effects of recombinant cMyBP-C N-terminal proteins. Supported by NIH HL080367 to SPH and a NSF Graduate Research Fellowship to JFS.

Regulation of Contraction by PKA Phosphorylation of Myosin Binding Protein C and Troponin I in Murine Skinned Myocardium

In skinned myocardium, cAMP-dependent protein kinase (PKA)-catalyzed phosphorylation of cardiac myosin binding protein-C (cMyBP-C) and troponin I (cTnI) leads to a decrease in myofilament Ca2+-sensitivity and an acceleration in the kinetics of cross-bridge cycling. To examine the relative roles of cTnI and cMyBP-C phosphorylation in altering contractile function, we determined the Ca2+-sensitivity of force (pCa50) and the rate of force redevelopment (ktr) in untreated and PKA-treated murine myocardium expressing: (1) phosphorylatable cTnI and cMyBP-C (WT), (2) non-phosphorylatable cTnI with serines23/24/43/45 and threonine144 residues mutated to alanines (cTnIala5), (3) phosphorylatable cTnI on a cMyBP-C null background (cMyBP-C-/-), and (4) non-phosphorylatable cTnI on a cMyBP-C null background (cTnIala5/cMyBP-C-/-). A novel aspect of this study was the use of 2,3-Butandione Monoxime (BDM) treatments to reduce the basal levels of myosin regulatory light chain (RLC) phosphorylation to near zero in order to more accurately define the functional consequences of removing cMyBP-C and/or cTnI phoshorylation in transgenic myocardium. Our results showed that in the absence of RLC phosphorylation, PKA-treatment decreased pCa50 in WT, cTnIala5, and cMyBP-C-/- myocardium by 0.13, 0.08 and 0.09 pCa units, respectively, but had no effect in cTnIala5/cMyBP-C-/- myocardium. In WT and cTnIala5 myocardium, PKA treatment increased ktr at submaximal levels of activation; however, treatment did not have an effect on ktr in cMyBP-C-/- and cTnIala5/cMyBP-C-/- myocardium. Together, these results indicate that the attenuation of the myofilament force response following PKA treatment is due to phosphorylation of both cTnI and cMyBP-C and that the reduced Ca2+-sensitivity of force mediated by phosphorylation of cMyBP-C is most likely due to an increased rate constant of cross-bridge detachment that also contributes to an acceleration of cross-bridge cycling kinetics.

Phosphorylation of Myosin Binding Protein-C Alters the Proximity of Cross-Bridges to Actin and Accelerates Myocardial Twitch Kinetics

The strength and kinetics of cardiac contraction vary on a beat-to-beat basis in efforts to match cardiac output in response to changing circulatory demands. In living myocardium, the beta-adrenoreceptor agonist dobutamine initiates protein kinase A (PKA)-mediated phosphorylations of Ca2+ handling proteins and contractile proteins including cardiac myosin binding protein-C (cMyBP-C) and cardiac troponin I (cTnI), which leads to potentiation of twitch force and faster kinetics of force development and relaxation. Our previous studies in skinned myocardium suggest that PKA phosphorylation of cMyBP-C disrupts its interaction with myosin subfragment 2 (S2), which relieves the tether-like constraint of myosin heads imposed by cMyBP-C, and thereby accelerates cross-bridge cycling kinetics. To examine the relative role of cMyBP-C phosphorylation in altering twitch kinetics, we recorded twitch force and low-angle x-ray diffraction patterns in between twitches and near maximum twitch force in intact trabeculae isolated from murine myocardium electrically stimulated at 0.5 Hz in the presence and absence of dobutamine. Our data suggest that phosphorylation of cMyBP-C caused a radial or azimuthal displacement of cross-bridges towards the thin filament in vivo prior to the twitch, which contributes to the accelerated contraction kinetics following twitch stimulation. These results suggest that interactions between cMyBP-C and the S2-domain of myosin heavy chain are dynamically regulated by phosphorylation of cMyBP-C and function to modulate the availability and cooperative binding of cross-bridges to actin during the myocardial twitch.

Species-specific differences in the Pro-Ala rich region of cardiac myosin binding protein-C

Cardiac myosin binding protein-C (cMyBP-C) is an accessory protein found in the A-bands of vertebrate sarcomeres and mutations in the cMyBP-C gene are a leading cause of familial hypertrophic cardiomyopathy. The regulatory functions of cMyBP-C have been attributed to the N-terminus of the protein, which is composed of tandem immunoglobulin (Ig)-like domains (C0, C1, and C2), a region rich in proline and alanine residues (the Pro-Ala rich region) that links C0 and C1, and a unique sequence referred to as the MyBP-C motif, or M-domain, that links C1 and C2. Recombinant proteins that contain various combinations of the N-terminal domains of cMyBP-C can activate actomyosin interactions in the absence of Ca2+, but the specific sequences required for these effects differ between species; the Pro-Ala region has been implicated in human cMyBP-C whereas the C1 and M-domains appear important in mouse cMyBP-C. To investigate whether species-specific differences in sequence can account for the observed differences in function, we compared sequences of the Pro-Ala rich region in cMyBP-C isoforms from different species. Here we report that the number of proline and alanine residues in the Pro-Ala rich region varies significantly between different species and that the number correlates directly with mammalian body size and inversely with heart rate. Thus, systematic sequence differences in the Pro-Ala rich region of cMyBP-C may contribute to observed functional differences in human versus mouse cMyBP-C isoforms and suggest that the Pro-Ala region may be important in matching contractile speed to cardiac function across species.

The cAMP binding protein Epac regulates cardiac myofilament function

In the heart, cAMP is a key regulator of excitation-contraction coupling and its biological effects are mainly associated with the activity of protein kinase A (PKA). The aim of this study was to investigate the contribution of the cAMP-binding protein Epac (Exchange protein directly activated by cAMP) in the regulation of the contractile properties of rat ventricular cardiac myocytes. We report that both PKA and Epac increased cardiac sarcomere contraction but through opposite mechanisms. Differently from PKA, selective Epac activation by the cAMP analog 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT) reduced Ca(2+) transient amplitude and increased cell shortening in intact cardiomyocytes and myofilament Ca(2+) sensitivity in permeabilized cardiomyocytes. Moreover, ventricular myocytes, which were infected in vivo with a constitutively active form of Epac, showed enhanced myofilament Ca(2+) sensitivity compared to control cells infected with green fluorescent protein (GFP) alone. At the molecular level, Epac increased phosphorylation of 2 key sarcomeric proteins, cardiac Troponin I (cTnI) and cardiac Myosin Binding Protein-C (cMyBP-C). The effects of Epac activation on myofilament Ca(2+) sensitivity and on cTnI and cMyBP-C phosphorylation were independent of PKA and were blocked by protein kinase C (PKC) and Ca(2+) calmodulin kinase II (CaMKII) inhibitors. Altogether these findings identify Epac as a new regulator of myofilament function.

Expanded Spectrum of Gene Causing Both Hypertrophic Cardiomyopathy and Dilated Cardiomyopathy

See related articles, pages 375–382 Cardiomyopathy can be classified into at least four main forms, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), restrictive cardiomyopathy, and left ventricular noncompaction. HCM produces ventricular wall thickening, especially in the interventricular septum, with decrease in ventricular chamber volumes. DCM produces a prominent increase in chamber volumes without ventricular wall thickening. In HCM, systolic function is increased or at least preserved, whereas diastolic function is impaired in part because of the hypertrophy itself, interstitial fibrosis, and/or myocyte disarrays. Diastolic dysfunction is thought to be responsible for symptoms of heart failure and premature sudden cardiac death of HCM patients. In contrast, DCM is characterized by systolic dysfunction, which leads to congestive heart failure requiring cardiac transplantation. Restrictive cardiomyopathy is characterized by restrictive diastolic dysfunction (restrictive filling and reduced diastolic volume of either or both ventricles) with normal or near normal systolic function and wall thickness. Left ventricular noncompaction is characterized by deep trabeculation in the ventricular wall with systolic and diastolic dysfunction, arrhythmias, and thromboembolic events. More than 400 mutations that cause HCM, DCM, restrictive cardiomyopathy, and left ventricular noncompaction have been found in the genes for proteins constituting the sarcomere of cardiac muscle in human, including α- and β-myosin heavy chains, α-cardiac actin, cardiac troponin (cTn)T, cTnI, cTnC, α-tropomyosin (αTM), cardiac myosin-binding protein-C, cardiac myosin essential light chain, cardiac myosin regulatory light chain, and cardiac titin/connectin. Most mutations cause cardiomyopathies in an autosomal dominant manner. Interestingly, the same sarcomeric gene was found to be simultaneously responsible for different forms of cardiomyopathy.1,2 For example, many mutations in cTnT, cTnC, α-tropomyosin, α-cardiac actin, β-myosin heavy chain, and titin have been found in both HCM and primary DCM that is clearly distinguished from an end-stage …

Evidence From Human Myectomy Samples That MYBPC3 Mutations Cause Hypertrophic Cardiomyopathy Through Haploinsufficiency

Most sarcomere gene mutations that cause hypertrophic cardiomyopathy are missense alleles that encode dominant negative proteins. The potential exceptions are mutations in the MYBPC3 gene (encoding cardiac myosin-binding protein-C [MyBP-C]), which frequently encode truncated proteins. We sought to determine whether there was evidence of haploinsufficiency in hypertrophic cardiomyopathy caused by MYBPC3 mutations by comparing left ventricular muscle from patients undergoing surgical myectomy with samples from donor hearts. MyBP-C protein and mRNA levels were quantitated using immunoblotting and RT-PCR. Nine of 37 myectomy samples had mutations in MYBPC3: 2 missense alleles (Glu258Lys, Arg502Trp) and 7 premature terminations. No specific truncated MyBP-C peptides were detected in whole muscle homogenates of hypertrophic cardiomyopathy tissue. However, the overall level of MyBP-C in myofibrils was significantly reduced (P<0.0005) in tissue containing either a truncation or missense MYBPC3 mutation: 0.76+/-0.03 compared with 1.00+/-0.05 in donor and 1.01+/-0.06 in non-MYBPC3 mutant myectomies. The absence of any detectable truncated MyBP-C argues against its incorporation in the myofiber and any dominant negative effect. In contrast, the lowered relative level of full length protein in both truncation and missense MYBPC3 mutations argues strongly that haploinsufficiency is sufficient to cause the disease.

Carbon-Nanotube-Based Materials for Protein Crystallization

We report on the first use of carbon-nanotube-based films to produce crystals of proteins. The crystals nucleate on the surface of the film. The difficulty of crystallizing proteins is a major bottleneck in the determination of the structure and function of biological molecules. The crystallization of two model proteins and two medically relevant proteins was studied. Quantitative data on the crystallization times of the model protein lysozyme are also presented. Two types of nanotube films, one made with the surfactant Triton X-100 (TX-100) and one with gelatin, were tested. Both induce nucleation of the crystal phase at supersaturations at which the protein solution would otherwise remain clear; however, the gelatin-based film induced nucleation down to much lower supersaturations for the two model proteins with which it was used. It appears that the interactions of gelatin with the protein molecules are particularly favorable to nucleation. Crystals of the C1 domain of the human cardiac myosin-binding protein-C that diffracted to a resolution of 1.6 A were obtained on the TX-100 film. This is far superior to the best crystals obtained using standard techniques, which only diffracted to 3.0 A. Thus, both of our nanotube-based films are very promising candidates for future work on crystallizing difficult-to-crystallize target proteins.

Cardiac Myosin Binding Protein-C Is Essential for Thick-Filament Stability and Flexural Rigidity

Using atomic force microscopy, we examined the contribution of cardiac myosin binding protein-C (cMyBP-C) to thick-filament length and flexural rigidity. Native thick filaments were isolated from the hearts of transgenic mice bearing a truncation mutation of cMyBP-C (t/t) that results in no detectable cMyBP-C and from age-matched wild-type controls (+/+). Atomic force microscopy images of these filaments were evaluated with an automated analysis algorithm that identified filament position and shape. The t/t thick-filament length (1.48 ± 0.02 μm) was significantly (P < 0.01) shorter than +/+ (1.56 ± 0.02 μm). This 5%-shorter thick-filament length in the t/t was reflected in 4% significantly shorter sarcomere lengths of relaxed isolated cardiomyocytes of the t/t (1.97 ± 0.01 μm) compared to +/+ (2.05 ± 0.01 μm). To determine if cMyBP-C contributes to the mechanical properties of thick filaments, we used statistical polymer chain mechanics to calculate a per-filament-specific persistence length, an index of flexural rigidity directly proportional to Young's modulus. Thick-filament-specific persistence length in the t/t (373 ± 62 μm) was significantly lower than in +/+ (639 ± 101 μm). Accordingly, Young's modulus of t/t thick filaments was ∼60% of +/+. These results provide what we consider a new understanding for the critical role of cMyBP-C in defining normal cardiac output by sustaining force and muscle stiffness.

Determination of AMP-activated protein kinase phosphorylation sites in recombinant protein expressed using the pET28a vector: A cautionary tale

AMP-activated protein kinase (AMPK) is responsible for sensing of the cell’s energetic status and it phosphorylates numerous substrates involved in anabolic and catabolic processes as well as interacting with signaling cascades. Mutations in the gene encoding the γ2 regulatory subunit have been shown to cause hypertrophic cardiomyopathy (HCM) with conduction abnormalities. As part of a study to examine the role of AMPK in the heart, we tested whether specific domains of the thick filament component cardiac myosin binding protein-C (cMyBP-C) were good in vitro AMPK substrates. The commercially available pET28a expression vector was used to generate a recombinant form of the cMyBP-C C8 domain as a fusion protein with a hexahistidine tag. In vitro phosphorylation with activated kinase showed that the purified fusion protein was a good AMPK substrate, phosphorylated at a similar rate to the control SAMS peptide and with phosphate incorporation specifically in serine residues. However, subsequent analysis of alanine replacement mutants and thrombin digestion revealed that the strong AMPK phosphorylation site was contained within the thrombin cleavage sequence encoded by the vector. As this sequence is common to many commercial pET vectors, caution is advised in the mapping of AMPK phosphorylation sites when this sequence is present.

J002 The cAMP binding protein Epac regulates cardiac myofilament function

In the heart, cAMP is a key regulator of excitation—contraction coupling and its biological effects are mainly associated with the activity of protein kinase A (PKA). The aim of this study was to investigate the contribution of the cAMP-binding protein Epac (Exchange protein directly activated by cAMP) in the regulation of the contractile properties of rat ventricular cardiac myocytes. We report that both PKA and Epac increased cardiac sarcomere contraction but through opposite mechanisms. Differently from PKA, selective Epac activation by the cAMP analog 8-pCPT reduced Ca2+ transient amplitude and increased cell shortening in intact cardiomyocytes as well as myofilament Ca2+ sensitivity in permeabilized cardiomyocytes. Moreover, ventricular myocytes, which were infected in vivo with a constitutively active form of Epac, showed enhanced myofilament Ca2+ sensitivity compared to control cells infected with GFP alone. At the molecular level, Epac increased phosphorylation of two key sarcomeric proteins, cardiac Troponin I (cTnI) and cardiac Myosin Binding Protein-C (cMyBP-C). The effects of Epac activation on myofilament Ca2+ sensitivity and on cTnI and cMyBP-C phosphorylation were independent of PKA, and were blocked by protein kinase C (PKC) and Ca2+ calmodulin kinase II (CaMKII) inhibitors. Altogether these findings identify Epac as a new regulator of myofilament function.

Cardiac Myosin Binding Protein-C Phosphorylation in a β-Myosin Heavy Chain Background

Cardiac myosin binding protein-C (cMyBP-C) phosphorylation modulates cardiac contractility. When expressed in cMyBP-C-null (cMyBP-C((t/t))) hearts, a cMyBP-C phosphomimetic (cMyBP-C(AllP+)) rescued cardiac dysfunction and protected the hearts from ischemia/reperfusion injury. However, cMyBP-C function may be dependent on the myosin isoform type. Because these replacements were performed in the mouse heart, which contains predominantly alpha-myosin heavy chain (alpha-MyHC), the applicability of the data to humans, whose cardiomyocytes contain predominantly beta-MyHC, is unclear. We determined the effect(s) of cMyBP-C phosphorylation in a beta-MyHC transgenic mouse heart in which >80% of the alpha-MyHC was replaced by beta-MyHC, which is the predominant myosin isoform in human cardiac muscle. To determine the effects of cMyBP-C phosphorylation in a beta-MyHC background, transgenic mice expressing normal cMyBP-C (cMyBP-C(WT)), nonphosphorylatable cMyBP-C (cMyBP-C(AllP)(-)), or cMyBP-C(AllP+) were bred into the beta-MyHC background (beta). These mice were then crossed into the cMyBP-C((t/t)) background to ensure the absence of endogenous cMyBP-C. cMyBP-C((t/t)/beta) and cMyBP-C(AllP)(-)(:(t/t)/beta) mice died prematurely because of heart failure, confirming that cMyBP-C phosphorylation is essential in the beta-MyHC background. cMyBP-C(AllP+:(t/t)/beta) and cMyBP-C(WT:(t/t)/beta) hearts showed no morbidity and mortality, and cMyBP-C(AllP+:(t/t)/beta) hearts were significantly cardioprotected from ischemia/reperfusion injury. cMyBP-C phosphorylation is necessary for basal myocardial function in the beta-MyHC background and can preserve function after ischemia/reperfusion injury. Our studies justify exploration of cMyBP-C phosphorylation as a therapeutic target in the human heart.