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Cardiac myosin binding protein-C (cMyBP-C) located in the C-zone of myocyte sarcomere is involved in the regulation of myocardial contraction. Its N-terminal domains C0, C1, C2, and the m-motif between C1 and C2 can bind to the myosin head and actin of the thin filament and affect the characteristics of their interaction. Measurements using an optical trap showed that the C0-C2 fragment of cMyBP-C increases the interaction time of cardiac myosin with the actin filament, while in an in vitro motility assay, it dose-dependently reduces the sliding velocity of actin filaments. Thus, it was found that the N-terminal part of cMyBP-C affects the kinetics of the myosin cross-bridge., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

The dosing and efficacy of chemotherapeutic drugs can be limited by toxicity caused by off-pathway reactions. One hypothesis for how such toxicity arises is via metal-catalyzed oxidative damage of cardiac myosin binding protein C (cMyBP-C) found in cardiac tissue. Previous research indicates that metal ion mediated reactive oxygen species induce high levels of protein carbonylation, changing the structure and function of this protein. In this work, we use long timescale all-atom molecular dynamics simulations to investigate the ion environment surrounding the C0 and C1 subunits of cMyBP-C responsible for actin binding. We show that divalent cations are co-localized with protein carbonylation-prone amino acid residues and that carbonylation of these residues can lead to site-specific interruption to the actin-cMyBP-C binding., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

Background: Metastatic prostate cancer is a fatal disease despite multiple new approvals in recent years. Recent studies revealed that circular RNAs (circRNAs) can be involved in cancer metastasis. Defining the role of circRNAs in prostate cancer metastasis and discovering therapeutic targets that block cancer metastasis is of great significance for the treatment of prostate cancer., Methods: The circSOBP levels in prostate cancer (PCa) were determined by qRT-PCR. We evaluated the function of circSOBP using a transwell assay and nude mice lung metastasis models. Immunofluorescence assay and electron microscopic assay were applied to determine the phenotypes of prostate cancer cells' migration. We used fluorescence in situ hybridization assay to determine the localization of RNAs. Dual luciferase and rescue assays were applied to verify the interactions between circSOBP, miR-141-3p, MYPT1, and phosphomyosin light chain (p-MLC2)., Results: We observed that circSOBP level was significantly lower in PCa specimens compared with adjacent noncancerous prostate specimens, and was correlated with the grade group of PCa. Overexpression of circSOBP suppressed PCa migration and invasion in vitro and metastasis in vivo. CircSOBP depletion increased migration and invasion and induced amoeboid migration of PCa cells. Mechanistically, circSOBP bound miR-141-3p and regulated the MYPT1/p-MLC2 axis. Moreover, the depletion of MYPT1 reversed the inhibitory effect of circSOBP on the migration and invasion of PCa cells. Complementary intronic Alu elements induced but were not necessary for the formation of circSOBP. The nuclear export of circSOBP was mediated by URH49., Conclusion: Our results suggest that circSOBP suppresses amoeboid migration of PCa cells and inhibits migration and invasion through sponging miR-141-3p and regulating the MYPT1/p-MLC2 axis., (© 2021 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiomyopathy and is characterized by asymmetric left ventricular hypertrophy and diastolic dysfunction, and a frequent cause of sudden cardiac death at young age. Pharmacological treatment to prevent or reverse HCM is lacking. This may be partly explained by the variety of underlying disease causes. Over 1500 mutations have been associated with HCM, of which the majority reside in genes encoding sarcomere proteins, the cardiac contractile building blocks. Several mutation-mediated disease mechanisms have been identified, with proof for gene- and mutation-specific cellular perturbations. In line with mutation-specific changes in cellular pathology, the response to treatment may depend on the underlying sarcomere gene mutation. In this review, we will discuss evidence for mutation-specific pathology and treatment responses in HCM patients, mouse models and engineered heart tissue. The pros and cons of these experimental models for studying mutation-specific HCM pathology and therapies will be outlined., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Cardiac myosin-binding protein C (cMyBP-C) is a thick filament-associated protein that influences actin-myosin interactions. cMyBP-C alters myofilament structure and contractile properties in a protein kinase A (PKA) phosphorylation-dependent manner. To determine the effects of cMyBP-C and its phosphorylation on the microsecond rotational dynamics of actin filaments, we attached a phosphorescent probe to F-actin at Cys-374 and performed transient phosphorescence anisotropy (TPA) experiments. Binding of cMyBP-C N-terminal domains (C0-C2) to labeled F-actin reduced rotational flexibility by 20-25°, indicated by increased final anisotropy of the TPA decay. The effects of C0-C2 on actin TPA were highly cooperative ( n = ∼8), suggesting that the cMyBP-C N terminus impacts the rotational dynamics of actin spanning seven monomers ( i.e. the length of tropomyosin). PKA-mediated phosphorylation of C0-C2 eliminated the cooperative effects on actin flexibility and modestly increased actin rotational rates. Effects of Ser to Asp phosphomimetic substitutions in the M-domain of C0-C2 on actin dynamics only partially recapitulated the phosphorylation effects. C0-C1 (lacking M-domain/C2) similarly exhibited reduced cooperativity, but not as reduced as by phosphorylated C0-C2. These results suggest an important regulatory role of the M-domain in cMyBP-C effects on actin structural dynamics. In contrast, phosphomimetic substitution of the glycogen synthase kinase (GSK3β) site in the Pro/Ala-rich linker of C0-C2 did not significantly affect the TPA results. We conclude that cMyBP-C binding and PKA-mediated phosphorylation can modulate actin dynamics. We propose that these N-terminal cMyBP-C-induced changes in actin dynamics help explain the functional effects of cMyBP-C phosphorylation on actin-myosin interactions., (© 2019 Bunch et al.)

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) accelerates cardiac contractility. However, the mechanisms by which cMyBP-C phosphorylation increases contractile kinetics have not been fully elucidated. In this study, we tested the hypothesis that phosphorylation of cMyBP-C releases myosin heads from the inhibited super-relaxed state (SRX), thereby determining the fraction of myosin available for contraction. Mice with various alanine (A) or aspartic acid (D) substitutions of the three main phosphorylatable serines of cMyBP-C (serines 273, 282, and 302) were used to address the association between cMyBP-C phosphorylation and SRX. Single-nucleotide turnover in skinned ventricular preparations demonstrated that phosphomimetic cMyBP-C destabilized SRX, whereas phospho-ablated cMyBP-C had a stabilizing effect on SRX. Strikingly, phosphorylation at serine 282 site was found to play a critical role in regulating the SRX. Treatment of WT preparations with protein kinase A (PKA) reduced the SRX, whereas, in nonphosphorylatable cMyBP-C preparations, PKA had no detectable effect. Mice with stable SRX exhibited reduced force production. Phosphomimetic cMyBP-C with reduced SRX exhibited increased rates of tension redevelopment and reduced binding to myosin. We also used recombinant myosin subfragment-2 to disrupt the endogenous interaction between cMyBP-C and myosin and observed a significant reduction in the population of SRX myosin. This peptide also increased force generation and rate of tension redevelopment in skinned fibers. Taken together, this study demonstrates that the phosphorylation-dependent interaction between cMyBP-C and myosin is a determinant of the fraction of myosin available for contraction. Furthermore, the binding between cMyBP-C and myosin may be targeted to improve contractile function., Competing Interests: Conflict of interest statement: S.S. provided consulting and collaborative services to AstraZeneca, Merck, and Amgen unrelated to the content of this manuscript. No other disclosures are reported.

The loss of cardiac myosin binding protein C (cMyBP-C) results in left ventricular dilation, cardiac hypertrophy, and impaired ventricular function in both constitutive and conditional cMyBP-C knockout ( MYBPC3 null) mice. It remains unclear whether the structural and functional phenotypes expressed in the MYBPC3 null mouse are reversible, which is an important question, since reduced expression of cMyBP-C is an important cause of hypertrophic cardiomyopathy in humans. To investigate this question, we generated a cardiac-specific transgenic mouse model using a Tet-Off inducible system to permit the controlled expression of WT cMyBP-C on the MYBPC3 null background. Functional Tet-Off mice expressing WT cMyBP-C (FT-WT) were generated by crossing tetracycline transactivator mice with responder mice carrying the WT cMyBP-C transgene. Prior to dietary doxycycline administration, cMyBP-C was expressed at normal levels in FT-WT myocardium, which exhibited similar levels of steady-state force and in vivo left ventricular function as WT mice. Introduction of dietary doxycycline for four weeks resulted in a partial knockdown of cMyBP-C expression and commensurate impairment of systolic and diastolic function to levels approaching those observed in MYBPC 3 null mice. Subsequent withdrawal of doxycycline from the diet resulted in the reexpression of cMyBP-C to levels comparable to those observed in WT mice, along with near-complete recovery of in vivo ventricular function. These results show that the cardiac phenotypes associated with MYBPC3 null mice are reversible. Our work also validates the use of the Tet-Off inducible system as a means to study the mechanisms underlying hypertrophic cardiomyopathy., (© 2018 Giles et al.)

Hypertrophic cardiomyopathy (HCM) is primarily caused by mutations in β-cardiac myosin and myosin-binding protein-C (MyBP-C). Changes in the contractile parameters of myosin measured so far do not explain the clinical hypercontractility caused by such mutations. We propose that hypercontractility is due to an increase in the number of myosin heads (S1) that are accessible for force production. In support of this hypothesis, we demonstrate myosin tail (S2)-dependent functional regulation of actin-activated human β-cardiac myosin ATPase. In addition, we show that both S2 and MyBP-C bind to S1 and that phosphorylation of either S1 or MyBP-C weakens these interactions. Importantly, the S1-S2 interaction is also weakened by four myosin HCM-causing mutations but not by two other mutations. To explain these experimental results, we propose a working structural model involving multiple interactions, including those with myosin's own S2 and MyBP-C, that hold myosin in a sequestered state.

Cardiac myosin binding protein C (cMyBP-C) has a key regulatory role in cardiac contraction, but the mechanism by which changes in phosphorylation of cMyBP-C accelerate cross-bridge kinetics remains unknown. In this study, we isolated thick filaments from the hearts of mice in which the three serine residues (Ser273, Ser282, and Ser302) that are phosphorylated by protein kinase A in the m-domain of cMyBP-C were replaced by either alanine or aspartic acid, mimicking the fully nonphosphorylated and the fully phosphorylated state of cMyBP-C, respectively. We found that thick filaments from the cMyBP-C phospho-deficient hearts had highly ordered cross-bridge arrays, whereas the filaments from the cMyBP-C phospho-mimetic hearts showed a strong tendency toward disorder. Our results support the hypothesis that dephosphorylation of cMyBP-C promotes or stabilizes the relaxed/superrelaxed quasi-helical ordering of the myosin heads on the filament surface, whereas phosphorylation weakens this stabilization and binding of the heads to the backbone. Such structural changes would modulate the probability of myosin binding to actin and could help explain the acceleration of cross-bridge interactions with actin when cMyBP-C is phosphorylated because of, for example, activation of β 1 -adrenergic receptors in myocardium.

Background: Hypertrophic cardiomyopathy (HCM) is a common cardiac genetic disorder associated with heart failure and sudden death. Mutations in the cardiac sarcomere genes are found in approximately half of HCM patients and are more common among cases with a family history of the disease. Data about the mutational spectrum of the sarcomeric genes in HCM patients from Northern Africa are limited. The population of Tunisia is particularly interesting due to its Berber genetic background. As founder mutations have been reported in other disorders., Methods: We performed semiconductor chip (Ion Torrent PGM) next generation sequencing of the nine main sarcomeric genes (MYH7, MYBPC3, TNNT2, TNNI3, ACTC1, TNNC1, MYL2, MYL3, TPM1) as well as the recently identified as an HCM gene, FLNC, in 45 Tunisian HCM patients., Results: We found sarcomere gene polymorphisms in 12 patients (27%), with MYBPC3 and MYH7 representing 83% (10/12) of the mutations. One patient was homozygous for a new MYL3 mutation and two were double MYBPC3 + MYH7 mutation carriers. Screening of the FLNC gene identified three new mutations, which points to FLNC mutations as an important cause of HCM among Tunisians., Conclusion: The mutational background of HCM in Tunisia is heterogeneous. Unlike other Mendelian disorders, there were no highly prevalent mutations that could explain most of the cases. Our study also suggested that FLNC mutations may play a role on the risk for HCM among Tunisians.

Cardiac myosin binding protein-C (cMyBP-C) is a structural and regulatory component of cardiac thick filaments. It is observed in electron micrographs as seven to nine transverse stripes in the central portion of each half of the A band. Its C-terminus binds tightly to the myosin rod and contributes to thick filament structure, while the N-terminus can bind both myosin S2 and actin, influencing their structure and function. Mutations in the MYBPC3 gene (encoding cMyBP-C) are commonly associated with hypertrophic cardiomyopathy (HCM). In cardiac cells there exists a population of myosin heads in the super-relaxed (SRX) state, which are bound to the thick filament core with a highly inhibited ATPase activity. This report examines the role cMyBP-C plays in regulating the population of the SRX state of cardiac myosin by using an assay that measures single ATP turnover of myosin. We report a significant decrease in the proportion of myosin heads in the SRX state in homozygous cMyBP-C knockout mice, however heterozygous cMyBP-C knockout mice do not significantly differ from the wild type. A smaller, non-significant decrease is observed when thoracic aortic constriction is used to induce cardiac hypertrophy in mutation negative mice. These results support the proposal that cMyBP-C stabilises the thick filament and that the loss of cMyBP-C results in an untethering of myosin heads. This results in an increased myosin ATP turnover, further consolidating the relationship between thick filament structure and the myosin ATPase., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

We tested whether cardiac myosin binding protein-C (cMyBP-C) affects myosin cross-bridge kinetics in the two cardiac myosin heavy chain (MyHC) isoforms. Mice lacking cMyBP-C (t/t) and transgenic controls (WT(t/t)) were fed L-thyroxine (T4) to induce 90/10% expression of α/β-MyHC. Non-transgenic (NTG) and t/t mice were fed 6-n-propyl-2-thiouracil (PTU) to induce 100% expression of β-MyHC. Ca(2+)-activated, chemically-skinned myocardium underwent length perturbation analysis with varying [MgATP] to estimate the MgADP release rate (k(-ADP)) and MgATP binding rate (k(+ATP)). Values for (k(-ADP)) were not significantly different between t/t(T4) (102.2 ± 7.0 s(-1)) and WT(t/t)(T4) (91.3 ± 8.9 s(-1)), but k(+ATP)) was lower in t/t(T4) (165.9 ± 12.5 mM(-1) s(-1)) compared to WT(t/t)(T4) (298.6 ± 15.7 mM(-1) s(-1), P < 0.01). In myocardium expressing β-MyHC, values for k(-ADP) were higher in t/t(PTU) (24.8 ± 1.0 s(-1)) compared to NTG(PTU) (15.6 ± 1.3 s(-1), P < 0.01), and k(+ATP) was not different. At saturating [MgATP], myosin detachment rate approximates k(-ADP), and detachment rate decreased as sarcomere length (SL) was increased in both t/t(T4) and WT(t/t)(T4) with similar sensitivities to SL. In myocardium expressing β-MyHC, detachment rate decreased more as SL increased in t/t(PTU) (21.5 ± 1.3 s(-1) at 2.2 μm and 13.3 ± 0.9 s(-1) at 3.3 μm) compared to NTGPTU (15.8 ± 0.3 s(-1) at 2.2 μm and 10.9 ± 0.3 s(-1) at 3.3 μm) as detected by repeated-measures ANOVA (P < 0.01). These findings suggest that cMyBP-C reduces MgADP release rate for β-MyHC, but not for α-MyHC, even as the number of cMyBP-C that overlap with the thin filament is reduced to zero. Therefore, cMyBP-C appears to affect β-MyHC kinetics independent of its interaction with the thin filament.

Pyruvate kinase M2 (PKM2) is expressed at high levels during embryonic development and tumour progression and is important for cell growth. However, it is not known whether it directly controls cell division. Here, we found that Aurora B phosphorylates PKM2, but not PKM1, at T45; this phosphorylation is required for PKM2's localization and interaction with myosin light chain 2 (MLC2) in the contractile ring region of mitotic cells during cytokinesis. PKM2 phosphorylates MLC2 at Y118, which primes the binding of ROCK2 to MLC2 and subsequent ROCK2-dependent MLC2 S15 phosphorylation. PKM2-regulated MLC2 phosphorylation, which is greatly enhanced by EGF stimulation or EGFRvIII, K-Ras G12V and B-Raf V600E mutant expression, plays a pivotal role in cytokinesis, cell proliferation and brain tumour development. These findings underscore the instrumental function of PKM2 in oncogenic EGFR-, K-Ras- and B-Raf-regulated cytokinesis and tumorigenesis.

Aims: Disease mechanisms regarding hypertrophic cardiomyopathy (HCM) are largely unknown and disease onset varies. Sarcomere mutations might induce energy depletion for which until now there is no direct evidence at sarcomere level in human HCM. This study investigated if mutations in genes encoding myosin-binding protein C (MYBPC3) and myosin heavy chain (MYH7) underlie changes in the energetic cost of contraction in the development of human HCM disease., Methods and Results: Energetic cost of contraction was studied in vitro by measurements of force development and ATPase activity in cardiac muscle strips from 26 manifest HCM patients (11 MYBPC3mut, 9 MYH7mut, and 6 sarcomere mutation-negative, HCMsmn). In addition, in vivo, the ratio between external work (EW) and myocardial oxygen consumption (MVO2) to obtain myocardial external efficiency (MEE) was determined in 28 pre-hypertrophic mutation carriers (14 MYBPC3mut and 14 MYH7mut) and 14 healthy controls using [(11)C]-acetate positron emission tomography and cardiovascular magnetic resonance imaging. Tension cost (TC), i.e. ATPase activity during force development, was higher in MYBPC3mut and MYH7mut compared with HCMsmn at saturating [Ca(2+)]. TC was also significantly higher in MYH7mut at submaximal, more physiological [Ca(2+)]. EW was significantly lower in both mutation carrier groups, while MVO2 did not differ. MEE was significantly lower in both mutation carrier groups compared with controls, showing the lowest efficiency in MYH7 mutation carriers., Conclusion: We provide direct evidence that sarcomere mutations perturb the energetic cost of cardiac contraction. Gene-specific severity of cardiac abnormalities may underlie differences in disease onset and suggests that early initiation of metabolic treatment may be beneficial, in particular, in MYH7 mutation carriers., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

Background: Hypertrophic cardiomyopathy (HC) is the most prevalent genetic cardiac disease caused by a mutation in sarcomeres, Z-disks, or calcium-handling genes and is characterized by unexplained left ventricular hypertrophy. The aim of this study was to determine the genetic profile of Brazilian patients with HC and correlate the genotype with the phenotype., Methods: We included 268 index patients from São Paulo city and 3 other cities in Brazil and extracted their DNA from whole blood. We amplified the coding sequencing of MYH7, MYBPC3, and TNNT2 genes and sequenced them with an automatic sequencer., Results: We identified causal mutations in 131 patients (48.8%). Seventy-eight (59.5%) were in the MYH7 gene, 50 (38.2%) in the MYBPC3 gene, and 3 (2.3%) in the TNNT2 gene. We identified 69 mutations, 24 not previously described. Patients with an identified mutation were younger at diagnosis and at current age, had a higher mean heart rate and higher nonsustained ventricular tachycardia frequency compared with those without a mutation. Patients with MYH7 gene mutations had a larger left atrium and higher frequency of atrial fibrillation than did patients with MYBPC3 gene mutations., Conclusion: The presence of a mutation in one of the genes suggests a worse prognosis. Mutations in the MYH7 gene, rather than in the MYBPC3 gene, were also related to a worse prognosis. This is the first work characterizing HC molecular epidemiology in the Brazilian population for the 3 most important genes., (© 2013.)

Genetic mutations account for a significant percentage of cardiomyopathies, which are a leading cause of congestive heart failure. In hypertrophic cardiomyopathy (HCM), cardiac output is limited by the thickened myocardium through impaired filling and outflow. Mutations in the genes encoding the thick filament components myosin heavy chain and myosin binding protein C (MYH7 and MYBPC3) together explain 75% of inherited HCMs, leading to the observation that HCM is a disease of the sarcomere. Many mutations are "private" or rare variants, often unique to families. In contrast, dilated cardiomyopathy (DCM) is far more genetically heterogeneous, with mutations in genes encoding cytoskeletal, nucleoskeletal, mitochondrial, and calcium-handling proteins. DCM is characterized by enlarged ventricular dimensions and impaired systolic and diastolic function. Private mutations account for most DCMs, with few hotspots or recurring mutations. More than 50 single genes are linked to inherited DCM, including many genes that also link to HCM. Relatively few clinical clues guide the diagnosis of inherited DCM, but emerging evidence supports the use of genetic testing to identify those patients at risk for faster disease progression, congestive heart failure, and arrhythmia.

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

Cardiac myosin binding protein-C (cMyBP-C) plays a role in sarcomeric structure and stability, as well as modulating heart muscle contraction. The 150 kDa full-length (FL) cMyBP-C has been shown to undergo proteolytic cleavage during ischemia-reperfusion injury, producing an N-terminal 40 kDa fragment (mass 29 kDa) that is predominantly associated with post-ischemic contractile dysfunction. Thus far, the pathogenic properties of such truncated cMyBP-C proteins have not been elucidated. In the present study, we hypothesized that the presence of these 40 kDa fragments is toxic to cardiomyocytes, compared to the 110 kDa C-terminal fragment and FL cMyBP-C. To test this hypothesis, we infected neonatal rat ventricular cardiomyocytes and adult rabbit ventricular cardiomyocytes with adenoviruses expressing the FL, 110 and 40 kDa fragments of cMyBP-C, and measured cytotoxicity, Ca(2+) transients, contractility, and protein-protein interactions. Here we show that expression of 40 kDa fragments in neonatal rat ventricular cardiomyocytes significantly increases LDH release and caspase 3 activity, significantly reduces cell viability, and impairs Ca(2+) handling. Adult cardiomyocytes expressing 40 kDa fragments exhibited similar impairment of Ca(2+) handling along with a significant reduction of sarcomere length shortening, relaxation velocity, and contraction velocity. Pull-down assays using recombinant proteins showed that the 40 kDa fragment binds significantly to sarcomeric actin, comparable to C0-C2 domains. In addition, we discovered several acetylation sites within the 40 kDa fragment that could potentially affect actomyosin function. Altogether, our data demonstrate that the 40 kDa cleavage fragments of cMyBP-C are toxic to cardiomyocytes and significantly impair contractility and Ca(2+) handling via inhibition of actomyosin function. By elucidating the deleterious effects of endogenously expressed cMyBP-C N-terminal fragments on sarcomere function, these data contribute to the understanding of contractile dysfunction following myocardial injury.

β-Adrenergic stimulation of the mammalian heart increases heart rate, the strength of contraction as well as the kinetics of force generation and relaxation. These effects are due to the phosphorylation of select membrane and thin filament proteins by cAMP-activated protein kinase (PKA). At the level of the sarcomere, it is typically the phosphorylation of cardiac myosin binding protein C (cMyBP-C) and cardiac troponin I (cTnI) that is responsible for the change in the kinetics of contraction and relaxation. Trout cTnI (ScTnI) lacks two critical PKA targets within the N-terminus of the protein that, when phosphorylated in mammalian cTnI, cause a reduction in myofilament Ca(2+) affinity. To determine what role the contractile element plays in the response of the trout heart to β-adrenergic stimulation, we characterized the influence of PKA treatment on the Ca(2+) activation of skinned preparations dissected from ventricular trabeculae. In these experiments, isometric force generation and the rate of force development were measured over a range of Ca(2+) concentrations. The results demonstrate that PKA treatment does not influence the Ca(2+) sensitivity of force generation but it decreases maximum force generation by 25% and the rate of force re-development at maximal activation by 46%. Analysis of the trabeculae preparations for phosphoproteins revealed that PKA treatment phosphorylated myosin light chain 2 but not cTnI or cMyBP-C. These results indicate that the function of the trout cardiac contractile element is altered by PKA phosphorylation but in a manner different from that in mammalian heart.

Rationale and Objective: The M-band represents a transverse structure in the center of the sarcomeric A-band and provides an anchor for the myosin-containing thick filaments. In contrast to other sarcomeric structures, eg, the Z-disc, only few M-band-specific proteins have been identified to date, and its exact molecular composition remains unclear., Methods and Results: Using a bioinformatic approach to identify novel heart- and muscle-specific genes, we found a leucine rich protein, myomasp (Myosin-interacting, M-band-associated stress-responsive protein)/LRRC39. RT-PCR and Northern and Western blot analyses confirmed a cardiac-enriched expression pattern, and immunolocalization of myomasp revealed a strong and specific signal at the sarcomeric M-band. Yeast 2-hybrid screens, as well as coimmunoprecipitation experiments, identified the C terminus of myosin heavy chain (MYH)7 as an interaction partner for myomasp. Knockdown of myomasp in neonatal rat ventricular myocytes (NRVCMs) led to a significant upregulation of the stretch-sensitive genes GDF-15 and BNP. Conversely, the expression of MYH7 and the M-band proteins myomesin-1 and -2 was found to be markedly reduced. Mechanistically, knockdown of myomasp in NRVCM led to a dose-dependent suppression of serum response factor-dependent gene expression, consistent with earlier observations linking the M-band to serum response factor-mediated signaling. Finally, downregulation of myomasp/LRRC39 in spontaneously beating engineered heart tissue constructs resulted in significantly lower force generation and reduced fractional shortening. Likewise, knockdown of the myomasp/LRRC39 ortholog in zebrafish resulted in severely impaired heart function and cardiomyopathy in vivo., Conclusions: These findings reveal myomasp as a previously unrecognized component of an M-band-associated signaling pathway that regulates cardiomyocyte gene expression in response to biomechanical stress.

Modulatory role of whole cardiac myosin binding protein-C (сMyBP-C) in regulation of cardiac muscle contractility was studied in the in vitro motility assay with rabbit cardiac myosin as a motor protein. The effects of cMyBP-C on the interaction of cardiac myosin with regulated thin filament were tested in both in vitro motility and ATPase assays. We demonstrate that the addition of cMyBP-C increases calcium regulated Mg-ATPase activity of cardiac myosin at submaximal calcium. The Hill coefficient for 'pCa-velocity' relation in the in vitro motility assay decreased and the calcium sensitivity increased when сMyBP-C was added. Results of our experiments testifies in favor of the hypothesis that сMyBP-C slows down cross-bridge kinetics when binding to actin., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Mutations in the thick filament associated protein cardiac myosin binding protein-C (cMyBP-C) are a major cause of familial hypertrophic cardiomyopathy. Although cMyBP-C is thought to play both a structural and a regulatory role in the contraction of cardiac muscle, detailed information about the role of this protein in stability of the thick filament and maintenance of the ordered helical arrangement of the myosin cross-bridges is limited. To address these questions, the structure of myosin thick filaments isolated from the hearts of wild-type mice containing cMyBP-C (cMyBP-C(+/+)) were compared to those of cMyBP-C knockout mice lacking this protein (cMyBp-C(-/-)). The filaments from the knockout mice hearts lacking cMyBP-C are stable and similar in length and appearance to filaments from the wild-type mice hearts containing cMyBP-C. Both wild-type and many of the cMyBP-C(-/-) filaments display a distinct 43 nm periodicity. Fourier transforms of electron microscope images typically show helical layer lines to the sixth layer line, confirming the well-ordered arrangement of the cross-bridges in both sets of filaments. However, the "forbidden" meridional reflections, thought to derive from a perturbation from helical symmetry in the wild-type filament, are weaker or absent in the transforms of the cMyBP-C(-/-) myocardial thick filaments. In addition, the cross-bridge array in the absence of cMyBP-C appears more easily disordered.

Although absence or abnormality of cardiac myosin binding protein C (cMyBP-C) produces serious structural and functional abnormalities of the heart, function of the protein itself is not clearly understood, and the cause of the abnormalities, unidentified. Here we report that a major function of cMyBP-C may be regulating the stability of the myosin-containing contractile filaments through phosphorylation of cMyBP-C. Antibodies were raised against three different regions of cMyBP-C to detect changes in structure within the molecule, and loss of myosin heavy chain was used to monitor degradation of the thick filament. Results from Western blotting and polyacrylamide gel electrophoresis indicate that cMyBP-C can exist in two different forms that produce, respectively, stable and unstable thick filaments. The stable form has well-ordered myosin heads and requires phosphorylation of the cMyBP-C. The unstable form has disordered myosin heads. In tissue with intact cardiac cells, the unstable unphosphorylated cMyBP-C is more easily proteolyzed, causing thick filaments first to release cMyBP-C and/or its proteolytic peptides and then myosin. Filaments deficient in cMyBP-C are fragmented by shear force well tolerated by the stable form. We hypothesize that modulation of filament stability can be coupled at the molecular level with the strength of contraction by the sensitivity of each to the concentration of calcium ions.

Congenital heart disease (CHD) is a major clinical manifestation of Down syndrome (DS). We recently showed that chimeric mice containing a human chromosome 21 (Chr 21) exhibited phenotypic traits of DS, including CHD. Our previous study showed that myosin light chain-2a (mlc2a) expression was reduced in the hearts of chimeric mice and DS patients. We found that phosphatidylethanolamine binding protein (PEBP) was also downregulated in Chr 21 chimeras in this study. As mlc2a is involved in heart morphogenesis, and PEBP controls the proliferation and differentiation of different cell types, these genes are candidates for involvement in DS-CHD. The DS-CHD candidate region has been suggested to span between PFKL and D21S3, which is the STS marker near the ETS2 loci. To identify gene(s) or a gene cluster on Chr 21 responsible for the downregulation of mlc2a and PEBP, we fragmented Chr 21 at the EST2 loci, by telomere-directed chromosome truncation in homologous recombination-proficient chicken DT40 cells. The modified Chr 21 was transferred to mouse ES cells by microcell-mediated chromosome transfer (MMCT), via CHO cells. We used ES cell lines retaining the Chr 21 truncated at the ETS2 locus (Chr 21E) to produce chimeric mice and compared overall protein expression patterns in hearts of the chimeras containing the intact and the fragmented Chr 21 by two-dimensional electrophoresis. While mouse mlc2a and PEBP expression was downregulated in the chimeras containing the intact Chr 21, the expression was not affected in the Chr 21E chimeras. Therefore, we suggest that Chr 21 gene(s) distal from the ETS2 locus reduce mouse mlc2a and PEBP expression in DS model mice and DS. Thus, this chromosome engineering technology is a useful tool for identification or mapping of genes that contribute to the DS phenotypes.