Mutations In Myosin Binding Protein Research Articles

Nonsense mediated decay factor UPF3B is associated with cMyBP-C haploinsufficiency in hypertrophic cardiomyopathy patients

Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited cardiac disease. Up to 40% of cases are associated with heterozygous mutations in myosin binding protein C (cMyBP-C, MYBPC3). Most of these mutations lead to premature termination codons (PTC) and patients show reduction of functional cMyBP-C. This so-called haploinsufficiency most likely contributes to disease development.We analyzed mechanisms underlying haploinsufficiency using cardiac tissue from HCM-patients with truncation mutations in MYBPC3 (MYBPC3trunc). We compared transcriptional activity, mRNA and protein expression to donor controls. To differentiate between HCM-specific and general hypertrophy-induced mechanisms we used patients with left ventricular hypertrophy due to aortic stenosis (AS) as an additional control. We show that cMyBP-C haploinsufficiency starts at the mRNA level, despite hypertrophy-induced increased transcriptional activity. Gene set enrichment analysis (GSEA) of RNA-sequencing data revealed an increased expression of NMD-components. Among them, Up-frameshift protein UPF3B, a regulator of NMD was upregulated in MYBPC3trunc patients and not in AS-patients. Strikingly, we show that in sarcomeres UPF3B but not UPF1 and UPF2 are localized to the Z-discs, the presumed location of sarcomeric protein translation. Our data suggest that cMyBP-C haploinsufficiency in HCM-patients is established by UPF3B-dependent NMD during the initial translation round at the Z-disc.

Novel MYBPC3 Mutations in Indian Population with Cardiomyopathies.

Mutations in Myosin Binding Protein C (MYBPC3) are one of the most frequent causes of cardiomyopathies in the world, but not much data are available in India. We carried out targeted direct sequencing of MYBPC3in 115 hypertrophic (HCM) and 127 dilated (DCM) cardiomyopathies against 197 ethnically matched healthy controls from India. We detected 34 single nucleotide variations in MYBPC3, of which 19 were novel. We found a splice site mutation [(IVS6+2T) T>G] and 16 missense mutations in Indian cardiomyopathies [5 in HCM; E258K, T262S, H287L, R408M, V483A: 4 in DCM; T146N, V321L, A392T, E393K and 7 in both HCM and DCM; L104M, V158M, S236G, R272C, T290A, G522E, A626V], but those were absent in 197 normal healthy controls. Interestingly, we found 7 out of 16 missense mutations (V158M, E258K, R272C, A392T, V483A, G522E, and A626V) in MYBPC3 were altering the evolutionarily conserved native amino acids, accounted for 8.7% and 6.3% in HCM and DCM, respectively. The bioinformatic tools predicted that those 7 missense mutations were pathogenic. Moreover, the co-segregation of those 7 mutations in families further confirmed their pathogenicity. Remarkably, we also identified compound mutations within the MYBPC3 gene of 6 cardiomyopathy patients (5%) with more severe disease phenotype; of which, 3 were HCM (2.6%) [(1. K244K + E258K + (IVS6+2T) T>G); (2. L104M + G522E + A626V); (3. P186P + G522E + A626V]; and 3 were DCM (2.4%) [(1. 5'UTR + A392T; 2. V158M+G522E; and 3.V158M + T262T + A626V]. The present comprehensive study on MYBPC3 has revealed both single and compound mutations in MYBPC3 and their association with disease in Indian Population with Cardiomyopathies. Our findings may perhaps help in initiating diagnostic strategies and eventually recognizing the targets for therapeutic interventions.

Abstract P1121: Substrate Rigidity And Mybpc3 Genotype Regulate Development Of Hypertrophic Cardiomyopathy Structural And Functional Phenotypes In Human Ipsc-derived Micro-heart Muscle Arrays

Introduction: We hypothesized that mechanical loading is a key factor in the early stages of hypertrophic remodeling associated with pathogenesis of hypertrophic cardiomyopathy (HCM). We tested this hypothesis in vitro with human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) bearing heterozygous loss-of-function mutations in Myosin Binding Protein C (MYBPC3). Cardiomyocytes were cultured in engineered micro-heart muscle arrays that were loaded by changing substrate rigidity. Methods: Isogenic control and MYBPC3 +/- hiPSC-CM were seeded into dogbone-shaped molds to form micro-heart muscle atop silicone rubber substrates with rigidity levels ranging from levels of embryonic (0.4 kPa) to fibrotic adult heart (114 kPa). Spontaneously contracting micro-tissues formed within 48 hours. 10 days after formation, tissues were monitored for action potentials (voltage sensitive dye, BeRST-1), Ca 2+ transients (GCaMP6f reporter), and contractility (substrate deflection). Cell and tissue architecture were assessed in 8 μm cryosections, and RNA transcription via qRT-PCR. Results: MYBPC3 +/- and control cardiomyocytes had similar physiology and morphology in 2D monolayers. However, MYBPC3 +/- tissues exhibited marked changes in Ca 2+ handling, which were exacerbated when substrate stiffness increased to the level of healthy heart muscle (15 kPa). This was concurrent with hypertrophy of MYBPC3 +/- cardiomyocytes at 15 kPa. MYBPC3 +/- tissue Ca 2+ handling abnormalities were linked to enhanced Ca 2+ intake rather than SERCA deficiency or excessive myofilament buffering. MYBPC3 +/- tissues exhibited mis-regulation of cardiac troponins on the RNA and protein localization levels, which may underlie the observed structural and physiological changes. Conclusions: Changes in the rigidity of the substrate, mimicking changes in heart muscle caused by blood pressure, pushed hiPSC-cardiac micro-tissues with MYBPC3 mutations to recapitulate key structural and electrophysiological characteristics of HCM. In contrast, control tissues exhibited hypertrophy and abnormal calcium handling, but only at extreme values of substrate stiffness. This suggests that HCM mutations may exaggerate load-dependent hypertrophic remodeling.

Contractility and Calcium Transient Maturation in the Human iPSC-Derived Cardiac Microfibers.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are considered immature in the sarcomere organization, contractile machinery, calcium transient, and transcriptome profile, which prevent them from further applications in modeling and studying cardiac development and disease. To improve the maturity of hiPSC-CMs, here, we engineered the hiPSC-CMs into cardiac microfibers (iCMFs) by a stencil-based micropatterning method, which enables the hiPSC-CMs to be aligned in an end-to-end connection for prolonged culture on the hydrogel of physiological stiffness. A series of characterization approaches were performed to evaluate the maturation in iCMFs on both structural and functional levels, including immunohistochemistry, calcium transient, reverse-transcription quantitative PCR, cardiac contractility, and electrical pacing analysis. Our results demonstrate an improved cardiac maturation of hiPSC-CMs in iCMFs compared to micropatterned or random single hiPSC-CMs and hiPSC-CMs in a random cluster at the same cell number of iCMFs. We found an increased sarcomere length, better regularity and alignment of sarcomeres, enhanced contractility, matured calcium transient, and T-tubule formation and improved adherens junction and gap junction formation. The hiPSC-CMs in iCMFs showed a robust calcium cycling in response to the programmed and continuous electrical pacing from 0.5 to 7 Hz. Moreover, we generated the iCMFs with hiPSC-CMs with mutations in myosin-binding protein C (MYBPC3) to have a proof-of-concept of iCMFs in modeling cardiac hypertrophic phenotype. These findings suggest that the multipatterned iCMF connection of hiPSC-CMs boosts the cardiac maturation structurally and functionally, which will reveal the full potential of the application of hiPSC-CM models in disease modeling of cardiomyopathy and cardiac regenerative medicine.

Alterations of sarcomeric stoichiometry by nonsense mediated decay of MYBPC3-mRNA in hypertrophic cardiomyopathy patients with truncation mutations

Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited cardiac disease with up to 40% of mutation-positive cases associated with heterozygous mutations in myosin binding protein C (cMyBP-C, MYBPC3). Most mutations in MYBPC3 lead to premature termination codons. This presumably induces nonsense mediated decay (NMD) of mutated MYBPC3-mRNA, resulting in reduction of functional cMyBP-C. This so-called haploinsufficiency disrupts the physiological stoichiometry of sarcomeric proteins and most likely contributes to disease development.

Heat shock proteins and small nucleolar RNAs are dysregulated in a Drosophila model for feline hypertrophic cardiomyopathy.

In cats, mutations in myosin binding protein C (encoded by the MYBPC3 gene) have been associated with hypertrophic cardiomyopathy (HCM). However, the molecular mechanisms linking these mutations to HCM remain unknown. Here, we establish Drosophila melanogaster as a model to understand this connection by generating flies harboring MYBPC3 missense mutations (A31P and R820W) associated with feline HCM. The A31P and R820W flies displayed cardiovascular defects in their heart rates and exercise endurance. We used RNA-seq to determine which processes are misregulated in the presence of mutant MYBPC3 alleles. Transcriptome analysis revealed significant downregulation of genes encoding small nucleolar RNA (snoRNAs) in exercised female flies harboring the mutant alleles compared to flies that harbor the wild-type allele. Other processes that were affected included the unfolded protein response and immune/defense responses. These data show that mutant MYBPC3 proteins have widespread effects on the transcriptome of co-regulated genes. Transcriptionally differentially expressed genes are also candidate genes for future evaluation as genetic modifiers of HCM as well as candidate genes for genotype by exercise environment interaction effects on the manifestation of HCM; in cats as well as humans.

Two novel mutations in myosin binding protein C slow causing distal arthrogryposis type 2 in two large Han Chinese families may suggest important functional role of immunoglobulin domain C2.

Distal arthrogryposes (DAs) are a group of disorders that mainly involve the distal parts of the limbs and at least ten different DAs have been described to date. DAs are mostly described as autosomal dominant disorders with variable expressivity and incomplete penetrance, but recently autosomal recessive pattern was reported in distal arthrogryposis type 5D. Mutations in the contractile genes are found in about 50% of all DA patients. Of these genes, mutations in the gene encoding myosin binding protein C slow MYBPC1 were recently identified in two families with distal arthrogryposis type 1B. Here, we described two large Chinese families with autosomal dominant distal arthrogryposis type 2(DA2) with incomplete penetrance and variable expressivity. Some unique overextension contractures of the lower limbs and some distinctive facial features were present in our DA2 pedigrees. We performed follow-up DNA sequencing after linkage mapping and first identified two novel MYBPC1 mutations (c.1075G>A [p.E359K] and c.956C>T [p.P319L]) responsible for these Chinese DA2 families of which one introduced by germline mosacism. Each mutation was found to cosegregate with the DA2 phenotype in each family but not in population controls. Both substitutions occur within C2 immunoglobulin domain, which together with C1 and the M motif constitute the binding site for the S2 subfragment of myosin. Our results expand the phenotypic spectrum of MYBPC1-related arthrogryposis multiplex congenita (AMC). We also proposed the possible molecular mechanisms that may underlie the pathogenesis of DA2 myopathy associated with these two substitutions in MYBPC1.

A New Way to Examine the Function of Mutant MYBPC3 Expression in Cardiomyocytes of Mice

Autosomal dominant mutations in myosin binding protein C (MYBPC3) account for up to 30% of myofibrillar mutations causing Hypertrophic Cardiomyopathy (HCM). We used SPontaneous Oscillatory Contractions (SPOC) to compare the performance of isolated cardiomyocytes from heterozygous MYBPC (+/-) and homozygous MYBPC (-/-) and wild type (+/+) mice. Our aim is to identify changes in their contractile parameters. SPOC is a physiological state that is intermediate to full contraction and relaxation. The stable auto-oscillatory properties of SPOC are well suited to precise measurements of contraction and relaxation.Preliminary evaluations reveal there is: (1) a progressive prolongation in both the relative lengthening and shortening periods from MYBPC+/+ to MYBPC+/- and MYBPC-/-; (2) MYBPC+/- exhibits faster rates of lengthening than MYBPC+/+; (3) MYBPC-/- displays significantly depressed rates of shortening compared to MYBPC+/- and MYBPC+/+. These findings suggest significant systolic dysfunction in MYBPC-/- associated with severe hypertrophic remodelling. Perhaps, more importantly MYBPC+/- exhibits diastolic dysfunction consistent with previous reports examining SPOC in human HCM at the 56th Biophysical Society Meeting. We conclude that SPOC can objectively assess the functional state of heart muscle fibres in this mouse model.View Large Image | View Hi-Res Image | Download PowerPoint Slide

073 Evaluation of clinical markers of early disease expression and the ability to predict genotype in families with HCM and mutations in cardiac myosin binding protein C

IntroductionFamilial evaluation for the presence of left ventricular hypertrophy (LVH) is an important part of management in hypertrophic cardiomyopathy. However due to incomplete penetrance, the presence of LVH does not...

Sarcomeric genotyping in hypertrophic cardiomyopathy.

To pool results from studies of patients with hypertrophic cardiomyopathy (HCM) to elucidate important phenotypic differences among genotypes. Data published from November 1998 through November 2004 were gathered and compared from unrelated study population genotyping studies from the Mayo Clinic (Rochester, Minn), Harvard Medical School (Boston, Mass), France, Germany, Sweden, Finland, and Spain. Standard statistical analysis techniques were used to pool and compare data across genotypes with respect to frequency of mutations, age at diagnosis, and degree of hypertrophy (left ventricular wall thickness). The French study population harbored the highest frequency of mutations (61%), followed by the Mayo Clinic (38%), Harvard Medical School (36%), and Swedish (30%) study populations. For every study population, mutations in myosin binding protein C (MYBPC3) were the most common cause of HCM. Patients with a family history of HCM had mutations more frequently than those without. This pooled analysis revealed no statistically significant differences in left ventricular wall thickness or in mean age at diagnosis across all genotypes. Differentiation of sarcomeric genotypes, such as MYBPC3-HCM and MYH7-HCM, is not possible on the basis of currently reported phenotypic data. A myriad of genetic and/or environmental modifiers in addition to the primary disease-causing genetic substrate must play an important role in determining a patient's particular phenotype.

Tissue Doppler Imaging in Maine Coon Cats with a Mutation of Myosin Binding Protein C with or without Hypertrophy

Background: The cardiac myosin binding protein C gene is mutated in Maine Coon (MC) cats with familial hypertrophic cardiomyopathy.Hypotheses: Early diastolic mitral annular velocity is incrementally reduced from normal cats to MC cats with only an abnormal genotype to MC cats with abnormal genotype and hypertrophy.Animals:Group 1 consisted of 6 normal domestic shorthair cats, group 2 of 6 MC cats with abnormal genotype but no hypertrophy, and group 3 of 15 MC cats with hypertrophy and abnormal genotype.Methods:The genotype and echocardiographic phenotype of cats were determined, and the cats were divided into the 3 groups. Tissue Doppler imaging (TDI) of the lateral mitral annulus from the left apical 4‐chamber view was performed. Five nonconsecutive measurements of early diastolic mitral annular velocity (EM) or summated early and late diastolic velocity (EAsum) and heart rate were averaged.Results:There was an ordered reduction in Em‐EAsum as group number increased (group 1, range 9.7–14.7 cm/s; group 2, range 7.5–13.2 cm/s; group 3, range 4.5–14.1 cm/s; P= .001). Using the lower prediction limit for normal Em‐EAsum, the proportion of cats with normal Em‐EAsum decreased as the group number increased (P= .001). However, Em‐EAsum was reduced in only 3 of 6 cats in group 2.Conclusion: The incremental reduction of Em‐EAsum as group severity increased indicates that diastolic dysfunction is an early abnormality that occurs before hypertrophy development. TDI measurement of Em or EAsum of the lateral mitral annulus is an insensitive screening test for identification of phenotypically normal, genotypically affected cats.

Tissue Doppler Imaging in Maine Coon Cats with a Mutation of Myosin Binding Protein C with or without Hypertrophy

The cardiac myosin binding protein C gene is mutated in Maine Coon (MC) cats with familial hypertrophic cardiomyopathy. Early diastolic mitral annular velocity is incrementally reduced from normal cats to MC cats with only an abnormal genotype to MC cats with abnormal genotype and hypertrophy. Group 1 consisted of 6 normal domestic shorthair cats, group 2 of 6 MC cats with abnormal genotype but no hypertrophy, and group 3 of 15 MC cats with hypertrophy and abnormal genotype. The genotype and echocardiographic phenotype of cats were determined, and the cats were divided into the 3 groups. Tissue Doppler imaging (TDI) of the lateral mitral annulus from the left apical 4-chamber view was performed. Five nonconsecutive measurements of early diastolic mitral annular velocity (EM) or summated early and late diastolic velocity (EAsum) and heart rate were averaged. There was an ordered reduction in Em-EAsum as group number increased (group 1, range 9.7-14.7 cm/s; group 2, range 7.5-13.2 cm/s; group 3, range 4.5-14.1 cm/s; P = .001). Using the lower prediction limit for normal Em-EAsum, the proportion of cats with normal Em-EAsum decreased as the group number increased (P = .001). However, Em-EAsum was reduced in only 3 of 6 cats in group 2. The incremental reduction of Em-EAsum as group severity increased indicates that diastolic dysfunction is an early abnormality that occurs before hypertrophy development. TDI measurement of Em or EAsum of the lateral mitral annulus is an insensitive screening test for identification of phenotypically normal, genotypically affected cats.

Heart muscle disease

<h2>Abstract</h2> Primary cardiomyopathies are heart muscle diseases intrinsic to the myocardium. This group includes dilated cardiomyopathy (DCM), arrhythmogenic cardiomyopathy (ARVC), hypertrophic cardiomyopathy (HCM), restrictive cardiomyopathy (RCM) and unclassified cardiomyopathy. The cardiomyopathies may be classified pathophysiologically and display unique pathological and clinical features. ARVC is characterized by fibroadipose substitution of right ventricular myocardium and a high risk of sudden cardiac death. HCM may be symmetrical or asymmetrical and histologically is associated with widespread myocyte disarray. The clinical presentation and phenotype varies according to which type of myofibrillary gene mutation is responsible for disease. For example, mutations in troponin T carry a poor prognosis and a high risk of sudden cardiac death, whilst mutations in myosin binding protein C are associated with mild disease and onset in middle-age or late adult life. DCM is characterized by an increase in ventricular mass and cavity dilatation with or without thrombi in the atrial appendages. The histological features of DCM are often non-specific but myofibrillary loss, when present, is a helpful diagnostic feature. DCM is the commonest type of cardiomyopathy and most cases are idiopathic. RCM is the least common type of cardiomyopathy and includes diseases that ‘stiffen' the myocardium by fibrosis or infiltration. This group includes Loeffler's endocarditis, tropical endomyocardial fibrosis as well as cardiac amyloidosis and storage disorders. The inflammatory cardiomyopathies include myocarditis, idiopathic giant cell myocarditis and sarcoidosis. Myocarditis has an acute onset and is often accompanied by febrile illness, whereas sarcoidosis has an insidious onset and often presents with heart block.

Sarcomeric Genotyping in Hypertrophic Cardiomyopathy

To pool results from studies of patients with hypertrophic cardiomyopathy (HCM) to elucidate important phenotypic differences among genotypes.Data published from November 1998 through November 2004 were gathered and compared from unrelated study population genotyping studies from the Mayo Clinic (Rochester, Minn), Harvard Medical School (Boston, Mass), France, Germany, Sweden, Finland, and Spain. Standard statistical analysis techniques were used to pool and compare data across genotypes with respect to frequency of mutations, age at diagnosis, and degree of hypertrophy (left ventricular wall thickness).The French study population harbored the highest frequency of mutations (61%), followed by the Mayo Clinic (38%), Harvard Medical School (36%), and Swedish (30%) study populations. For every study population, mutations in myosin binding protein C (MYBPC3) were the most common cause of HCM. Patients with a family history of HCM had mutations more frequently than those without. This pooled analysis revealed no statistically significant differences in left ventricular wall thickness or in mean age at diagnosis across all genotypes.Differentiation of sarcomeric genotypes, such as MYBPC3-HCM and MYH7-HCM, is not possible on the basis of currently reported phenotypic data. A myriad of genetic and/or environmental modifiers in addition to the primary disease-causing genetic substrate must play an important role in determining a patient's particular phenotype.

Myosin binding protein C mutations and compound heterozygosity in hypertrophic cardiomyopathy

We sought to determine the frequency and phenotype of mutations in myosin binding protein C (MYBPC3) in a large outpatient cohort of patients with hypertrophic cardiomyopathy (HCM) seen at our tertiary referral center. Mutations in MYBPC3 are one of the most frequent genetic causes of HCM and have been associated with variable onset of disease and prognosis. However, the frequency of mutations and associated clinical presentation have not been established in a large, unrelated cohort of patients. Using deoxyribonucleic acid from 389 unrelated patients with HCM, each protein coding exon of MYBPC3 was analyzed for mutations by polymerase chain reaction, denaturing high-performance liquid chromatography, and direct deoxyribonucleic acid sequencing. Clinical data were extracted from patient records blinded to patient genotype. Of 389 patients with HCM, 71 (18%) had mutations in MYBPC3. In all, 46 mutations were identified, 33 of which were novel (72%). Patients with MYBPC3 mutations did not differ significantly from patients with thick filament-HCM, thin filament-HCM, or genotype-negative HCM with respect to age at diagnosis, degree of hypertrophy, incidence of myectomy, or family history of HCM or sudden death. Patients with multiple mutations (n = 10, 2.6%) had the most severe disease presentation. This study defines the frequency and associated phenotype for MYBPC3 and/or multiple mutations in HCM in the largest cohort to date. In this cohort, unrelated patients with MYBPC3-HCM virtually mimicked the phenotype of those with mutations in the beta-myosin heavy chain. Patients with multiple mutations had the most severe phenotype.

Hypertrophic cardiomyopathy linked to homozygosity for a new mutation in the myosin-binding protein C gene (A627V) suggests a dosage effect

Mutations in the cardiac myosin-binding protein C gene (MYBPC3) are responsible for up to 50% of familial cases with hypertrophic cardiomyopathy (HC). Compared to patients with mutations in other sarcomeric genes, patients with MYBPC3 mutations would have a milder form of the disease, with a lower incidence of sudden cardiac death. Because most of the mutations have been found in only one family, it is currently difficult to establish a correlation between a particular mutation and the HC phenotype. The aim of our study was to contribute to understanding of the role of MYBPC3 mutations in HC. We analysed the MYBPC3 exons and intron flanking regions in 10 patients from 10 families with at least two HC cases. After direct sequencing of polymerase chain reaction (PCR) fragments, we found three new mutations in three families (V771M, V342D, and A627V). These changes affected evolutionary conserved amino acids and were not found in 100 healthy controls. The Ala 627>Val was found homozygous in a 47-year-old patient with a severe form of HC, while his mother and a nephew were heterozygous carriers and asymptomatic. This fact suggests a dosage effect for mutations at the MYPBC3 gene.

Hypertrophic Cardiomyopathy

ypertrophic cardiomyopathy (HCM) is a fascinatingdisease that has intrigued and challenged cardiolo-gists for decades. Initially a diagnosis made bymaster clinicians adept at translating bedside dynamic aus-cultation into hemodynamic pathophysiology, HCM contin-ues to challenge the frontiers of diagnosis and technology.The development of 2D and Doppler echocardiography pro-vided the greatest increment in understanding the complexityof HCM. While clinicians at large became skilled at thebedside detection of dynamic left ventricular outflow obstruc-tion, echocardiography revealed that many more patients hadunexplained left ventricular hypertrophy (LVH) consistentwith HCM but without evidence of obstruction. In fact, thenonobstructive, “silent” forms are more frequent than theobstructive, “noisy” variants. The pattern and severity ofLVH are now appreciated as heterogeneous and diastolicdysfunction as highly prevalent.