Abstract 3412: Molecular and Cellular Characterization of Splice Site Mutations in MYBPC3 -encoded Myosin Binding Protein C

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Jeanne L Theis ◽  
J M Bos ◽  
Steve R Ommen ◽  
Michael J Ackerman
Keyword(s):  
Splice Site ◽  
Protein C ◽  
Binding Protein ◽  
Donor Site ◽  
Splice Donor Site ◽  
Splice Donor ◽  
Myosin Binding Protein ◽  
Myosin Binding Protein C ◽  
Alternatively Spliced ◽  
Myosin Binding

Background: Mutations in MYBPC3 -encoded myosin binding protein C underlie the most common genotype in hypertrophic cardiomyopathy (HCM). Compared to most HCM-associated mutations which are primarily missense mutations, MYBPC3-HCM is often caused by insertions, deletions, nonsense mutations or mutations involving the canonical splice site. However, the effects of most mutations that prematurely truncate MYBPC3 are unknown. Access to cardiac specific mRNA and tissue has permitted a molecular and cellular elucidation of the consequences of two splice site mutations. Methods: Mutational analysis of MYBPC3 revealed 2 HCM-susceptibility mutations involving the splice donor sites of exons 7 and 30. Reverse transcription was performed on cDNA generated from RNA extracted from cardiac tissue obtained following surgical myectomy. Western blot analysis and immunohistochemistry (IHC) of the myofilaments were performed to further assess the impact of the splice-site mutations. Results: Both splice donor mutations produced abnormal RNA splicing of MYBPC3. The c.821+1 g>a mutation in the splice donor site of exon 7 generated two alternatively-spliced mutant transcripts resulting in two frame-shifted, premature truncations (H257 fs/37 and H257 fs/15). The c.3330+2 t>g mutation in exon 30’s splice donor site produced a single alternatively-spliced mutant transcript that translated into a frame-shifted premature truncation (V1063 fs/37). Expression levels of wild type myosin binding protein C were decreased in the myectomy specimen from the patient with the exon 7 splice donor site mutation. By IHC, the spatial organization of myosin binding protein C was disrupted severely in both patients. Conclusions: This study provides the first molecular and cellular characterization of HCM-causing mutations involving canonical splice-site motifs within the intron. Loss of function of the splice site resulted in exon skipping and generation of frame-shifted and prematurely truncated myosin binding protein C and disruption of the distribution and organization of myosin binding protein C in the heart tissue. This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).

Biological variation of cardiac myosin-binding protein C in healthy individuals

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Bashir Alaour ◽  
Torbjørn Omland ◽  
Janniche Torsvik ◽  
Thomas E. Kaier ◽  
Marit S. Sylte ◽  
...  
Keyword(s):  
Risk Stratification ◽  
Protein C ◽  
Myocardial Injury ◽  
Binding Protein ◽  
Biological Variation ◽  
Cardiac Myosin ◽  
Analytical Quality ◽  
Myosin Binding Protein ◽  
Myosin Binding Protein C ◽  
Myosin Binding

Abstract Objectives Cardiac myosin-binding protein C (cMyC) is a novel biomarker of myocardial injury, with a promising role in the triage and risk stratification of patients presenting with acute cardiac disease. In this study, we assess the weekly biological variation of cMyC, to examine its potential in monitoring chronic myocardial injury, and to suggest analytical quality specification for routine use of the test in clinical practice. Methods Thirty healthy volunteers were included. Non-fasting samples were obtained once a week for ten consecutive weeks. Samples were tested in duplicate on the Erenna® platform by EMD Millipore Corporation. Outlying measurements and subjects were identified and excluded systematically, and homogeneity of analytical and within-subject variances was achieved before calculating the biological variability (CVI and CVG), reference change values (RCV) and index of individuality (II). Results Mean age was 38 (range, 21–64) years, and 16 participants were women (53%). The biological variation, RCV and II with 95% confidence interval (CI) were: CVA (%) 19.5 (17.8–21.6), CVI (%) 17.8 (14.8–21.0), CVG (%) 66.9 (50.4–109.9), RCV (%) 106.7 (96.6–120.1)/−51.6 (−54.6 to −49.1) and II 0.42 (0.29–0.56). There was a trend for women to have lower CVG. The calculated RCVs were comparable between genders. Conclusions cMyC exhibits acceptable RCV and low II suggesting that it could be suitable for disease monitoring, risk stratification and prognostication if measured serially. Analytical quality specifications based on biological variation are similar to those for cardiac troponin and should be achievable at clinically relevant concentrations.

scholarly journals The Interplay between S-glutathionylation and Phosphorylation of Cardiac Troponin I and Myosin Binding Protein C in End-Stage Human Failing Hearts

Antioxidants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1134
Author(s):  
Heidi Budde ◽  
Roua Hassoun ◽  
Melina Tangos ◽  
Saltanat Zhazykbayeva ◽  
Melissa Herwig ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Protein C ◽  
Troponin I ◽  
Reduced Glutathione ◽  
Binding Protein ◽  
Enzyme Treatment ◽  
Myosin Binding Protein ◽  
Myosin Binding Protein C ◽  
Myosin Binding ◽  
End Stage

Oxidative stress is defined as an imbalance between the antioxidant defense system and the production of reactive oxygen species (ROS). At low levels, ROS are involved in the regulation of redox signaling for cell protection. However, upon chronical increase in oxidative stress, cell damage occurs, due to protein, DNA and lipid oxidation. Here, we investigated the oxidative modifications of myofilament proteins, and their role in modulating cardiomyocyte function in end-stage human failing hearts. We found altered maximum Ca2+-activated tension and Ca2+ sensitivity of force production of skinned single cardiomyocytes in end-stage human failing hearts compared to non-failing hearts, which was corrected upon treatment with reduced glutathione enzyme. This was accompanied by the increased oxidation of troponin I and myosin binding protein C, and decreased levels of protein kinases A (PKA)- and C (PKC)-mediated phosphorylation of both proteins. The Ca2+ sensitivity and maximal tension correlated strongly with the myofilament oxidation levels, hypo-phosphorylation, and oxidative stress parameters that were measured in all the samples. Furthermore, we detected elevated titin-based myocardial stiffness in HF myocytes, which was reversed by PKA and reduced glutathione enzyme treatment. Finally, many oxidative stress and inflammation parameters were significantly elevated in failing hearts compared to non-failing hearts, and corrected upon treatment with the anti-oxidant GSH enzyme. Here, we provide evidence that the altered mechanical properties of failing human cardiomyocytes are partially due to phosphorylation, S-glutathionylation, and the interplay between the two post-translational modifications, which contribute to the development of heart failure.

scholarly journals Cardiac Myosin-Binding Protein C Modulates the Tuning of the Molecular Motor in the Heart

2008 ◽  
Vol 95 (2) ◽  
pp. 720-728 ◽  
Author(s):  
Yves Lecarpentier ◽  
Nicolas Vignier ◽  
Patricia Oliviero ◽  
Aziz Guellich ◽  
Lucie Carrier ◽  
...  
Keyword(s):  
Protein C ◽  
Molecular Motor ◽  
Binding Protein ◽  
Cardiac Myosin ◽  
Myosin Binding Protein ◽  
Myosin Binding Protein C ◽  
Myosin Binding

scholarly journals Phosphorylation of cardiac myosin binding protein C releases myosin heads from the surface of cardiac thick filaments

2017 ◽  
Vol 114 (8) ◽  
pp. E1355-E1364 ◽  
Author(s):  
Robert W. Kensler ◽  
Roger Craig ◽  
Richard L. Moss
Keyword(s):  
Protein C ◽  
Structural Changes ◽  
Binding Protein ◽  
Cardiac Contraction ◽  
Cardiac Myosin ◽  
Cross Bridge ◽  
Myosin Binding Protein ◽  
Myosin Binding Protein C ◽  
Thick Filaments ◽  
Myosin Binding

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 β1-adrenergic receptors in myocardium.

scholarly journals S ‐glutathiolation impairs phosphoregulation and function of cardiac myosin‐binding protein C in human heart failure

2016 ◽  
Vol 30 (5) ◽  
pp. 1849-1864 ◽  
Author(s):  
Konstantina Stathopoulou ◽  
Ilka Wittig ◽  
Juliana Heidler ◽  
Angelika Piasecki ◽  
Florian Richter ◽  
...  
Keyword(s):  
Heart Failure ◽  
Protein C ◽  
Human Heart ◽  
Binding Protein ◽  
Cardiac Myosin ◽  
Human Heart Failure ◽  
Myosin Binding Protein ◽  
Myosin Binding Protein C ◽  
Myosin Binding ◽  
And Function

scholarly journals Calcium Handling Derangement is Associated with Conditional Cardiac Myosin Binding Protein C Knock Out

2012 ◽  
Vol 102 (3) ◽  
pp. 226a-227a
Author(s):  
Erin M. Capes ◽  
Randall Loaiza ◽  
Peter P. Chen ◽  
Daniel P. Fitzsimons ◽  
Hector H. Valdivia ◽  
...  
Keyword(s):  
Protein C ◽  
Binding Protein ◽  
Calcium Handling ◽  
Cardiac Myosin ◽  
Knock Out ◽  
Myosin Binding Protein ◽  
Myosin Binding Protein C ◽  
Myosin Binding

Abstract 391: The Pathophysiological Role of MYBPHL in Dilated Cardiomyopathy

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
David Y Barefield ◽  
Megan J Puckelwartz ◽  
Lisa Dellefave-Castillo ◽  
Elizabeth M McNally
Keyword(s):  
Cardiac Function ◽  
Protein C ◽  
Stop Codon ◽  
Binding Protein ◽  
Premature Stop Codon ◽  
Myosin Binding Protein ◽  
Myosin Binding Protein C ◽  
Pathophysiological Role ◽  
Myosin Binding

Background: Cardiomyopathy is a leading cause of heart failure and is highly heritable. One common form of cardiomyopathy is dilated cardiomyopathy (DCM), which currently has over 70 identified genes that have been described as causative for the disease. Genetic testing for DCM employs gene panels and has a sensitivity of mutation detection less than 50%, indicating that additional genes contribute to DCM. Here, we employed whole genome sequencing (WGS) in a family with DCM and heart block who had previously undergone unrevealing genetic testing. We identified a premature stop codon in the MYBPHL gene, a gene that has not previously been linked to DCM as a likely cause of DCM in this family. Myosin binding protein H Like (MyBP-HL) is a muscle-expressed protein bearing structural similarity to myosin binding protein C (MyBP-C), which is commonly mutated gene in cardiomyopathies. Objective: Determine the physiological and pathophysiological role of Mybphl . Results: RNA-seq and qPCR from mouse hearts revealed that Mybphl is highly expressed in the right and left atria with lower expression in the ventricle and virtually no expression in skeletal muscle. As MyBP-HL shares a high homology with the myofilament proteins cardiac myosin binding protein-C and H, we investigated if MyBP-HL is also myofilament-associated. We determined that MyBP-HL protein is myofilament-associated in the atria although not clearly so in ventricle. To assess the requirement of MyBP-HL in cardiac function, we used a mouse model with an insertional disruption of the Mybphl gene. These mice have deficits in in vivo cardiac function, with reduced fractional shortening. In addition, ECG recordings from the Mybphl null mice show conduction system abnormalities affecting atrioventricular conduction. Conclusions: WGS identified a premature stop codon in MYBPHL in human DCM. A mouse model with a disrupted Mybphl gene showed similar pathophysiological features as the humans with reduced ventricular function and cardiac conduction system abnormalities. MyBP-HL is an important protein for normal cardiac function.

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