In Vivo Cardiac Myosin Binding Protein C Gene Transfer Rescues Myofilament Contractile Dysfunction in Cardiac Myosin Binding Protein C Null Mice

Abstract Aims A 25-base pair deletion in the cardiac myosin binding protein-C (cMyBP-C) gene (MYBPC3), proposed to skip exon 33, modifies the C10 domain (cMyBP-CΔC10mut) and is associated with hypertrophic cardiomyopathy (HCM) and heart failure, affecting approximately 100 million South Asians. However, the molecular mechanisms underlying the pathogenicity of cMyBP-CΔC10mutin vivo are unknown. We hypothesized that expression of cMyBP-CΔC10mut exerts a poison polypeptide effect leading to improper assembly of cardiac sarcomeres and the development of HCM. Methods and results To determine whether expression of cMyBP-CΔC10mut is sufficient to cause HCM and contractile dysfunction in vivo, we generated transgenic (TG) mice having cardiac-specific protein expression of cMyBP-CΔC10mut at approximately half the level of endogenous cMyBP-C. At 12 weeks of age, significant hypertrophy was observed in TG mice expressing cMyBP-CΔC10mut (heart weight/body weight ratio: 4.43 ± 0.11 mg/g non-transgenic (NTG) vs. 5.34 ± 0.25 mg/g cMyBP-CΔC10mut, P < 0.05). Furthermore, haematoxylin and eosin, Masson’s trichrome staining, as well as second-harmonic generation imaging revealed the presence of significant fibrosis and a greater relative nuclear area in cMyBP-CΔC10mut hearts compared with NTG controls. M-mode echocardiography analysis revealed hypercontractile hearts (EF: 53.4%±2.9% NTG vs. 66.4% ± 4.7% cMyBP-CΔC10mut; P < 0.05) and early diastolic dysfunction (E/E′: 28.7 ± 3.7 NTG vs. 46.3 ± 8.4 cMyBP-CΔC10mut; P < 0.05), indicating the presence of an HCM phenotype. To assess whether these changes manifested at the myofilament level, contractile function of single skinned cardiomyocytes was measured. Preserved maximum force generation and increased Ca2+-sensitivity of force generation were observed in cardiomyocytes from cMyBP-CΔC10mut mice compared with NTG controls (EC50: 3.6 ± 0.02 µM NTG vs. 2.90 ± 0.01 µM cMyBP-CΔC10mut; P < 0.0001). Conclusion Expression of cMyBP-C protein with a modified C10 domain is sufficient to cause contractile dysfunction and HCM in vivo.

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Abstract 376: Amino Terminal C0-C1f Region of Cardiac Myosin Binding Protein-C is Essential for Normal Cardiac Function

Circulation Research ◽

10.1161/res.117.suppl_1.376 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Thomas L Lynch ◽

Diederik W Kuster ◽

David Barefield ◽

Mayandi Sivaguru ◽

Michael J Previs ◽

...

Keyword(s):

Cardiac Function ◽

Protein C ◽

Binding Protein ◽

Cardiac Myosin ◽

Amino Terminal ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Thick Filaments ◽

Myosin Binding

Rationale: Cardiac myosin binding protein-C (cMyBP-C) is a trans-filament protein that has been shown to regulate cardiac function via its amino terminal (N’) regions. However, it is unknown whether the first 271 residues (C0-C1f region) are necessary to regulate contractile function in vivo. Hypothesis: The N’-region of cMyBP-C is critical for proper cardiac function in vivo. Methods and Results: Transgenic mice with approximately 80% expression of mutant truncated cMyBP-C missing C0-C1f (cMyBP-C 110kDa ), compared to endogenous cMyBP-C, were generated and characterized at 3-months of age. cMyBP-C 110kDa hearts had significantly elevated heart weight/body weight ratio, fibrosis, nuclear area and collagen content compared to hearts from non-transgenic (NTG) littermates. Electron microscopic analysis revealed normal sarcomere structure in cMyBP-C 110kDa hearts but with apparently weaker cMyBP-C stripes. Furthermore, the ability of cMyBP-C to slow actin-filament sliding within the C-zone of native thick filaments isolated from NTG hearts was lost on thick filaments from cMyBP-C 110kDa hearts. Short axis M-mode echocardiography revealed a significant increase in left ventricular (LV) internal diameter during diastole in cMyBP-C 110kDa hearts. Importantly, cMyBP-C 110kDa hearts displayed a significant reduction in fractional shortening compared to hearts from NTG littermates. We further observed a decrease in the thickness of the LV interventricular septum and free wall during systole in cMyBP-C 110kDa hearts. Strain analysis using images acquired from ECG-Gated Kilohertz Visualization identified a significant deficit in global longitudinal strain in cMyBP-C 110kDa hearts compared to NTG hearts. Conclusion: The N’-region of cMyBP-C is indispensable for maintaining normal cardiac morphology and function and loss of this region promotes contractile dysfunction both at the molecular and tissue levels.

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Serine (Ser) 302 of Cardiac Myosin Binding Protein C (cMyBPC) Is a Unique PKCɛ Phosphorylation Site Both In Vivo and In Vitro

Journal of Cardiac Failure ◽

10.1016/j.cardfail.2006.06.149 ◽

2006 ◽

Vol 12 (6) ◽

pp. S42

Author(s):

Qiong Zhao ◽

Lei Xiao ◽

Yanmei Du ◽

Chao Yuan ◽

R. John Solaro ◽

...

Keyword(s):

Protein C ◽

Binding Protein ◽

Phosphorylation Site ◽

Cardiac Myosin ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Myosin Binding

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Phosphomimetic cardiac myosin-binding protein C partially rescues a cardiomyopathy phenotype in murine engineered heart tissue

Scientific Reports ◽

10.1038/s41598-019-54665-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 5

Author(s):

Alexander Dutsch ◽

Paul J. M. Wijnker ◽

Saskia Schlossarek ◽

Felix W. Friedrich ◽

Elisabeth Krämer ◽

...

Keyword(s):

Gene Transfer ◽

Protein C ◽

Binding Protein ◽

Heart Tissue ◽

Cardiac Myosin ◽

Wild Type ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Myosin Binding

AbstractPhosphorylation of cardiac myosin-binding protein C (cMyBP-C), encoded by MYBPC3, increases the availability of myosin heads for interaction with actin thus enhancing contraction. cMyBP-C phosphorylation level is lower in septal myectomies of patients with hypertrophic cardiomyopathy (HCM) than in non-failing hearts. Here we compared the effect of phosphomimetic (D282) and wild-type (S282) cMyBP-C gene transfer on the HCM phenotype of engineered heart tissues (EHTs) generated from a mouse model carrying a Mybpc3 mutation (KI). KI EHTs showed lower levels of mutant Mybpc3 mRNA and protein, and altered gene expression compared with wild-type (WT) EHTs. Furthermore, KI EHTs exhibited faster spontaneous contractions and higher maximal force and sensitivity to external [Ca2+] under pacing. Adeno-associated virus-mediated gene transfer of D282 and S282 similarly restored Mybpc3 mRNA and protein levels and suppressed mutant Mybpc3 transcripts. Moreover, both exogenous cMyBP-C proteins were properly incorporated in the sarcomere. KI EHTs hypercontractility was similarly prevented by both treatments, but S282 had a stronger effect than D282 to normalize the force-Ca2+-relationship and the expression of dysregulated genes. These findings in an in vitro model indicate that S282 is a better choice than D282 to restore the HCM EHT phenotype. To which extent the results apply to human HCM remains to be seen.

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ID: 136: N-TERMINAL REGION OF CARDIAC MYOSIN BINDING PROTEIN-C IS NECESSARY FOR CARDIAC FUNCTION

Journal of Investigative Medicine ◽

10.1136/jim-2016-000120.2 ◽

2016 ◽

Vol 64 (4) ◽

pp. 911.2-912

Author(s):

M Sivaguru ◽

TL Lynch ◽

DW Kuster ◽

S Govindan ◽

S Sadayappan ◽

...

Keyword(s):

Cardiac Function ◽

Actin Filament ◽

Protein C ◽

Binding Protein ◽

Cardiac Myosin ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Thick Filaments ◽

Myosin Binding

RationaleCardiac myosin binding protein-C (cMyBP-C) is a trans-filament protein that has been shown to regulate cardiac function via its amino terminal (N′) region. In vitro studies have suggested the importance of the first 271 N′-residues of cMyBP-C (C0-C1f region) in slowing actin filament sliding over myosin to regulate cross-bridge cycling kinetics within the cardiac sarcomere. However, the role and necessity of the C0-C1f region of cMyBP-C in regulating contractile and cardiac function in vivo have not been elucidated.HypothesisThe N′-C0-C1f region of cMyBP-C is critical for proper cardiac function in vivo.Methods and ResultsTransgenic mice with approximately 95% expression of a mutant truncated cMyBP-C missing the N′-C0-C1f region (cMyBP-C110 kDa), compared to endogenous cMyBP-C, were generated and characterized at 3-months of age. cMyBP-C110 kDa hearts had significantly elevated heart weight/body weight ratio, fibrosis, nuclear area and collagen content compared to hearts from non-transgenic (NTG) littermates. Electron microscopic analysis revealed normal sarcomere structure in cMyBP-C110 kDa hearts but with apparently weaker cMyBP-C stripes. Furthermore, the ability of cMyBP-C to slow actin-filament sliding within the C-zone of native thick filaments isolated from NTG hearts was lost on thick filaments from cMyBP-C110 kDa hearts. Short axis M-mode echocardiography revealed a significant increase in left ventricular (LV) internal diameter during diastole in cMyBP-C110 kDa hearts. Importantly, cMyBP-C110 kDa hearts displayed a significant reduction in fractional shortening compared to hearts from NTG mice. We further observed a decrease in the thickness of the LV interventricular septum and free wall during systole in cMyBP-C110 kDa hearts. Strain analysis using images acquired from ECG-Gated Kilohertz Visualization identified a significant deficit in global longitudinal strain in cMyBP-C110 kDa hearts compared to NTG hearts. Consistent with cardiac hypertrophy, we observed a significant increase in the expression of the hypertrophic genes MYH7 and NPPA by real-time PCR analysis. As expected, the expression levels of the MYBPC3 gene were significantly elevated in cMyBP-C110 kDa hearts compared to NTG hearts. Surprisingly, our Western blot analyses revealed no significant difference in total cMyBP-C levels between NTG and cMyBP-C110 kDa heart homogenates. However, intriguingly, we observed a significant elevation in cMyBP-C phosphorylation at Ser-273, Ser-282, and Ser-302, sites important for cMyBP-C's regulation of actomyosin interaction, in cMyBP-C110 kDa heart homogenates compared to those from NTG mice.ConclusionThe N′-C0-C1f region of cMyBP-C is essential for maintaining normal cardiac morphology and function in vivo and loss of this region promotes contractile dysfunction both at the molecular and tissue level.

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PKCε Increases Phosphorylation of the Cardiac Myosin Binding Protein C at Serine 302 both in Vitro and in Vivo†

Biochemistry ◽

10.1021/bi700467k ◽

2007 ◽

Vol 46 (23) ◽

pp. 7054-7061 ◽

Cited By ~ 31

Author(s):

Lei Xiao ◽

Qiong Zhao ◽

Yanmei Du ◽

Chao Yuan ◽

R. John Solaro ◽

...

Keyword(s):

Protein C ◽

Binding Protein ◽

Cardiac Myosin ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Myosin Binding

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117 Use of drosophila to approach the in vivo function of human cardiac myosin binding protein C

European Journal of Heart Failure Supplements ◽

10.1016/s1567-4215(03)90051-8 ◽

2003 ◽

Vol 2 (1) ◽

pp. 17

Author(s):

L RODER

Keyword(s):

Protein C ◽

Binding Protein ◽

Cardiac Myosin ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Myosin Binding

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

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2021-0306 ◽

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.

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