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

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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Abstract 389: Amino Terminal Region of Cardiac Myosin Binding Protein-C is Necessary for Cardiac Function

Circulation Research ◽

10.1161/res.119.suppl_1.389 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Thomas L Lynch ◽

Diederik W Kuster ◽

Mayandi Sivaguru ◽

Michael J Previs ◽

Kyounghwan Lee ◽

...

Keyword(s):

Cardiac Function ◽

Protein C ◽

Cardiac Myosin ◽

Amino Terminal ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Thick Filaments ◽

Myosin Binding ◽

Normal Cardiac Function

Rationale: Cardiac myosin binding protein-C (cMyBP-C) is a thick filament-associated protein that has been suggested to regulate cardiac contraction via its amino terminal (N’) region. However, the necessity of the N’-C0-C1f region (domains C0 through C1 and the first 17 residues of the M-domain) of cMyBP-C in regulating cardiac function in vivo has not been elucidated. Hypothesis: The N’-C0-C1f region of cMyBP-C is critical for normal cardiac function in vivo . Methods and Results: Transgenic mice with 80±4% expression of a truncated cMyBP-C missing the N’-C0-C1f region (cMyBP-C 110kDa ) were generated and characterized at 3-months of age. cMyBP-C 110kDa animals exhibited cardiac hypertrophy as suggested by an increased heart weight to body weight ratio (5.0±0.1 mg/g NTG vs. 6.9±0.1 mg/g cMyBP-C 110kDa , p<0.0001) and an elevation in pathological hypertrophy markers determined by real-time PCR. Histopathological analysis showed increased cardiac fibrosis in cMyBP-C 110kDa hearts compared to hearts from non-transgenic (NTG) littermates. Intriguingly, increased phosphorylation of cMyBP-C at Ser-282 and Ser-302, sites important for cMyBP-C’s regulation of actomyosin interactions, was observed in cMyBP-C 110kDa hearts compared to controls. Electron microscopy 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 indicated a significant elevation in left ventricular internal diameter and a significant reduction in fractional shortening (31±5% NTG vs. 16±3% cMyBP-C 110kDa , p=0.0003) in cMyBP-C 110kDa hearts compared to controls. Finally, global longitudinal strain analysis revealed abnormal wall motion in cMyBP-C 110kDa hearts. Based upon these data, we propose that the N’-region of cMyBP-C is a critical regulator of actomyosin interactions and controls aberrant contraction kinetics within the cardiac sarcomere. Conclusion: The N’-C0-C1f region of cMyBP-C regulates cardiac contractility and is necessary for maintaining normal cardiac function in vivo .

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1614020114 ◽

2017 ◽

Vol 114 (8) ◽

pp. E1355-E1364 ◽

Cited By ~ 35

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.

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Interaction between cardiac myosin-binding protein C and formin Fhod3

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1716498115 ◽

2018 ◽

Vol 115 (19) ◽

pp. E4386-E4395 ◽

Cited By ~ 7

Author(s):

Sho Matsuyama ◽

Yohko Kage ◽

Noriko Fujimoto ◽

Tomoki Ushijima ◽

Toshihiro Tsuruda ◽

...

Keyword(s):

Hypertrophic Cardiomyopathy ◽

Cardiac Function ◽

Protein C ◽

Binding Protein ◽

Direct Interaction ◽

Familial Hypertrophic Cardiomyopathy ◽

Cardiac Myosin ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Myosin Binding

Mutations in cardiac myosin-binding protein C (cMyBP-C) are a major cause of familial hypertrophic cardiomyopathy. Although cMyBP-C has been considered to regulate the cardiac function via cross-bridge arrangement at the C-zone of the myosin-containing A-band, the mechanism by which cMyBP-C functions remains unclear. We identified formin Fhod3, an actin organizer essential for the formation and maintenance of cardiac sarcomeres, as a cMyBP-C–binding protein. The cardiac-specific N-terminal Ig-like domain of cMyBP-C directly interacts with the cardiac-specific N-terminal region of Fhod3. The interaction seems to direct the localization of Fhod3 to the C-zone, since a noncardiac Fhod3 variant lacking the cMyBP-C–binding region failed to localize to the C-zone. Conversely, the cardiac variant of Fhod3 failed to localize to the C-zone in the cMyBP-C–null mice, which display a phenotype of hypertrophic cardiomyopathy. The cardiomyopathic phenotype of cMyBP-C–null mice was further exacerbated by Fhod3 overexpression with a defect of sarcomere integrity, whereas that was partially ameliorated by a reduction in the Fhod3 protein levels, suggesting that Fhod3 has a deleterious effect on cardiac function under cMyBP-C–null conditions where Fhod3 is aberrantly mislocalized. Together, these findings suggest the possibility that Fhod3 contributes to the pathogenesis of cMyBP-C–related cardiomyopathy and that Fhod3 is critically involved in cMyBP-C–mediated regulation of cardiac function via direct interaction.

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Altered C10 domain in cardiac myosin binding protein-C results in hypertrophic cardiomyopathy

Cardiovascular Research ◽

10.1093/cvr/cvz111 ◽

2019 ◽

Vol 115 (14) ◽

pp. 1986-1997 ◽

Cited By ~ 5

Author(s):

Diederik W D Kuster ◽

Thomas L Lynch ◽

David Y Barefield ◽

Mayandi Sivaguru ◽

Gina Kuffel ◽

...

Keyword(s):

Hypertrophic Cardiomyopathy ◽

Protein C ◽

Binding Protein ◽

Force Generation ◽

Contractile Dysfunction ◽

Cardiac Myosin ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Myosin Binding

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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