Amino terminus of cardiac myosin binding protein-C regulates cardiac contractility

Thomas L Lynch, IV; Mohit Kumar; James W McNamara; Diederik WD Kuster; Mayandi Sivaguru; Rohit R Singh; Michael J Previs; Kyoung Hwan Lee; Gina Kuffel; Michael J Zilliox; Brian Leei Lin; Weikang Ma; Aaron M Gibson; Burns C Blaxall; Michelle L Nieman; John N Lorenz; Dana M Leichter; Owen P Leary; Paul ML Janssen; Pieter P de Tombe; Richard J Gilbert; Roger Craig; Thomas Irving; David M Warshaw; Sakthivel Sadayappan

doi:10.1016/j.yjmcc.2021.03.009

. Author manuscript; available in PMC: 2022 Jul 1.

Published in final edited form as: J Mol Cell Cardiol. 2021 Mar 26;156:33–44. doi: 10.1016/j.yjmcc.2021.03.009

Amino terminus of cardiac myosin binding protein-C regulates cardiac contractility

Thomas L Lynch IV ^a,^1,², Mohit Kumar ^a,^b,², James W McNamara ^b,³, Diederik WD Kuster ^a,^c, Mayandi Sivaguru ^d, Rohit R Singh ^b, Michael J Previs ^e, Kyoung Hwan Lee ^f,⁴, Gina Kuffel ^g, Michael J Zilliox ^g, Brian Leei Lin ^a, Weikang Ma ^h, Aaron M Gibson ⁱ, Burns C Blaxall ^i,⁵, Michelle L Nieman ^j, John N Lorenz ^j, Dana M Leichter ^k, Owen P Leary ^k, Paul ML Janssen ^l, Pieter P de Tombe ^a,^m,ⁿ, Richard J Gilbert ^k, Roger Craig ^f, Thomas Irving ^h, David M Warshaw ^e, Sakthivel Sadayappan ^a,^b,^*

^aDepartment of Cell and Molecular Physiology, Loyola University Chicago, Maywood, IL 60153, USA

^bHeart, Lung and Vascular Institute, Division of Cardiovascular Health and Disease, Department of Internal Medicine, University of Cincinnati, Cincinnati, OH 45267, USA

^cDepartment of Physiology, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam Cardiovascular Sciences, Amsterdam, the Netherlands

^dInstitute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA

^eDepartment of Molecular Physiology and Biophysics, Cardiovascular Research Institute, University of Vermont, Burlington VT 05405, USA

^fDivision of Cell Biology and Imaging, Department of Radiology, University of Massachusetts Medical School, Worcester, MA 01655, USA

^gDepartment of Public Health Sciences, Loyola University Chicago, Maywood, IL 60153, USA

^hCenter for Synchrotron Radiation Research and Instrumentation and Department of Biological Sciences, Illinois Institute of Technology, Chicago, IL 60616, USA

ⁱDepartment of Pediatrics, Heart Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, 45229, USA

^jDepartment of Pharmacology and Systems Physiology, University of Cincinnati College of Medicine, Cincinnati, OH, USA

^kResearch Service, Providence VA Medical Center, Providence, RI 02908, USA

^lDepartment of Physiology and Cell Biology, The Ohio State University, Columbus, OH 43210, USA

^mDepartment of Physiology, University of Illinois at Chicago, Chicago 60612, USA

ⁿPhymedexp, Université de Montpellier, Inserm, CNRS, Montpellier, France

AbbVie, Drug Metabolism and Pharmacokinetics - Bioanalysis, North Chicago, IL 60064-1802.

These authors contributed equally to this work.

Murdoch Children’s Research Institute, The Royal Children’s Hospital, Parkville, Victoria 3052 and Department of Physiology, School of Biomedical Sciences, The University of Melbourne, Parkville, Victoria, 3010, Australia.

⁴

Massachusetts Facility for High-Resolution Electron Cryo-microscopy, University of Massachusetts Medical School, Worcester MA 01655, USA.

⁵

Personalized Medicine, The Christ Hospital Health Network, 2139 Auburn Ave, Cincinnati, OH 45219, USA.

Corresponding author at: Department of Internal Medicine, University of Cincinnati, Cincinnati, OH 45267-0575, USA. sadayasl@ucmail.uc.edu

Author contributions

TLLIV, MK, JWM, DWDK, MS, RRS, MJP, KHL, GN, MJZ, BLL, WM, DML, OPL, RJG, RC, TI, DMW and SS conceived and designed the study. TLLIV, MK, JWM, DWDK, MS, MJP, KHL, GN, BLL, WM, MLN, JNL, DML, OPL RJG, RC, TI, DMW and SS developed the methods. TLLIV, MK, JWM, DWDK, MS, RRS, MJP, KHL, GN, BLL, WM, MLN, DML and OPL conducted experiments and acquired data. AMG, BCB, JNL, PMLJ and PPdeT provided reagents and technical expertise. TLLIV, MK, JWM, DWDK, MS, RRS, MJP, KHL, GN, BLL, WM, MLN, JNL, DML, OPL RJG, RC, TI, DMW and SS analyzed and interpreted the data. TLLIV, MK, JWM, DWDK, MS, MJP, KHL, GN, MJZ, BLL, BCB, MLN, JNL, DML, OPL, RJG, RC, TI, DMW and SS wrote, reviewed, and revised the manuscript.

PMCID: PMC8217138 NIHMSID: NIHMS1689212 PMID: 33781820

Abstract

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein’s amino terminal (N’)-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C^ΔC0-C1f), displayed dilated cardiomyopathy, underscoring the importance of the N’-region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, likely contributing to normal sarcomere morphology and myoarchitecture. These results led us to hypothesize that restoration of the N’-region of cMyBP-C would return actomyosin interaction to its steady state. Accordingly, we administered recombinant C0-C2 (rC0-C2) to permeabilized cardiomyocytes from transgenic, cMyBP-C null, and human heart failure biopsies, and we found that normal regulation of actomyosin interaction and contractility was restored. Overall, these data provide a unique picture of selective perturbations of the cardiac sarcomere that either lead to injury or adaptation to injury in the myocardium.

Keywords: Heart Failure, MYBPC3, cMyBP-C phosphorylation, Myofilament, Sarcomere

Graphical Abstract

graphic file with name nihms-1689212-f0007.jpg

1. Introduction

Cardiac myosin binding protein-C (cMyBP-C), a 150-kDa, multi-domain (C0-C10; Fig. 1A) myofilament protein, regulates cardiac muscle sarcomere structure and function [1, 2]. Mutations in this protein have been linked to familial hypertrophic cardiomyopathy in more than 40% of patients [2]. cMyBP-C is considered a trans-filament protein based on its ability to interact with both thick (myosin S2, myosin RLC) and thin filament (actin and α-tropomyosin) proteins via its N’-terminal region [3]. cMyBP-C interacts with actin via the C0, C1 and M domains and with myosin via the C0, C1, M, and C2 domains. The proline-alanine-rich (P/A)-rich region interacts with α-tropomyosin [3]. Additionally, both actin and myosin interactions are regulated by M-domain phosphorylation [1]. Three cMyBP-C serines (Ser-273, Ser-282 and Ser-302) in the M domain of cMyBP-C are substrates for protein kinase A [1], and their phosphorylation accelerates cross-bridge kinetics. cMyBP-C phosphorylation activates the thin filament and increases contractility [4]. By contrast, dephosphorylated cMyBP-C has improved myosin interaction, consequently limiting actomyosin interaction and depressing contractility [5, 6]. Thus, it can be concluded that the N’-region acts a molecular throttle to control actomyosin interactions and to modulate sarcomere structure and function [7, 8]. While it has been demonstrated that the N’-region of cMyBP-C can modulate contractility and regulate sarcomere sliding velocity in vitro [7], its necessity in regulating contractile function in vivo has not been demonstrated.

The first 271 residues of the amino terminal (N’)-region of cMyBP-C, consisting of the Ig-like domains C0 and C1, the P/A-rich linker between them, and the first 17 residues of the M domain (i.e., C0-C1f), are cleaved from the full-length cMyBP-C protein in association with myocardial infarction, ischemia-reperfusion (I-R) injury and in certain models and forms of heart failure (HF) [9]. Therefore, to elucidate the pathophysiological role of cMyBP-C in cardiac disease, we and others have focused our investigations on this N’-terminal region. We have shown that cMyBP-C dephosphorylation is directly associated with its degradation [10], providing a condition that results in cleavage of the C0-C1f protein, which, in turn, causes contractile dysfunction [11]. We recently reported that inhibiting calpain-mediated proteolysis of this cMyBP-C region limited infarct size and preserved cardiac function following I-R injury [9]. Although proteolytic cleavage of this region has known pathogenic consequences on sarcomere mechanics [7] and contractility [3], it is not clear how the loss of the C0-C1f region affects sarcomere and cardiac function in vivo. Specifically, the molecular mechanisms by which the N’-region of cMyBP-C regulates thin and thick filament interactions remain unknown.

This study examines the necessity of the N’-region of cMyBP-C, particularly, a region known as C0-C1f, in maintaining normal cardiac contractile function in vivo. The N’-C0-C1f region of cMyBP-C is a 29 kilodalton (kDa) peptide consisting of the first 271 amino acid residues of cMyBP-C and incorporating domains C0 through C1 and the first 17 residues of the M-domain [3, 10–14]. However, it migrates on sodium dodecyl sulfate polyacrylamide gel electrophoresis at around 40 kDa [13]. The importance of the N’-C0-C1f region of cMyBP-C is several-fold. Initially, our group and others identified this region as a predominant N’-cleavage fragment of cMyBP-C following the dephosphorylation of cMyBP-C during conditions of hypoxia or ischemic injury [10, 15]. In two subsequent reports, we reported that C0-C1f was sufficient to cause contractile dysfunction in isolated rat ventricular myocytes [13] and that it could impair function in human cardiac myofilaments [3]. A later study by Razzaque et al. demonstrated that endogenous expression of C0-C1f was sufficient to cause HCM, heart failure and death in mice [11]. Others have reported the ability of C0-C1f to inhibit actin filament sliding over myosin [12] and that the first 17 residues of the M-domain (the “f” region of C0-C1f) are crucial to this process [16]. However, the novelty of the present work stems from directly associating the necessity of the N’-C0-C1f region of cMyBP-C with its role in maintaining steady-state myofilament function.

Given its functional importance, as suggested by in vitro [3, 7] and in vivo [11] studies, our hypothesis holds that the N’-C0-C1f region of cMyBP-C is vital for regulating cardiac function in vivo, but that perturbations to its biology, such as mutations, lead to pathological mechanics and cardiac remodeling. To test this hypothesis, we generated cardiac-specific transgenic (TG) mice expressing N’-truncated cMyBP-C proteins lacking the C0-C1f region (cMyBP-C^ΔC0-C1f). These mice showed decreased fractional shortening and cardiac enlargement with dilation, compared to littermate non-transgenic (NTG) mice carrying the full-length cMyBP-C protein. In vitro studies using isolated TG skinned myofibrils displayed a higher rate of crossbridge cycling, as well as faster actin sliding velocity through the C-zone. In contrast, compensatory hyper-phosphorylation of the unablated phosphoregulatory sites was associated with stabilization of sarcomere structure and tissue myoarchitecture in agreement with previous reports that cMyBP-C phosphorylation can restore sarcomere dysregulation [17] and myoarchitecture [18]. However, we wanted to further test the sufficiency of the C0-C2 region of cMyBP-C in rescuing normal actomyosin interactions [19, 20] and, hence, contractile function. To accomplish this, rC0-C2 was applied to permeabilized skinned cardiomyocytes from TG and cMyBP-C null mice, as well as biopsy of tissue from human HF, and force-pCa relationships were compared to those from NTG mice. Indeed, normal regulation of actomyosin interaction was restored with improved contractile function in C0-C2-treated TG, cMyBP-C null, and human HF myocytes ex vivo. These results suggest that the N-terminal domain of cMyBP-C plays a critical role in the regulation of sarcomere structure and function under normal and pathological conditions and that its restitution following ablation may emulate physiological restoration of contractile function following its pathological degradation.

2. Materials and Methods

Further experimental details are described in SI Materials and Methods.

2.1. Transgenic cMyBP-C^ΔC0-C1f Mouse Model

cMyBP-C cDNA lacking the first 813 nucleotides (~3 kb) was made by polymerase chain reaction (PCR) and cloned downstream of the α-MHC promoter. An N’-terminal cMyc tag was included for recognition. The construct was used to generate multiple TG founders on an NTG background having cardiomyocyte-specific replacement of endogenous cMyBP-C with the cMyBP-C^ΔC0-C1f protein (C57BL/6 strain). An NTG background was chosen based on its physiological relevance to HF in which both cleaved C0-C1f and full-length cMyBP-C have been detected in animals and patients [7, 9, 11, 15]. Furthermore, we did not choose the cMyBP-C null (t/t) background as the t/t mouse itself develops a DCM phenotype, which could impact the phenotype of cMyBP-C^ΔC0-C1f [17, 21, 22]. The t/t mouse model was previously generated and characterized in the literature [17, 21]. All experiments were performed on animals between 3 and 8 months of age with age- and sex-matched controls. All experiments using animals detailed in this work were approved by the Institutional Animal Care and Use Committees at Loyola University Chicago and University of Cincinnati and followed the policies of the Guide for the Use and Care of Laboratory Animals published by the National Institutes of Health.

2.2. Molecular Analyses

Myofilament-enriched protein fractions from frozen mouse hearts were isolated and subjected to SDS-PAGE and Western blot analysis for total and phosphorylated cMyBP-C and cardiac troponin I as described previously [10, 17]. Total RNA isolation, quantitative expression of MYBPC3 and hypertrophic gene markers, and RNA-seq were performed as previously described [10, 17, 23].

2.3. Biophysical Experiments

Cardiomyocytes were harvested from frozen TG and NTG heart tissue, and isometric force development was recorded at varying calcium concentrations (pCa 10.0 to pCa 4.5) as previously described [3]. The rate of tension redevelopment (k_tr) was measured using isolated skinned cardiomyocytes from TG and NTG hearts in activating solution at pCa 4.5. Isolated fresh cardiomyocytes were then used to measure contractile parameters and calcium transients and kinetics, as recently described [24]. Native thick and thin filaments from TG mice were prepared and used on the thick filament sliding assay as described previously [7]. SRX measurements were performed on detergent-skinned muscle from TG and NTG hearts as previously described [6, 25].

2.4. X-Ray Diffraction Analysis

X-ray diffraction patterns were collected using fresh t-clipped, skinned left ventricular papillary tissue set at a length of 2.1 μm in relaxing solution at pCa 9.0 and the small-angle diffraction instrument on the BioCAT beamline 18 ID at the Advanced Photon Source, Argonne National Laboratory, as described previously [3].

2.5. Q-space MRI

High angular resolution diffusion-weighted (generalized Q-Space, b=750 s/mm², 512 gradient directions) and conventional T2-weighted MRI images were acquired on a 7T MR scanner equipped with a Cryoprobe apparatus (Bruker, MA) for transgenic (n=4) and wild-type (WT) (n=4) excised hearts. Regional inter-voxel directional coherence was reconstructed visually in the form of diffusion tractography in DSI Studio (Feb. 2019 build) with an angular threshold of 30° and a length constraint of 0–10 mm. Six radial spherical regions of interest (ROIs) = 1.00mm were positioned to span the transmural depth of the left ventricular wall from the epicardium to the endocardium at a total of 15 locations for each heart, and mean data were obtained for each condition and lateral transmural position (3 transmural ROI arrays per lateral position) across the myocardium [9, 18].

2.6. Statistical Analysis

All data are represented as the mean ± standard error of the mean (SEM) unless otherwise indicated. Statistical analyses were performed using GraphPad Prism (version 6.0). Normality testing of data distribution was performed with a D’Agostino & Pearson test. Data were analyzed using a one- or two-way ANOVA with a Tukey’s post-hoc test or a Student’s t-test for parametric data and a Kruskal-Wallis with a Dunn’s post-hoc test or a Mann-Whitney U test for nonparametric data where appropriate. Statistical significance was defined as P < 0.05.

3.0. Results

3.1. cMyBP-C^ΔC0-C1f incorporates normally into the sarcomere

The primary aim of this study was to determine the necessity of the C0-C1f region in the maintenance of regular cardiac structure and function. To accomplish this, we generated cardiac-specific TG mice expressing truncated cMyBP-C in which the N’-terminal C0-C1f region was removed (cMyBP-C^ΔC0-C1f) (Fig. 1A). In this truncated protein (110 kDa molecular weight), the PKA recognition sequence for Ser-273 motif is deleted, but the Ser-282 and -302 residues are preserved, compared to the full-length, endogenous cMyBP-C (150 kDa). Expression of cMyBP-C^ΔC0-C1f was determined by SDS-PAGE (Online Supplemental Fig. 1A) and Western blot analysis using C’-specific anti-cMyBP-C antibodies (Fig. 1B and C). Based on the C’ antibody, cMyBP-C^ΔC0-C1f comprised 81 ± 2% of total cMyBP-C expression in TG mouse hearts. Mass spectrometry analyses demonstrated that the myosin to cMyBP-C ratio was 10.6 ± 1.8 to 1, equal to that in NTG mouse hearts [7], and the level of cMyBP-C^ΔC0-C1f was 85 ± 6% in TG mouse hearts, confirming our Western blot data. However, total cMyBP-C protein content, including both endogenous and TG, was not significantly different between TG and NTG hearts, confirming that the total stoichiometry of cMyBP-C was maintained (Fig. 1C). Immunofluorescence staining of isolated cardiomyocytes from TG hearts confirmed proper localization of cMyBP-C^ΔC0-C1f to the classical doublet pattern on either side of the sarcomere Z-disk where endogenous cMyBP-C also localizes (Fig. 1D, and Online Supplemental Fig. 2). To further validate whether cMyBP-C^ΔC0-C1f protein had integrated into the myofilaments, cardiac proteins were fractionated as total, soluble and myofilamentous from TG and NTG hearts, and Western blot analysis was performed with newly generated C5- and C8-domain specific cMyBP-C antibodies (Online Supplemental Fig. 3). Results confirmed that the cMyBP-C^ΔC0-C1f protein was fully integrated into the myofilaments, similar to endogenous cMyBP-C (Online Supplemental Fig. 4).

Phospho-specific antibodies demonstrated hyperphosphorylation of both endogenous cMyBP-C and exogenous cMyBP-C^ΔC0-C1f in TG hearts at Ser-273 and -302 compared to NTG controls (p < 0.01, Online Supplemental Fig. 1B, D and F). Owing to disruption of the serine 273 PKA recognition motif, no phosphorylation was observed at this residue in cMyBP-C^ΔC0-C1f, while phosphorylation of Ser-282 and -302 was significantly elevated (Online Supplemental Fig. 1B, D, E and F). No changes in Ser-23 and -24 of cardiac troponin I was observed (Online Supplemental Fig. 1 B and C). Collectively, these data show that cMyBP-C^ΔC0-C1f readily incorporates into the myofilament, resulting in the hyperphosphorylation of specific serine residues within the M-domain in TG mouse hearts, possibly as a compensatory mechanism to improve contractility.

3.2. The C0-C1f region of cMyBP-C is essential to maintain cardiac function

Next, we compared whole heart size, morphology, and function between TG and NTG mice. Isolated hearts from TG mice were appreciably larger than NTG hearts, as measured by heart/tibia length ratio (P<0.01) and 2D-confocal imaging of the perfused hearts, indicating global cardiac enlargement (Fig. 2A and B, and Online Supplemental Fig. 5). Furthermore, to determine the molecular changes that result from cMyBP-C^ΔC0-C1f expression in TG hearts, RNA-Seq analyses were performed, followed by quantitative PCR for selected genes on mice 7–8 months of age, a time when the phenotype was well established in TG hearts. RNA-Seq analysis revealed the upregulation of several genes involved in the biology of cardiac remodeling, contraction and calcium signaling in TG, compared to NTG, hearts (Online Supplemental Fig. 6A and B). As expected, quantitative PCR analysis determined that total cMyBP-C gene expression was elevated by 6-fold (p < 0.001) in TG hearts compared to NTG hearts, confirming our RNA-Seq data (Online Supplemental Fig. 6C). Elevation of the cardiac hypertrophic markers MYH7 and NPPA observed in RNA-Seq was also verified by quantitative PCR (Online Supplemental Fig. 6D and E). RNA-sequencing further revealed a significant upregulation in several sarcomeric proteins, including many skeletal isoforms of these proteins. Notably, the expression levels of MYBPC2, which encodes the fast-skeletal isoform of MyBP-C, was found to be upregulated in TG hearts, compared to NTG hearts (Online Supplemental Fig. 6A and B). The expression of fast-skeletal MyBP-C has been previously detected in cMyBP-C null hearts undergoing remodeling [26].

Cardiac function was then measured by echocardiography and revealed a significant decrease (p < 0.05) in left ventricular ejection fraction (%EF) and fractional shortening (%FS) in TG hearts, compared to NTG hearts at 3 months of age, and a further deterioration at 6 months (p < 0.05), indicating progressive HF (Fig. 2C and D, and Online Supplemental Table 1). Changes in chamber size, fractional shortening and global longitudinal strain were also consistent with progressing dilated HF in TG hearts at both 3 and 6 months of age (p < 0.01, Online Supplemental Table 1). Additionally, pressure-volume (PV) loop analyses revealed a significant decrease in dP/dt_max (p < 0.05) and increase in dP/dt_min, P_ed, and Tau (p < 0.01) in TG, compared to NTG, hearts at 3 months of age (Online Supplemental Fig. 7A-F and Online Supplemental Table 2), suggesting impaired contractility and relaxation in TG hearts. No change in heart rate between NTG and TG hearts was observed (Online Supplemental Fig. 7G).

Both Masson’s Trichrome staining and second-harmonic imaging revealed increased fibrosis in TG hearts (Fig. 2E and F, and Online Supplemental Fig. 8). Also, WGA staining revealed increased cross-sectional area of cardiomyocytes from TG, compared to NTG, hearts (Online Supplemental Fig. 9). These data suggest that the cardiac dysfunction induced by loss of C0-C1f leads to fibrotic remodeling and cardiomyocyte hypertrophy in TG hearts. In contrast to the above findings, electron microscopic imaging of TG and NTG cardiac sections and negatively stained myofibrils revealed normal sarcomere ultrastructure, noting, however, that C-zone stripes were generally weaker in TG hearts (Online Supplemental Fig. 10, and Online Supplemental Table 3). Assessment of myoarchitecture in the excised whole heart with generalized Q-space MRI revealed a normal distribution of myofiber helix angles across the ventricular wall (Fig. 3) in TG and NTG samples. These data indicate that physiological effects of C0-C1f loss described in this report are not attributable either to alterations of sarcomere morphology or effects on the shape and distribution of myocytes in the ventricular wall. However, larger myocytes were observed in TG hearts. Collectively, these data demonstrate the necessity of the C0-C1f region in maintaining both normal cardiac structure and function, thus supporting our central hypothesis.

Fig. 3. — We employed Q-space MRI, with 512 gradient directions, B value of 750 s/mm², and a voxel size of 100 × 100 × 300 μm to define cardiac myoarchitectural features in excised NTG and TG hearts (mixed sex). Transverse sections with fibers colorized based on helix angle were shown for the NTG (A) and TG (B) hearts. Using a flywheel architectural depiction, we showed a similar transmural helix angle distribution, viewed from the endocardium to epicardium, in both the NTG (C) and TG (D) hearts. Average transmural helix angle distribution in NTG and TG was shown in panel E. Both NTG (n=3) and TG (n=3) hearts displayed a similar linearized average transmural helix angle slope (E), p=0.3.

3.3. Ablation of C0-C1f in cMyBP-C results in faster thin filament sliding in the C-zone

Adding exogenous C0-C1f proteins to an in vitro motility assay slows down actin filament velocity generated by mouse cardiac myosin [7]. Therefore, we employed a total internal reflection fluorescence microscopy assay to determine if genetic deletion of the C0-C1f region would eliminate slowing of actin velocity in the C-zone of native thick filaments [7]. Displacement-time graphs demonstrated constant velocity (Fig. 4 A and B) for actin filaments sliding along thick filaments isolated from the TG hearts. The subsequent slower phase of actin sliding velocity previously observed within the C-zone of native thick filaments isolated from NTG mouse hearts (Fig. 4 A and B, dotted lines taken from Previs MJ et al. [7]) was not observed on thick filaments from TG mice. When fitting our data with a two-phase regression, <10% of the velocities in TG samples could be fit with two phases. When fit with one phase, the mean velocity was approximately 1.7 μm/s in TG hearts, which is indeed slower than that in NTG hearts. Therefore, some slowing is likely in the C-zone in TG, compared to NTG hearts; however, this is at the limit of detection in the assay. This confirmed the earlier report that interactions between C0-C1f domains and either actin or the myosin head are responsible for slowing actin sliding velocity within the thick filament C- zone (Fig. 4 C and D) (10).

Fig. 4. — (A) Displacement-time graphs for thin filaments sliding on thick filaments having cMyBP-C^ΔC0-C1f, demonstrating a single fast velocity phase in TG thick filaments with cMyBP-C^ΔC0-C1f (n = 6); dotted line indicates expected results with slowing as occurs in the presence of native cMyBP-C [7]. Inset for 20 cMyBP-C^ΔC0-C1f filaments with distance traveled during fast velocity phase identified (raw data). (B) As in (A), but with a frequency-velocity plot. (**C & D**) Illustration of a thin-filament shard traveling over one-half of a thick filament isolated from control sarcomere with cMyBP-C showing two phases of velocity (C, fast and slow) and a single fast velocity phase in the sarcomere expressing cMyBP-C^ΔC0-C1f (D).

3.4. Effect of C0-C1f loss on thick and thin filament mass distribution in the sarcomere

To determine whether loss of the C0-C1f region alters the distribution of cross-bridge mass between thick and thin filaments, we performed X-ray diffraction studies on fresh, detergent-skinned papillary muscles from TG and NTG mice under resting conditions (Fig. 5A). The ratio of the 1,0 and 1,1 equatorial intensities (I_1,1/I_1,0) can be used to determine shifts of molecular mass (cross-bridges) from the thick to thin filament [27, 28]. Here, the I_1,1/I_1,0 intensity ratio in skinned papillary muscle from TG hearts was significantly lower (p < 0.05) (Fig. 5B and C), while interfilament lattice spacing was significantly higher (p < 0.01) (Fig. 5B and C) than that of NTG controls. We next measured the proportion of myosin heads in the super-relaxed (SRX) state in TG vs. NTG skinned cardiac muscle fibers. No significant difference in the proportion of myosin heads in SRX (~15%) was observed between these groups (Online Supplemental Fig. 11). In the resting myocardium, these results suggest that loss of the C0-C1f region leads to a net transfer of myosin head mass closer to the thick filament backbone, without significant enrichment of the population in the SRX state, and greater interfilament spacing.

Fig. 5. — (A) Representative X-ray patterns from skinned TG and NTG papillary muscle fibers in relaxing solution with 1,0 (inner two spots, arising from thick filament-associated mass) and 1,1 (outer two spots, arising from thick and thin filament-associated mass) equatorial spots labeled. (B) Intensity traces along the equator from X-ray patterns of TG and NTG skinned papillary muscles. (C) Left, ratio of intensities of the 1,1 and 1,0 equatorial X-ray reflections (I_1,1/I_1,0) (n = 10 TG and 7 NTG fibers, mixed sex). Right, interfilament spacing (IFS) in TG or NTG papillary muscle fibers (n = 8 fibers per genotype). Data are expressed as mean ± S.E.M., and statistical analyses were performed by unpaired t-test. †p< 0.05, *p < 0.01.

3.5. N’-terminal ablation of cMyBP-C reduces sarcomere contractility, but does not alter calcium handling

Detergent-skinned cardiomyocytes set to a 1.9 μm sarcomere length from TG and cMyBP-C null (t/t) hearts, which show dilated cardiomyopathy [22], were used to test for mechanical deficits in the myofilaments resulting from the loss of the C0-C1f region and cMyBP-C, respectively, compared to NTG controls (Online Supplemental Table 4). At pCa 4.5, we found that skinned TG cardiomyocytes had a significant deficit in maximal force development (p < 0.0001), while calcium sensitivity was significantly decreased (p < 0.0001), and the Hill slope was steeper (p < 0.01), compared to NTG controls (Fig. 6A-C). These mechanical deficits further demonstrate the necessity of C0-C1f in maintaining normal contractile function. Interestingly, while maximal force was significantly reduced in skinned TG cardiomyocytes compared to NTG controls, maximal force was still further reduced in skinned cardiomyocytes from t/t hearts (Online Supplemental Fig. 12, and Online Supplemental Table 4). The rate of tension redevelopment (k_tr) was significantly faster in k_tr from TG skinned cardiomyocytes compared to NTG skinned cardiomyocytes (p < 0.0001, Fig. 6D). In a parallel experiment, preincubation with an antibody targeted to the C0-domain of cMyBP-C significantly reduced maximal tension in NTG, but had no effect in skinned TG cardiomyocytes, confirming the importance of the N’-region in maintaining contractile function (Online Supplemental Fig. 13).

Fig. 6. — (A) Maximal force generation in NTG (white bar) and TG (red bar) cardiomyocytes at maximal calcium concentration, pCa 4.5 (n = 9 cardiomyocytes from 8 animals per group). (**B-C**) Calcium sensitivity indexed as pCa₅₀ and Hill coefficient indexed as nH (n= 9 cardiomyocytes from 3 animals per group). (D) Rate of tension redevelopment (*k_tr*) in single cardiomyocytes from TG and NTG hearts at maximal calcium concentration, pCa 4.5 (n = 9 cardiomyocytes from 8 animals per group, mixed sex). (E) Maximal force generation in nonfailing (light blue) and failing (purple) human cardiomyocytes in the presence or absence of 10μM human rC0-C2 (n = 9 cardiomyocytes from 3 hearts per group). (**F-G**) pCa₅₀ and nH from nonfailing and failing human cardiomyocytes (n= 9 cardiomyocytes from 3 hearts per group). (H) Rate of tension redevelopment (*k_tr*) nonfailing and failing human cardiomyocytes at maximal calcium concentration (n = 9 cardiomyocytes from 3 hearts per group). Data are expressed as mean ± S.E.M., and statistical analyses were performed by ordinary Two-way ANOVA, followed by Tukey’s multiple comparison test. †p < 0.05, *p < 0.01.

To further determine whether reduced contractile properties in the TG skinned myofibrils are associated with altered calcium handling, functional analyses of contractile mechanics and calcium kinetics were performed in isolated fresh cardiomyocytes from TG and NTG hearts at 3 months of age, using the IonOptix system [24]. Results from this experiment showed that relaxed sarcomere length at baseline was significantly shortened in TG cardiomyocytes (1.57 ± 0.01 μm, p <0.001), as compared with NTG cardiomyocytes (1.70 ± 0.01 μm) (Online Supplemental Fig. 14 and Online Supplemental Table 5). Similarly, the rate of contraction (p < 0.01) and the time to 50% contraction (p <0.001) werealso significantly different in TG vs. NTG cardiomyocytes. The contractile defects in TG cardiomyocytes correlate with cardiac dysfunction observed at the whole organ level. However, we observed no change in calcium kinetics. Taken together, these results point to functional deficits in TG cardiomyocytes, consistent with reduced cardiac function, as previously observed by echocardiography, indicating that the N’-region of cMyBP-C is a key regulator of contractility.

3.6. Restoration of previously ablated C0-C2 promotes normal thick and thin filament interactions

The C0-C2 region alone has been previously shown to rescue biochemical defects of cMyBP-C deficiency in neonates in vivo [20]. Thus, we next hypothesized that the C0-C2 region alone, instead of full-length cMyBP-C, could restore thick and thin filament interactions in TG and t/t adult cardiomyocytes. Since the presence of C0-C1f proteins in the myofilament is pathogenic and can impair contractile function in vitro and in vivo [3, 11, 13], we used rC0-C2 to determine whether TG contractile function could be restored. To perform this experiment, TG and t/t skinned cardiomyocytes were treated with 10 μM mouse rC0-C2 protein [22]. Addition of rC0-C2 increased force production and calcium sensitivity in TG skinned cardiomyocytes (Fig. 6A and B) and t/t groups (Online Supplemental Fig. 12), compared to NTG group. Notably, maximal force in TG skinned cardiomyocytes treated with rC0-C2 was no different from that of untreated NTG skinned cardiomyocytes. Both TG and NTG skinned cardiomyocytes exhibited similarly increased k_tr upon rC0-C2 treatment. Next, to translate these findings to treat human HF, we used skinned cardiomyocytes from three non-failing and three non-ischemic failing human hearts (Online Supplemental Table 6). Before we used these samples for functional studies, we assessed the quality of the samples and presence of cMyBP-C proteolysis by Western blot analyses. As described previously [11], the C0-C1f region of cMyBP-C was cleaved to varying levels from the full-length protein in failing human heart samples, an observation that was not detected in samples from nonfailing hearts. We used novel anti-C5-domain and -C8-domain specific antibodies, compared to N’-specific antibodies, and observed an enrichment of C’-terminal cMyBP-C fragments at around 110 kDa in the failing samples with significant reduction in cMyBP-C phosphorylation, compared to nonfailing samples (Online Supplemental Fig. 15). Skinned cardiomyocytes from failing hearts showed a reduction in maximal force development and calcium sensitivity (p < 0.001) and increased Hill coefficient (p < 0.01) (Fig. 6E-H) in comparison to the nonfailing cardiomyocytes. Treatment of failing skinned cardiomyocytes with 10 μM of human rC0-C2 protein significantly (p < 0.0001) restored maximal force at pCa 4.5. Moreover, rC0-C2 protein treatment showed a significant improvement in calcium sensitivity (p < 0.0001) and Hill coefficient (p < 0.05) in failing skinned cardiomyocytes. Similar to the mouse model, k_tr values in both failing and nonfailing skinned cardiomyocytes were significantly increased (p < 0.0001), indicating restoration of thick and thin filament interactions.

4. Discussion

We demonstrate that the ablation of the N’-region of cMyBP-C, a molecular perturbation that partially emulates the pathological cleavage of cMyBP-C resulting from cardiac ischemia and HF, results in altered actomyosin dynamics, fibrogenesis and DCM. On the other hand, this modification is associated with compensatory hyper-phosphorylation of the unablated phosphoregulatory domains of cMyBP-C in the TG mouse model developed here. These observations provide substantial clarification of the physiological role of the N’-region of cMyBP-C in the regulation of actomyosin interactions and in the pathogenesis of ischemic cardiomyopathy and, potentially, certain forms of HF.

4.1. C0-C1f is essential for normal cardiac contractility

Since MYBPC3 mutations constitute the preeminent cause of genetic cardiomyopathies in humans, determining the structure-function relationship of cMyBP-C is clinically significant. It is well established in the literature that cMyBP-C is necessary for normal cardiac function, as a complete knockout of cMyBP-C in mice shows severe cardiac hypertrophy and HF [22, 29]. However, the structure-function relationship of the N’-region of cMyBP-C in regulating cardiac function in vivo remains unclear. Based on in vitro studies, the N’-region of cMyBP-C interacts with myosin regulatory light chain [30], actin [31], α-tropomyosin [3] and myosin S2 [32]. These interactions regulate and modulate sarcomere structure and function [7, 8]. Here, we observed hyperphosphorylation of cMyBP-C^ΔC0-C1f in TG hearts. As previously reported [10], we proposed that the hyperphosphorylation of cMyBP-C in TG hearts may act as a compensatory mechanism that attempts to increase or decrease cross-bridge interactions and cycling rates in a calcium-dependent manner. However, while this may be effective in NTG hearts, this effort is futile in TG hearts containing the cMyBP-C^ΔC0-C1f protein owing to the lack of actin-binding sites. That is, the phosphorylation of cMyBP-C^ΔC0-C1f at Ser-282 and Ser-302 likely has little, to no, effect owing to loss of the N’-region preventing binding to actin [33]. However, the exact mechanisms by which this would occur are difficult to determine and may include a temporal component to cMyBP-C phosphorylation during cardiac remodeling. It is currently thought that the phosphorylation of cMyBP-C at low Ca²⁺ relieves its constraint on myosin S2, thereby releasing myosin heads and potentially activating the thin filament to accelerate cross-bridge kinetics [34]. In contrast, at high Ca²⁺, cMyBP-C binding to the thin filament may slow thin filament sliding within the C-zone. Therefore, in the TG mouse model, hyperphosphorylation of the cMyBP-C^ΔC0-C1f protein may increase cross-bridge cycling kinetics at low Ca²⁺, accounting for part of the increased k_tr we observed, but may also act to slow cross-bridge cycling rate and increase cross-bridge lifetime at high Ca²⁺ levels by the increase of cMyBP-C molecules binding to actin. Again, however, this effort at high Ca²⁺ would be fruitless in an N’-truncated form of cMyBP-C, such as that demonstrated by the cMyBP-C^ΔC0-C1f protein, by the loss of actin binding sites and inability to bind to actin. Additionally, ablation of the PKA phosphorylation motif for phosphorylation of serine 273 in the cMyBP-C^ΔC0-C1f protein may play a role in the ability (or inability) of this protein to regulate contraction.

cMyBP-C is proteolyzed during MI [10] and HF [11] such that C0-C1f is cleaved from the full-length protein. The C0-C1f fragment itself was sufficient to cause contractile dysfunction in isolated rat ventricular myocytes and human myofilaments [3], activate macrophages [35] and cause HF in mice [11]. Furthermore, addition of C0-C1f in vitro inhibited actin filament sliding over myosin [7, 16]. A potential mechanism underlying the inhibition of velocity involved the interaction of C0-C1f with actin, but not the myosin S2 region [3, 16], thereby acting as a viscous load to impede actin motility. In contrast, the C0-C2 region of cMyBP-C can facilitate both actin and myosin interaction [3, 16] and, hence, bridge thin and thick filaments to modulate cardiomyocyte contractility. The data presented herein indicate that the C0-C1f region is necessary for normal cardiac function in vivo and that the C0-C2 region is sufficient to regulate normal contractile function in vitro. TG mice expressing the cMyBP-C^ΔC0-C1f protein developed cardiac enlargement, ventricular dilation and increased myocardial fibrosis.

Interestingly, TG mice developed significant deficits in cardiac function, as measured by ejection fraction, fractional shortening and global longitudinal strain. Taken together, these results indicate that the loss of C0-C1f can induce a dilated cardiomyopathy phenotype, confirming its importance in regulating cardiac function in vivo. It has been previously demonstrated that ablation of the C0-domain alone in mice results in hypercontractility [36], suggesting that the N’-region of cMyBP-C is a key region for the regulation of normal cardiac function. In contrast, ablation of the proline/alanine-rich region and C1 immunoglobulin domain in mice had no effect on cardiac pathology [37], adding further complexity to the N’-region, indicating that a thorough and systematic investigation is warranted to further define the role of the N-’ region of cMyBP-C.

4.2. C0-C1f is necessary to regulate sarcomere function

Since no major changes in cardiac ultrastructure or myoarchitecture of TG mice were identified, we propose that the observed structural and functional deficits are primarily driven by direct changes in actomyosin regulation by the cMyBP-C^ΔC0-C1f protein. Since the N-terminal domains of cMyBP-C can interact with both thin and thick filaments to regulate contractile function, loss of C0-C1f would be expected to result in aberrant actomyosin regulation. Indeed, in vitro motility assays measuring actin sliding velocity over native thick filaments demonstrated that TG thick filaments could not slow actin sliding over the C-zone, as had been observed in NTG thick filaments [7]. Importantly, this effect was also previously demonstrated in NTG thick filaments in which C0-C1f had been enzymatically cleaved with calpain [7, 9]. These findings are consistent with previous reports in the literature suggesting that the primary actin binding domains of cMyBP-C exist within the C0-C1f region [3]. Furthermore, the loss of actin binding domains may explain the weakening of the transverse cMyBP-C stripes in TG myofibrils, assuming that these stripes arise largely from perpendicular extension of cMyBP-C from thick to thin filaments, as suggested by electron tomography [38]. Our studies further indicate that the loss of C0-C1f directly affects actomyosin interactions, leading to reduced maximal force production of permeabilized muscle from TG compared to NTG. The loss of actin binding may promote interaction between cMyBP-C and myosin and inhibit cross-bridge formation within the C-zone [3]. This interaction would reduce the number of recruitable cross-bridges, thus contributing to the reduction in maximal force generation. Consistent with this proposed mechanism, X-ray diffraction demonstrated retention of myosin heads closer to the thick filament backbone in permeabilized TG papillary muscles than in NTG, interestingly, without significantly enriching the population of heads in the SRX state. When myosin heads are sequestered into the SRX, they are less available for contraction, as they are held farther away from the thin filament, and catalytic activity is shut down through multiple inter- and intra-molecular interactions, a process that is dependent on the interaction of cMyBP-C with myosin, likely through the N-terminus [25]. Our results suggest that interactions that hold the myosin heads close to the backbone can still occur in the absence of the cMyBP-C N-terminus, but that the N-terminus is required for establishment of the full SRX state with its greatly reduced ATPase-rate. This intriguing possibility needs to be followed up in later studies. Notably, recent data suggest the possibility that the N-terminus of cMyBP-C may bind to actin and myosin heads, but only when myosin heads are near actin filaments [39], which could explain why the loss of the N-terminus in cMyBP-C^ΔC0-C1f had no impact on the SRX state in TG, compared to NTG, fibers. Initial sarcomere length was controlled in our X-ray experiments; therefore, the difference in lattice spacing most likely does not result from a difference in sarcomere length, which is not expected to change under relaxed conditions. Increased lattice spacing could, however, explain a deficit in F_max and reduced calcium sensitivity in our TG model [40,41]. This suggests that increased lattice spacing might be a common feature of dilated cardiomyopathy, which could be verified in future experiments.

While maximal force production was reduced in myocytes from TG mice, it is interesting to note that ktr was significantly faster (Fig. 6). A similar acceleration of tension redevelopment has been demonstrated in cMyBP-C null myocytes [42]. Clearly, the sequestration of myosin heads discussed above cannot account for this. Rather, we propose that the loss of the viscous load applied to actin by the N’-terminal domains of cMyBP-C is responsible. Specifically, when actin moves through the C-zone, sliding velocity is reduced, a phenomenon which can be attributed to its interaction with the N’-terminal domains of cMyBP-C [7]. Our in vitro motility assays demonstrated that actin can move through the C-zone unimpeded in TG thick filaments, similar to findings previously published in which t/t thick filaments were used [7]. Both results highlight the importance of the C0-C1f region in regulating actomyosin interactions. However, we were concerned that the low level of endogenous full-length cMyBP-C expression in TG hearts might diminish the impact of cMyBP-C^ΔC0-C1f expression on contractile function. To address this concern, maximum force generation was measured in TG and NTG cardiomyocytes in the presence and absence of an N’-targeted antibody that binds only full-length cMyBP-C. Interestingly, incubation of NTG cardiomyocytes with this N’-targeted antibody reduced contractile function to a level similar to that of untreated TG cardiomyocytes. However, in TG myocytes, no further reduction in maximal force generation was observed, suggesting that the residual full-length cMyBP-C in TG hearts only minimally contributes to the contractile force. Thus, we conclude that the TG cMyBP-C model exhibits both increased contractile kinetics and reduced contractile force via loss of normal actomyosin regulation by C0-C1f.

4.3. Introduction of the C0-C2 region restores thin and thick filament interactions

When rC0-C2 protein was introduced into human and mouse skinned ventricular myocytes, the Kentish laboratory notably showed that rC0-C2 alone was sufficient to activate force production in the absence of calcium [19], indicating that C0-C2 is sufficient to regulate thick and thin filament interactions. The C0-C2 region (448 amino acids) includes C0, a Pro-Ala-rich region, and the C1, M, and C2 domains (Fig. 1). This region, unlike the C0-C1f region, interacts with both actin and the myosin S2 region [13, 16] and has been extensively characterized in vitro [8, 13, 16, 19, 31, 43–50]. Subsequently, numerous studies indicated that rC0-C2 can interact with both actin and myosin to modulate actomyosin contractility [3, 8, 16, 18, 31, 51]. Furthermore, in contrast to rC0-C1f, rC0-C2 is a myosin activator that increases contractility at physiological Ca2+ levels [8]. However, it is not known whether in vivo rC0-C2 expression in the heart at a level similar to that of full-length cMyBP-C results in therapeutic restoration of cardiac function. To address this issue, Li et al. demonstrated that AAV9 gene transfer of C0-C2 can regulate in vivo cardiac function, thereby increasing ejection fraction and reducing cross-bridge kinetics and the histopathology of cardiomyopathy [20]. These data suggest the increased therapeutic potential of the C0-C2 region for normalizing myofilament function during disease. Studies also showed that the Pro-Ala–rich region in the rC0-C2 protein interacts with α-TM to modulate sarcomere function [1, 8]. Since contractile deficits detected in the cMyBP-CΔC0-C1f TG model likely arise from direct changes in the actomyosin cycle, we hypothesized that the C0-C2 region of cMyBP-C is sufficient to modulate actomyosin interaction and rescue contractile dysfunction in TG and t/t cardiomyocytes. Thus, we tested whether incubation of rC0-C2, which can bind thick and thin filaments in permeabilized muscle, could restore contractile function. Treatment of TG myocytes with rC0-C2 improved maximal force production compared to that of untreated NTG controls and showed partial restoration of calcium sensitivity. However, while treatment of t/t myocytes with rC0-C2 increased maximal force, rC0-C2 could not raise force production to levels observed in TG myocytes, even though the magnitude of the increase was similar (Figs. 6 and S8). This again supports the hypothesis that both N-terminal domains and C-terminal domains of cMyBP- C are likely involved in modulating actomyosin force generation. Furthermore, when non-ischemic human HF samples were treated with rC0-C2, force production was similarly increased. Recombinant C0-C2 has been used in permeabilized muscle preparations in the past to understand cMyBP-C biology [19, 52], but to the best of our knowledge, this is the first study to show that the introduction of rC0-C2 domains may restore thick and thin filament interactions, resulting in improved myocardial force generation. Since we also observed an improvement in contractility in cMyBP-C null and failing human myofibrils upon treatment with rC0-C2, we postulate that the actin- and myosin-binding sites on rC0-C2 are mutually exclusive and that the dynamic regulation of these interactions modulate contractility.

4.4. Study Limitations

We recognize several limitations in this study. The data definitively demonstrate that cMyBP-C^ΔC0-C1f is expressed solely in the sarcomere with no detection in non-sarcomeric compartments (Online Supplemental Figure 4). As we sought to mimic a more physiological condition during certain forms of HF in which both cMyBP-C^ΔC0-C1f and endogenous cMyBP-C are present in cardiomyocytes (Online Supplemental Figure 15), we used an NTG background to develop our TG mice and not a cMyBP-C null background. The presence of bands at the Z-line by immunofluorescence analyses in Online Supplemental Figure 2C resulted from the imaging procedure, not a genuine expression of a cMyc-labeled protein at the Z-line, as shown in the image. The absence of an anti-cMyc-labeled protein in NTG hearts was also confirmed by Western blot analyses (Figure 1B and Online Supplemental Figure 4) in which only endogenous full-length cMyBP-C is expressed. Furthermore, the presence of weak bands in subcellular fractionation of TG in non-sarcomeric compartments resulted from the preparation procedure, not genuine expression in cytosolic compartments, as endogenous WT cMyBP-C is also present in the blot from NTG hearts (Online Supplemental Figure 4). Importantly, while we observed that treatment of rC0-C2 in TG hearts led to an increase in force production, when compared to untreated NTG controls, it is likely that rC0-C2 decorates the length of the sarcomere, as shown previously [19], rather than localizes specifically to the C-zone. Therefore, whether the impact on contractile force results from restoration of normal cMyBP-C, the addition of a viscous load, or another mechanism regulating contractility in vivo remains to be determined. Furthermore, regarding sarcomere ultrastructure obtained with negatively stained myofibrils, we cannot exclude the possibility that the weaker C-zone stripes result from fewer cMyBP-C molecules per stripe as we did not and could not measure the number of cMyBP-C molecules per stripe based on the EM images. Weakness of the stripes could possibly be a result of changed stoichiometry, or possibly result from different orientation of cMyBP-C on the filament owing to removal of the N-terminus, which may bind to actin in the NTG sarcomeres. Regarding the hyperphosphorylation of cMyBP-C and cMyBP-C^ΔC0-C1f relative to increased lattice spacing, as mentioned above, the observed hyperphosphorylation may serve as a compensatory mechanism during cardiac remodeling, or it may serve to alter k_tr in a calcium-dependent manner or based on the level of actomyosin interactions in the intact myocardium. Increasing cMyBP-C phosphorylation could be presumed to relieve the constraint of cMyBP-C on myosin heads, allowing for their movement closer to actin filaments. However, the X-ray and steady-state mechanical studies performed here were conducted on skinned fibers in the absence of kinases and phosphatases, and the X-ray studies were only performed under relaxing conditions. Therefore, the phosphorylation status of cMyBP-C could have been altered, compared to its status in vivo, or may have had no additional effect on the interaction of cMyBP-C^ΔC0-C1f with myosin by the loss of certain myosin-binding regions in cMyBP-C^ΔC0-C1f and the Ser-273 site. Thus, our conclusions are speculative and theoretical when relating the data to the in vivo condition in these areas. Lastly, a detailed analysis of the effects of altering the phosphorylation status of cMyBP-C^ΔC0-C1f, relative to lattice spacing and the functional effects on in vitro contractility, was outside of the scope of this study, but may prove valuable for future research.

4.5. Conclusions

Together, these studies establish the necessity of the C0-C1f and sufficiency of the C0-C2 N’-regions of cMyBP-C in regulating cardiac function. In particular, the results determine the critical role of the N’-region of cMyBP-C at the sarcomere and whole-heart levels. The interactions of the C0-C2 domains of cMyBP-C with the thick and thin filaments are critical to regulating contractility, leading to the development of potential cardioprotective therapeutic approaches to improve cardiac function in HF.

Supplementary Material

NIHMS1689212-supplement-1.pdf^{(9.9MB, pdf)}

HIGHLIGHTS.

Deletion of the N’-C0-C1f region of cMyBP-C results in actomyosin dysregulation, and, hence, contractile dysfunction, in combination with a form of dilated cardiomyopathy.
Administration of rC0-C2 to permeabilized cardiomyocytes from transgenic cMyBP-C^ΔC0-C1f mice, cMyBP-C null mice, and biopsy of tissue of human heart failure restored steady-state actomyosin interactions.
C0-C2 region of cMyBP-C augments contractility and thin-thick filament interactions, whereas its absence mimics ischemic- and non-ischemic-associated degradation of cMyBP-C and associated cardiomyopathy.

Acknowledgements

Dr. Sadayappan has received support from National Institutes of Health grants (R01 HL130356, R01 HL105826, R01 AR078001, R38 HL155775 and R01 HL143490), American Heart Association, Cardiovascular Genome-Phenome Study (15CVGPSD27020012), Catalyst (17CCRG33671128), Institutional Undergraduate Student (19UFEL34380251) and Transformation (19TPA34830084) awards, the PLN Foundation (PLN crazy idea) awards, as well as AstraZeneca, MyoKardia, Merck and Amgen. This work was further supported by an American Heart Association Predoctoral Fellowship to Dr. Lynch (15PRE22430028) and Dr. Kumar (17PRE33630192) and Postdoctoral Fellowship to Dr. Kuster (13POST17220009) and Dr. McNamara (17POST33630095). Additional support came from the National Institutes of Health to Dr. Previs (R00 HL124041) and Dr. Warshaw (R01 HL126909). Dr. Craig was supported by NIH grants P01 HL059408, R01 AR067279, and R01 HL139883. Dr. Janssen was supported by NIH grant R01 HL113084. Dr. de Tombe was supported by NIH grant PO1 HL62426. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357 and supported by grant P41 GM103622 from the National Institute of General Medical Sciences of the National Institutes of Health. The authors thank Christine E. Seidman and Jonathan G. Seidman, Harvard Medical School, Boston, MA 02115, USA, for providing the cMyBP-C(t/t) mouse model.

Abbreviations

cMyBP-C^FL: full-length cardiac myosin binding protein C
cMyBP-C^ΔC0-C1f: cMyBP-C lacking the C0-C1f region
TG: transgenic
WT: wild type
S2: subfragment 2
RLC: regulatory light chain
non-transgenic: NTG
C0-C1f: the first 271 residues of N’-region of cMyBP-C
I-R: ischemia-reperfusion
N’: amino terminal
t/t: cMyBP-C null homozygous mice
HF: heart failure
C0-C2: the first 448 residues of N’-region of cMyBP-C

Footnotes

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DISCLOSURE

Dr. Sadayappan provides consulting and collaborative research studies to the Leducq Foundation (Transatlantic Network 18CVD01, PLN-CURE), Red Saree Inc., Greater Cincinnati Tamil Sangam, AstraZeneca, MyoKardia, Merck and Amgen, but such work is unrelated to the content of this manuscript. Dr. Lynch is currently employed at AbbVie and performed all work related to the content of this manuscript prior to his employment at AbbVie. Dr. Singh has been a postdoctoral fellow of Amgen, starting from June 2019, and performs research at the University of Cincinnati. No other disclosures are reported. AbbVie and Amgen did not provide funding support and did not participate in the planning, research, data analysis and writing related to the content of this manuscript. No other disclosures are reported.

Supplementary data

Supplementary material

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18.Taylor EN, Hoffman MP, Barefield DY, Aninwene GE 2nd, Abrishamchi AD, Lynch TL t, et al. Alterations in Multi-Scale Cardiac Architecture in Association With Phosphorylation of Myosin Binding Protein-C. J Am Heart Assoc 2016; 5: e002836. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Herron TJ, Rostkova E, Kunst G, Chaturvedi R, Gautel M, Kentish JC. Activation of myocardial contraction by the N-terminal domains of myosin binding protein-C. Circ Res 2006; 98: 1290–8. [DOI] [PubMed] [Google Scholar]
20.Li J, Mamidi R, Doh CY, Holmes JB, Bharambe N, Ramachandran R, et al. AAV9 gene transfer of cMyBPC N-terminal domains ameliorates cardiomyopathy in cMyBPC-deficient mice. JCI Insight 2020; 5. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Lynch TLt Ismahil MA, Jegga AG Zilliox MJ, Troidl C Prabhu SD, et al. Cardiac inflammation in genetic dilated cardiomyopathy caused by MYBPC3 mutation. J Mol Cell Cardiol 2017; 102: 83–93. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.McConnell BK, Jones KA, Fatkin D, Arroyo LH, Lee RT, Aristizabal O, et al. Dilated cardiomyopathy in homozygous myosin binding protein-C mutant mice. J Clin Invest 1999; 104: 1235–44. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Kuster DWD, Lynch TL, Barefield DY, Sivaguru M, Kuffel G, Zilliox MJ, et al. Altered C10 domain in cardiac myosin binding protein-C results in hypertrophic cardiomyopathy. Cardiovasc Res 2019; 115: 1986–97. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Kumar M, Haghighi K, Kranias EG, Sadayappan S. Phosphorylation of cardiac myosin-binding protein-C contributes to calcium homeostasis. J Biol Chem 2020; 295: 11275–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.McNamara JW, Li A, Smith NJ, Lal S, Graham RM, Kooiker KB, et al. Ablation of cardiac myosin binding protein-C disrupts the super-relaxed state of myosin in murine cardiomyocytes. J Mol Cell Cardiol 2016; 94: 65–71. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Lin B, Govindan S, Lee K, Zhao P, Han R, Runte KE, et al. Cardiac myosin binding protein-C plays no regulatory role in skeletal muscle structure and function. PLoS One 2013; 8: e69671. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Colson BA, Bekyarova T, Locher MR, Fitzsimons DP, Irving TC, Moss RL. Protein kinase A-mediated phosphorylation of cMyBP-C increases proximity of myosin heads to actin in resting myocardium. Circ Res 2008; 103: 244–51. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Colson BA, Bekyarova T, Fitzsimons DP, Irving TC, Moss RL. Radial displacement of myosin cross-bridges in mouse myocardium due to ablation of myosin binding protein-C. J Mol Biol 2007; 367: 36–41. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Harris SP, Bartley CR, Hacker TA, McDonald KS, Douglas PS, Greaser ML, et al. Hypertrophic cardiomyopathy in cardiac myosin binding protein-C knockout mice. Circ Res 2002; 90: 594–601. [DOI] [PubMed] [Google Scholar]
30.Ratti J, Rostkova E, Gautel M, Pfuhl M. Structure and interactions of myosin binding protein-C domain C0: cardiac-specific regulation of myosin at its neck? J Biol Chem 2011; 286: 12650–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Bhuiyan MS, Gulick J, Osinska H, Gupta M, Robbins J. Determination of the critical residues responsible for cardiac myosin binding protein-C’s interactions. J Mol Cell Cardiol 2012; 53: 838–47. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Gruen M, Prinz H, Gautel M. cAPK-phosphorylation controls the interaction of the regulatory domain of cardiac myosin binding protein-C with myosin-S2 in an on-off fashion. FEBS Lett 1999; 453: 254–9. [DOI] [PubMed] [Google Scholar]
33.Bhuiyan MS, McLendon P, James J, Osinska H, Gulick J, Bhandary B, et al. In vivo definition of cardiac myosin-binding protein C’s critical interactions with myosin. Pflugers Archiv: European journal of physiology 2016; 468: 1685–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Tong CW, Stelzer JE, Greaser ML, Powers PA, Moss RL. Acceleration of cross bridge kinetics by protein kinase A phosphorylation of cardiac myosin binding protein C modulates cardiac function. Circ Res 2008; 103: 974–82. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Lipps C, Nguyen JH, Pyttel L, Lynch TLt, Liebetrau C, Aleshcheva G, et al. N-terminal fragment of cardiac myosin binding protein-C triggers pro-inflammatory responses in vitro. J Mol Cell Cardiol 2016; 99: 47–56. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Witt CC, Gerull B, Davies MJ, Centner T, Linke WA, Thierfelder L. Hypercontractile properties of cardiac muscle fibers in a knock-in mouse model of cardiac myosin-binding protein-C. J Biol Chem 2001; 276: 5353–9. [DOI] [PubMed] [Google Scholar]
37.van Dijk SJ, Witt CC, Harris SP. Normal cardiac contraction in mice lacking the proline-alanine rich region and C1 domain of cardiac myosin binding protein C. J Mol Cell Cardiol 2015; 88: 124–32. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Luther PK, Winkler H, Taylor K, Zoghbi ME, Craig R, Padron R, et al. Direct visualization of myosin-binding protein C bridging myosin and actin filaments in intact muscle. Proc Natl Acad Sci U S A 2011; 108: 11423–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Rahmanseresht S, Lee KH, OĽeary TS, McNamara JW, Sadayappan S, Robbins J, et al. The N terminus of myosin-binding protein C extends toward actin filaments in intact cardiac muscle. J Gen Physiol 2021; 153. [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Williams CD, Salcedo MK, Irving TC, Regnier M, Daniel TL. The length-tension curve in muscle depends on lattice spacing. Proc Biol Sci 2013; 280: 20130697. [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Fuchs F, Smith SH. Calcium, cross-bridges, and the Frank-Starling relationship. News Physiol Sci 2001; 16: 5–10. [DOI] [PubMed] [Google Scholar]
42.Stelzer JE, Dunning SB, Moss RL. Ablation of cardiac myosin-binding protein-C accelerates stretch activation in murine skinned myocardium. Circ Res 2006; 98: 1212–8. [DOI] [PubMed] [Google Scholar]
43.Kulikovskaya I, McClellan G, Levine R, Winegrad S. Effect of extraction of myosin binding protein-C on contractility of rat heart. Am J Physiol Heart Circ Physiol 2003; 285: H857–65. [DOI] [PubMed] [Google Scholar]
44.Razumova MV, Shaffer JF, Tu AY, Flint GV, Regnier M, Harris SP. Effects of the N-terminal domains of myosin binding protein-C in an in vitro motility assay: Evidence for long-l ived cross-bridges. J Biol Chem 2006; 281: 35846–54. [DOI] [PubMed] [Google Scholar]
45.Ababou A, Rostkova E, Mistry S, Le Masurier C, Gautel M, Pfuhl M. Myosin binding protein-C positioned to play a key role in regulation of muscle contraction: structure and interactions of domain C1. J Mol Biol 2008; 384: 615–30. [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Whitten AE, Jeffries CM, Harris SP, Trewhella J. Cardiac myosin binding protein-C decorates F-actin: implications for cardiac function. Proc Natl Acad Sci U S A 2008; 105: 18360–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Jeffries CM, Whitten AE, Harris SP, Trewhella J. Small-angle X-ray scattering reveals the N-terminal domain organization of cardiac myosin binding protein-C. J Mol Biol 2008; 377: 1186–99. [DOI] [PubMed] [Google Scholar]
48.Shaffer JF, Kensler RW, Harris SP. The myosin binding protein-C motif binds to F-actin in a phosphorylation-sensitive manner. J Biol Chem 2009; 284: 12318–27. [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Kensler RW, Shaffer JF, Harris SP. Binding of the N-terminal fragment C0-C2 of cardiac MyBP-C to cardiac F-actin. J Struct Biol 2011; 174: 44–51. [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Walcott S, Docken S, Harris SP. Effects of cardiac Myosin binding protein-C on actin motility are explained with a drag-activation-competition model. Biophys J 2015; 108: 10–3. [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Inchingolo AV, Previs SB, Previs MJ, Warshaw DM, Kad NM. Revealing the mechanism of how cardiac myosin-binding protein C N-terminal fragments sensitize thin filaments for myosin binding. Proc Natl Acad Sci U S A 2019; 116: 6828–35. [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Razumova MV, Bezold KL, Tu AY, Regnier M, Harris SP. Contribution of the myosin binding protein-C motif to functional effects in permeabilized rat trabeculae. J Gen Physiol 2008; 132: 575–85. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

NIHMS1689212-supplement-1.pdf^{(9.9MB, pdf)}

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[R17] 17.Sadayappan S, Osinska H, Klevitsky R, Lorenz JN, Sargent M, Molkentin JD, et al. Cardiac myosin binding protein-C phosphorylation is cardioprotective. Proc Natl Acad Sci U S A 2006; 103: 16918–23. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R18] 18.Taylor EN, Hoffman MP, Barefield DY, Aninwene GE 2nd, Abrishamchi AD, Lynch TL t, et al. Alterations in Multi-Scale Cardiac Architecture in Association With Phosphorylation of Myosin Binding Protein-C. J Am Heart Assoc 2016; 5: e002836. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R19] 19.Herron TJ, Rostkova E, Kunst G, Chaturvedi R, Gautel M, Kentish JC. Activation of myocardial contraction by the N-terminal domains of myosin binding protein-C. Circ Res 2006; 98: 1290–8. [DOI] [PubMed] [Google Scholar]

[R20] 20.Li J, Mamidi R, Doh CY, Holmes JB, Bharambe N, Ramachandran R, et al. AAV9 gene transfer of cMyBPC N-terminal domains ameliorates cardiomyopathy in cMyBPC-deficient mice. JCI Insight 2020; 5. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R21] 21.Lynch TLt Ismahil MA, Jegga AG Zilliox MJ, Troidl C Prabhu SD, et al. Cardiac inflammation in genetic dilated cardiomyopathy caused by MYBPC3 mutation. J Mol Cell Cardiol 2017; 102: 83–93. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R22] 22.McConnell BK, Jones KA, Fatkin D, Arroyo LH, Lee RT, Aristizabal O, et al. Dilated cardiomyopathy in homozygous myosin binding protein-C mutant mice. J Clin Invest 1999; 104: 1235–44. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R23] 23.Kuster DWD, Lynch TL, Barefield DY, Sivaguru M, Kuffel G, Zilliox MJ, et al. Altered C10 domain in cardiac myosin binding protein-C results in hypertrophic cardiomyopathy. Cardiovasc Res 2019; 115: 1986–97. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R24] 24.Kumar M, Haghighi K, Kranias EG, Sadayappan S. Phosphorylation of cardiac myosin-binding protein-C contributes to calcium homeostasis. J Biol Chem 2020; 295: 11275–91. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R25] 25.McNamara JW, Li A, Smith NJ, Lal S, Graham RM, Kooiker KB, et al. Ablation of cardiac myosin binding protein-C disrupts the super-relaxed state of myosin in murine cardiomyocytes. J Mol Cell Cardiol 2016; 94: 65–71. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R26] 26.Lin B, Govindan S, Lee K, Zhao P, Han R, Runte KE, et al. Cardiac myosin binding protein-C plays no regulatory role in skeletal muscle structure and function. PLoS One 2013; 8: e69671. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R27] 27.Colson BA, Bekyarova T, Locher MR, Fitzsimons DP, Irving TC, Moss RL. Protein kinase A-mediated phosphorylation of cMyBP-C increases proximity of myosin heads to actin in resting myocardium. Circ Res 2008; 103: 244–51. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R28] 28.Colson BA, Bekyarova T, Fitzsimons DP, Irving TC, Moss RL. Radial displacement of myosin cross-bridges in mouse myocardium due to ablation of myosin binding protein-C. J Mol Biol 2007; 367: 36–41. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R29] 29.Harris SP, Bartley CR, Hacker TA, McDonald KS, Douglas PS, Greaser ML, et al. Hypertrophic cardiomyopathy in cardiac myosin binding protein-C knockout mice. Circ Res 2002; 90: 594–601. [DOI] [PubMed] [Google Scholar]

[R30] 30.Ratti J, Rostkova E, Gautel M, Pfuhl M. Structure and interactions of myosin binding protein-C domain C0: cardiac-specific regulation of myosin at its neck? J Biol Chem 2011; 286: 12650–8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R31] 31.Bhuiyan MS, Gulick J, Osinska H, Gupta M, Robbins J. Determination of the critical residues responsible for cardiac myosin binding protein-C’s interactions. J Mol Cell Cardiol 2012; 53: 838–47. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R32] 32.Gruen M, Prinz H, Gautel M. cAPK-phosphorylation controls the interaction of the regulatory domain of cardiac myosin binding protein-C with myosin-S2 in an on-off fashion. FEBS Lett 1999; 453: 254–9. [DOI] [PubMed] [Google Scholar]

[R33] 33.Bhuiyan MS, McLendon P, James J, Osinska H, Gulick J, Bhandary B, et al. In vivo definition of cardiac myosin-binding protein C’s critical interactions with myosin. Pflugers Archiv: European journal of physiology 2016; 468: 1685–95. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R34] 34.Tong CW, Stelzer JE, Greaser ML, Powers PA, Moss RL. Acceleration of cross bridge kinetics by protein kinase A phosphorylation of cardiac myosin binding protein C modulates cardiac function. Circ Res 2008; 103: 974–82. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R35] 35.Lipps C, Nguyen JH, Pyttel L, Lynch TLt, Liebetrau C, Aleshcheva G, et al. N-terminal fragment of cardiac myosin binding protein-C triggers pro-inflammatory responses in vitro. J Mol Cell Cardiol 2016; 99: 47–56. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R36] 36.Witt CC, Gerull B, Davies MJ, Centner T, Linke WA, Thierfelder L. Hypercontractile properties of cardiac muscle fibers in a knock-in mouse model of cardiac myosin-binding protein-C. J Biol Chem 2001; 276: 5353–9. [DOI] [PubMed] [Google Scholar]

[R37] 37.van Dijk SJ, Witt CC, Harris SP. Normal cardiac contraction in mice lacking the proline-alanine rich region and C1 domain of cardiac myosin binding protein C. J Mol Cell Cardiol 2015; 88: 124–32. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R38] 38.Luther PK, Winkler H, Taylor K, Zoghbi ME, Craig R, Padron R, et al. Direct visualization of myosin-binding protein C bridging myosin and actin filaments in intact muscle. Proc Natl Acad Sci U S A 2011; 108: 11423–8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R39] 39.Rahmanseresht S, Lee KH, OĽeary TS, McNamara JW, Sadayappan S, Robbins J, et al. The N terminus of myosin-binding protein C extends toward actin filaments in intact cardiac muscle. J Gen Physiol 2021; 153. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R40] 40.Williams CD, Salcedo MK, Irving TC, Regnier M, Daniel TL. The length-tension curve in muscle depends on lattice spacing. Proc Biol Sci 2013; 280: 20130697. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R41] 41.Fuchs F, Smith SH. Calcium, cross-bridges, and the Frank-Starling relationship. News Physiol Sci 2001; 16: 5–10. [DOI] [PubMed] [Google Scholar]

[R42] 42.Stelzer JE, Dunning SB, Moss RL. Ablation of cardiac myosin-binding protein-C accelerates stretch activation in murine skinned myocardium. Circ Res 2006; 98: 1212–8. [DOI] [PubMed] [Google Scholar]

[R43] 43.Kulikovskaya I, McClellan G, Levine R, Winegrad S. Effect of extraction of myosin binding protein-C on contractility of rat heart. Am J Physiol Heart Circ Physiol 2003; 285: H857–65. [DOI] [PubMed] [Google Scholar]

[R44] 44.Razumova MV, Shaffer JF, Tu AY, Flint GV, Regnier M, Harris SP. Effects of the N-terminal domains of myosin binding protein-C in an in vitro motility assay: Evidence for long-l ived cross-bridges. J Biol Chem 2006; 281: 35846–54. [DOI] [PubMed] [Google Scholar]

[R45] 45.Ababou A, Rostkova E, Mistry S, Le Masurier C, Gautel M, Pfuhl M. Myosin binding protein-C positioned to play a key role in regulation of muscle contraction: structure and interactions of domain C1. J Mol Biol 2008; 384: 615–30. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R46] 46.Whitten AE, Jeffries CM, Harris SP, Trewhella J. Cardiac myosin binding protein-C decorates F-actin: implications for cardiac function. Proc Natl Acad Sci U S A 2008; 105: 18360–5. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R47] 47.Jeffries CM, Whitten AE, Harris SP, Trewhella J. Small-angle X-ray scattering reveals the N-terminal domain organization of cardiac myosin binding protein-C. J Mol Biol 2008; 377: 1186–99. [DOI] [PubMed] [Google Scholar]

[R48] 48.Shaffer JF, Kensler RW, Harris SP. The myosin binding protein-C motif binds to F-actin in a phosphorylation-sensitive manner. J Biol Chem 2009; 284: 12318–27. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R49] 49.Kensler RW, Shaffer JF, Harris SP. Binding of the N-terminal fragment C0-C2 of cardiac MyBP-C to cardiac F-actin. J Struct Biol 2011; 174: 44–51. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R50] 50.Walcott S, Docken S, Harris SP. Effects of cardiac Myosin binding protein-C on actin motility are explained with a drag-activation-competition model. Biophys J 2015; 108: 10–3. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R51] 51.Inchingolo AV, Previs SB, Previs MJ, Warshaw DM, Kad NM. Revealing the mechanism of how cardiac myosin-binding protein C N-terminal fragments sensitize thin filaments for myosin binding. Proc Natl Acad Sci U S A 2019; 116: 6828–35. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R52] 52.Razumova MV, Bezold KL, Tu AY, Regnier M, Harris SP. Contribution of the myosin binding protein-C motif to functional effects in permeabilized rat trabeculae. J Gen Physiol 2008; 132: 575–85. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Amino terminus of cardiac myosin binding protein-C regulates cardiac contractility

Thomas L Lynch IV

Mohit Kumar

James W McNamara

Diederik WD Kuster

Mayandi Sivaguru

Rohit R Singh

Michael J Previs

Kyoung Hwan Lee

Gina Kuffel

Michael J Zilliox

Brian Leei Lin

Weikang Ma

Aaron M Gibson

Burns C Blaxall

Michelle L Nieman

John N Lorenz

Dana M Leichter

Owen P Leary

Paul ML Janssen

Pieter P de Tombe

Richard J Gilbert

Roger Craig

Thomas Irving

David M Warshaw

Sakthivel Sadayappan

Abstract

Graphical Abstract

1. Introduction

Fig. 1. cMyBP-CΔC0-C1f transgenic mouse line characterization.

2. Materials and Methods

2.1. Transgenic cMyBP-CΔC0-C1f Mouse Model

2.2. Molecular Analyses

2.3. Biophysical Experiments

2.4. X-Ray Diffraction Analysis

2.5. Q-space MRI

2.6. Statistical Analysis

3.0. Results

3.1. cMyBP-CΔC0-C1f incorporates normally into the sarcomere

3.2. The C0-C1f region of cMyBP-C is essential to maintain cardiac function

Fig. 2. TG mice expressing cMyBP-CΔC0-C1f display cardiac enlargement, reduced cardiac function and increased fibrosis.

Fig. 3. Myoarchitectural effects of cMyBP-CΔC0-C1f.

3.3. Ablation of C0-C1f in cMyBP-C results in faster thin filament sliding in the C-zone

Fig. 4. Loss of C0-C1f enhances actin-sliding velocity.

3.4. Effect of C0-C1f loss on thick and thin filament mass distribution in the sarcomere

Fig. 5. X-ray diffraction analysis of cMyBP-CΔC0-C1f fibers.

3.5. N’-terminal ablation of cMyBP-C reduces sarcomere contractility, but does not alter calcium handling

Fig. 6. Mechanical analysis of skinned cardiomyocytes in the presence or absence of 10μM rC0-C2.

3.6. Restoration of previously ablated C0-C2 promotes normal thick and thin filament interactions

4. Discussion

4.1. C0-C1f is essential for normal cardiac contractility

4.2. C0-C1f is necessary to regulate sarcomere function

4.3. Introduction of the C0-C2 region restores thin and thick filament interactions

4.4. Study Limitations

4.5. Conclusions

Supplementary Material

HIGHLIGHTS.

Acknowledgements

Abbreviations

Footnotes

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Fig. 1. cMyBP-C^ΔC0-C1f transgenic mouse line characterization.

2.1. Transgenic cMyBP-C^ΔC0-C1f Mouse Model

3.1. cMyBP-C^ΔC0-C1f incorporates normally into the sarcomere

Fig. 2. TG mice expressing cMyBP-C^ΔC0-C1f display cardiac enlargement, reduced cardiac function and increased fibrosis.

Fig. 3. Myoarchitectural effects of cMyBP-C^ΔC0-C1f.

Fig. 5. X-ray diffraction analysis of cMyBP-C^ΔC0-C1f fibers.