A 25-basepair deletion variant of MYBPC3 occurs at high frequency in individuals of South Asian descent and is estimated to affect 55 million people worldwide, carrying an increased likelihood of cardiomyopathy. Since this variant is prevalent and severe in this subpopulation, quick and affordable screening to provide risk-assessment to guide treatment for these patients is critical. An RNaseH qPCR assay was developed to quickly and specifically diagnose the presence of the 25-basepair deletion variant in MYBPC3. RNAseH-blocked nucleotide primers were designed to identify the presence or absence of the wild type MYBPC3 allele or the genomic sequence containing the 25-basepair deletion. Using this assay, three blinded operators were able to accurately determine the genotype from human genomic DNA samples from blood and saliva using a qPCR thermocycler. Furthermore, positive variant subjects were examined by both electrocardiography and echocardiography for the presence of cardiomyopathy. A simple, robust assay was established, verified and validated that can be automated to detect the presence of the highly prevalent 25-basepair deletion MYBPC3 variant using both blood and saliva samples. The assay will provide quick and accurate prescreening of individuals at high risk for cardiomyopathies and allow for better clinical identification of 25-basepair deletion MYBPC3 carriers in large cohort epidemiological studies.
Hypertrophic cardiomyopathy; Genotype-phenotype; South Asian population; RNaseH qPCR; DNA diagnostic test
Pharmacologic activation of cGMP-dependent protein kinase I (PKGI) has emerged as a therapeutic strategy for humans with heart failure. However, PKG activating drugs have been limited by hypotension arising from PKG-induced vasodilation. PKGIα anti-remodeling substrates specific to the myocardium might provide targets to circumvent this limitation, but currently remain poorly understood.
Methods and Results
We performed a screen for myocardial proteins interacting with the PKGIα leucine zipper (LZ) binding domain to identify myocardial-specific PKGI anti-remodeling substrates. Our screen identified cardiac myosin binding protein C (cMyBP-C), a cardiac myocyte-specific protein, which has been demonstrated to inhibit cardiac remodeling in the phosphorylated state, and when mutated leads to hypertrophic cardiomyopathy in humans. GST-pulldowns and precipitations with cGMP-conjugated beads confirmed the PKGIα-cMyBP-C interaction in myocardial lysates. In vitro studies demonstrated that purified PKGIα phosphorylates the cMyBP-C M-domain at Ser-273, Ser-282, and Ser-302. cGMP induced cMyBP-C phosphorylation at these residues in COS cells transfected with PKGIα, but not in cells transfected with LZ mutant PKGIα containing mutations to disrupt LZ substrate binding. In mice subjected to LV pressure overload, PKGI activation with sildenafil increased cMyBP-C phosphorylation at Ser-273 compared with untreated mice. cGMP also induced cMyBP-C phosphorylation in isolated cardiac myocytes.
Taken together these data support that PKGIα and cMyBP-C interact in the heart, and that cMyBP-C is an anti-remodeling PKGIα kinase substrate. This study provides the first identification of a myocardial specific PKGIα LZ-dependent anti-remodeling substrate and supports further exploration of PKGIα myocardial LZ substrates as potential therapeutic targets for heart failure.
cGMP; cardiac remodeling; heart failure; myosin binding protein
The South Asian population, numbered at 1.8 billion, is estimated to comprise around 20% of the global population and 1% of the American population, and has one of the highest rates of cardiovascular disease. While South Asians show increased classical risk factors for developing heart failure, the role of population-specific genetic risk factors has not yet been examined for this group. Hypertrophic cardiomyopathy (HCM) is one of the major cardiac genetic disorders among South Asians, leading to contractile dysfunction, heart failure, and sudden cardiac death. This disease displays autosomal dominant inheritance, and it is associated with a large number of variants in both sarcomeric and non-sarcomeric proteins. The South Asians, a population with large ethnic diversity, potentially carries region-specific polymorphisms. There is high variability in disease penetrance and phenotypic expression of variants associated with HCM. Thus, extensive studies are required to decipher pathogenicity and the physiological mechanisms of these variants, as well as the contribution of modifier genes and environmental factors to disease phenotypes. Conducting genotype-phenotype correlation studies will lead to improved understanding of HCM and, consequently, improved treatment options for this high-risk population. The objective of this review is to report the history of cardiovascular disease and HCM in South Asians, present previously published pathogenic variants, and introduce current efforts to study HCM using induced pluripotent stem cell-derived cardiomyocytes, next-generation sequencing, and gene editing technologies. The authors ultimately hope that this review will stimulate further research, drive novel discoveries, and contribute to the development of personalized medicine with the aim of expanding therapeutic strategies for HCM.
hypertrophic cardiomyopathy; South Asians; β-myosin heavy chain; MYH7; cardiac myosin binding protein-C; MYPBC3
Mammalian hearts exhibit positive inotropic responses to β-adrenergic stimulation as a consequence of protein kinase A (PKA)-mediated phosphorylation or as a result of increased beat frequency (the Bowditch effect). Several membrane and myofibrillar proteins are phosphorylated under these conditions, but the relative contributions of these to increased contractility are not known. Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) by PKA accelerates the kinetics of force development in permeabilized heart muscle, but its role in vivo is unknown. Such understanding is important, since adrenergic responsiveness of the heart and the Bowditch effect are both depressed in heart failure.
Methods and Results
The roles of cMyBP-C phosphorylation were studied using mice in which either WT or nonphosphorylatable forms of cMyBP-C [ser273ala, ser282ala, ser302ala: cMyBP-C(t3SA)] were expressed at similar levels on a cMyBP-C null background. Force and [Ca2+]in measurements in isolated papillary muscles showed that the increased force and twitch kinetics due to increased pacing or β1-adrenergic stimulation were nearly absent in cMyBP-C(t3SA) myocardium, even though [Ca2+]intransients under each condition were similar to WT. Biochemical measurements confirmed that PKA phosphorylated ser273, ser282 and ser302 in WT cMyBP-C. In contrast, CaMKIIδ, which is activated by increased pacing, phosphorylated ser302 principally, ser282 to a lesser degree, and ser273 not at all.
Phosphorylation of cMyBP-C increases the force and kinetics of twitches in living cardiac muscle. Further, cMyBP-C is a principal mediator of increased contractility observed with β-adrenergic stimulation or increased pacing, due to PKA and CaMKIIδ phosphorylations of cMyB-C.
myosin binding protein-C; phosphorylation; contractility
The geometric organization of myocytes in the ventricular wall comprises the structural underpinnings of cardiac mechanical function. Cardiac myosin binding protein‐C (MYBPC3) is a sarcomeric protein, for which phosphorylation modulates myofilament binding, sarcomere morphology, and myocyte alignment in the ventricular wall. To elucidate the mechanisms by which MYBPC3 phospho‐regulation affects cardiac tissue organization, we studied ventricular myoarchitecture using generalized Q‐space imaging (GQI). GQI assessed geometric phenotype in excised hearts that had undergone transgenic (TG) modification of phospho‐regulatory serine sites to nonphosphorylatable alanines (MYBPC3AllP−/(t/t)) or phospho‐mimetic aspartic acids (MYBPC3AllP+/(t/t)).
Methods and Results
Myoarchitecture in the wild‐type (MYBPC3WT) left‐ventricle (LV) varied with transmural position, with helix angles ranging from −90/+90 degrees and contiguous circular orientation from the LV mid‐myocardium to the right ventricle (RV). Whereas MYBPC3AllP+/(t/t) hearts were not architecturally distinct from MYBPC3WT, MYBPC3AllP−/(t/t) hearts demonstrated a significant reduction in LV transmural helicity. Null MYBPC3(t/t) hearts, as constituted by a truncated MYBPC3 protein, demonstrated global architectural disarray and loss in helicity. Electron microscopy was performed to correlate the observed macroscopic architectural changes with sarcomere ultrastructure and demonstrated that impaired phosphorylation of MYBPC3 resulted in modifications of the sarcomere aspect ratio and shear angle. The mechanical effect of helicity loss was assessed through a geometric model relating cardiac work to ejection fraction, confirming the mechanical impairments observed with echocardiography.
We conclude that phosphorylation of MYBPC3 contributes to the genesis of ventricular wall geometry, linking myofilament biology with multiscale cardiac mechanics and myoarchitecture.
basic studies; echocardiography; genetically altered mice; heart failure; magnetic resonance imaging; quantitative modeling; Cardiomyopathy; Magnetic Resonance Imaging (MRI); Animal Models of Human Disease; Genetically Altered and Transgenic Models
Mutations in MYBPC3, the gene encoding cardiac myosin binding protein-C (cMyBP-C), account for ~40% of hypertrophic cardiomyopathy (HCM) cases. Most pathological MYBPC3 mutations encode truncated protein products not found in tissue. Reduced protein levels occur in symptomatic heterozygous human HCM carriers, suggesting haploinsufficiency as an underlying mechanism of disease. However, we do not know if reduced cMyBP-C content results from, or initiates the development of HCM. In previous studies, heterozygous (HET) mice with a MYBPC3 C’-terminal truncation mutation and normal cMyBP-C levels show altered contractile function prior to any overt hypertrophy. Therefore, this study aimed to test whether haploinsufficiency occurs, with decreased cMyBP-C content, following cardiac stress and whether the functional impairment in HET MYBPC3 hearts leads to worsened disease progression. To address these questions, transverse aortic constriction (TAC) was performed on three-month-old wild-type (WT) and HET MYBPC3-truncation mutant mice and then characterized at 4 and 12 weeks post-surgery. HET-TAC mice showed increased hypertrophy and reduced ejection fraction compared to WT-TAC mice. At 4 weeks post-surgery, HET myofilaments showed significantly reduced cMyBP-C content. Functionally, HET-TAC cardiomyocytes showed impaired force generation, higher Ca2+ sensitivity, and blunted length-dependent increase in force generation. RNA sequencing revealed several differentially regulated genes between HET and WT groups, including regulators of remodeling and hypertrophic response. Collectively, these results demonstrate that haploinsufficiency occurs in HET MYBPC3 mutant carriers following stress, causing, in turn, reduced cMyBP-C content and exacerbating the development of dysfunction at myofilament and whole-heart levels.
Cardiac myosin binding protein-C; Haploinsufficiency; Hypertrophic Cardiomyopathy; MYBPC3; Mouse Models
Myosin binding protein-C is a thick filament protein of vertebrate striated muscle. The cardiac isoform (cMyBP-C) is essential for normal cardiac function, and mutations in cMyBP-C cause cardiac muscle disease. The rod-shaped molecule is composed primarily of 11 immunoglobulin- or fibronectin-like domains, and is located at 9 sites, 43 nm apart, in each half of the A-band. To understand how cMyBP-C functions, it is important to know its structural organization in the sarcomere, as this will affect its ability to interact with other sarcomeric proteins. Several models have been proposed, in which cMyBP-C wraps around, extends radially from, or runs axially along the thick filament. Our goal was to define cMyBP-C orientation by determining the relative axial positions of different cMyBP-C domains. Immuno-electron microscopy was performed using mouse cardiac myofibrils labeled with antibodies specific to the N- and C-terminal domains and to the middle of cMyBP-C. Antibodies to all regions of the molecule, except the C-terminus, labeled at the same nine axial positions in each half A-band, consistent with a circumferential and/or radial rather than an axial orientation of the bulk of the molecule. The C-terminal antibody stripes were slightly displaced axially, demonstrating an axial orientation of the C-terminal 3 domains, with the C-terminus closer to the M-line. These results, combined with previous studies, suggest that the C-terminal domains of cMyBP-C run along the thick filament surface, while the N-terminus extends towards neighboring thin filaments. This organization provides a structural framework for understanding cMyBP-C’s modulation of cardiac muscle contraction.
cMyBP-C; cardiac muscle contraction; cardiac muscle disease; cardiac muscle structure; cardiac muscle regulation
Although ceramide accumulation in the heart is considered a major factor in promoting apoptosis and cardiac disorders, including heart failure, lipotoxicity and ischemia-reperfusion injury, little is known about ceramide’s role in mediating changes in contractility. In the present study, we measured the functional consequences of acute exposure of isolated field stimulated adult rat cardiomyocytes to C6-ceramide. Exogenous ceramide treatment depressed the peak amplitude and the maximal velocity of shortening without altering intracellular calcium levels or kinetics. The inactive ceramide analog C6-dihydroceramide had no effect on myocyte shortening or [Ca2+]i transients. Experiments testing a potential role for C6-ceramide-mediated effects on activation of protein kinase C (PKC) demonstrated evidence for signaling through the calcium-independent isoform, PKCε. We employed 2 dimensional electrophoresis and anti-phospho-peptide antibodies to test whether treatment of the cardiomyocytes with C6-ceramide altered myocyte shortening via PKC dependent phosphorylation of myofilament proteins. Compared to controls, myocytes treated with ceramide exhibited increased phosphorylation of myosin binding protein-C (cMyBP-C), specifically at Ser273 and Ser302, and troponin I (cTnI) at sites apart from Ser23/24, which could be attenuated with PKC inhibition. We conclude that the altered myofilament response to calcium resulting from multiple sites of PKC-dependent phosphorylation contributes to contractile dysfunction that is associated with cardiac diseases in which elevations in ceramides are present.
lipotoxicity; diabetes; troponin; myosin binding protein C
Cardiomyopathies can result from mutations in genes encoding sarcomere proteins including MYBPC3, which encodes cardiac myosin binding protein-C (cMyBP-C). However, whether oxidative stress is augmented due to contractile dysfunction and cardiomyocyte damage in MYBPC3-mutated cardiomyopathies has not been elucidated. To determine whether oxidative stress markers were elevated in MYBPC3-mutated cardiomyopathies, a previously characterized 3-month-old mouse model of dilated cardiomyopathy (DCM) expressing a homozygous MYBPC3 mutation (cMyBP-C(t/t)) was used, compared to wild-type (WT) mice. Echocardiography confirmed decreased percentage of fractional shortening in DCM versus WT hearts. Histopathological analysis indicated a significant increase in myocardial disarray and fibrosis while the second harmonic generation imaging revealed disorganized sarcomeric structure and myocyte damage in DCM hearts when compared to WT hearts. Intriguingly, DCM mouse heart homogenates had decreased glutathione (GSH/GSSG) ratio and increased protein carbonyl and lipid malondialdehyde content compared to WT heart homogenates, consistent with elevated oxidative stress. Importantly, a similar result was observed in human cardiomyopathy heart homogenate samples. These results were further supported by reduced signals for mitochondrial semiquinone radicals and Fe-S clusters in DCM mouse hearts measured using electron paramagnetic resonance spectroscopy. In conclusion, we demonstrate elevated oxidative stress in MYPBC3-mutated DCM mice, which may exacerbate the development of heart failure.
Cardiac myosin binding protein-C (cMyBP-C) is a regulatory protein of the contractile apparatus within the cardiac sarcomere. Ischemic injury to the heart during myocardial infarction (MI) results in the cleavage of cMyBP-C in a phosphorylation-dependent manner and release of an N-terminal fragment (C0C1f) into the circulation. C0C1f has been shown to be pathogenic within cardiac tissue, leading to the development of heart failure (HF). Based on its high levels and early release into the circulation post-MI, C0C1f may serve as a novel biomarker for diagnosing MI more effectively than current clinically used biomarkers. Over time, circulating C0C1f could trigger an autoimmune response leading to myocarditis and progressive cardiac dysfunction. Given the importance of cMyBP-C phosphorylation state in the context of proteolytic cleavage and release into the circulation post-MI, understanding the post-translational modifications (PTMs) of cMyBP-C would help in further elucidating the role of this protein in health and disease. Accordingly, recent studies have implemented the latest proteomics approaches to define the PTMs of cMyBP-C. The use of such proteomics assays may provide accurate quantitation of the levels of cMyBP-C in the circulation following MI, which could, in turn, demonstrate the efficacy of using plasma cMyBP-C as a cardiac-specific early biomarker of MI. In this review, we define the pathogenic and potential immunogenic effects of C0C1f on cardiac function in the post-MI heart. We also discuss the most advanced proteomics approaches now used to determine cMyBP-C PTMs with the aim of validating C0C1f as an early biomarker of MI.
Autoantibodies; Biomarkers; cMyBP-C; Dilated cardiomyopathy; Myocardial infarction; Myocarditis; Post-translational modification; Proteomics
Heterozygous mutations in sarcomere genes in hypertrophic cardiomyopathy (HCM) are proposed to exert their effect through gain-of-function for missense mutations or loss-of-function for truncating mutations. However, allelic expression from individual mutations has not been sufficiently characterized to support this exclusive distinction in human HCM.
Methods and Results
Sarcomere transcript and protein levels were analyzed in septal myectomy and transplant specimens from 46 genotyped HCM patients with or without sarcomere gene mutations and 10 control hearts. For truncating mutations in MYBPC3, the average ratio of mutant:wild-type transcripts was ~1:5, in contrast to ~1:1 for all sarcomere missense mutations, confirming that nonsense transcripts are uniquely unstable. However, total MYBPC3 mRNA was significantly increased by ~9 fold in HCM samples with MYBPC3 mutations compared to control hearts and to HCM samples without sarcomere gene mutations. Full-length MYBPC3 protein content was not different between MYBPC3 mutant HCM and control samples and no truncated proteins were detected. By absolute quantification of abundance (AQUA) with multiple reaction monitoring, stoichiometric ratios of mutant sarcomere proteins relative to wild-type were strikingly variable in a mutation-specific manner, with the fraction of mutant protein ranging from 30–84%.
These results challenge the concept that haploinsufficiency is a unifying mechanism for HCM caused by MYBPC3 truncating mutations. The range of allelic imbalance for several missense sarcomere mutations suggests that certain mutant proteins may be more or less stable, or incorporate more or less efficiently into the sarcomere than wild-type proteins. These mutation-specific properties may distinctly influence disease phenotypes.
hypertrophic cardiomyopathy; sarcomere; gene expression; human; proteomics; expression/regulation
Heart failure is associated with pump dysfunction and remodeling but it is not yet known if the condition affects different transmural regions of the heart in the same way. We tested the hypotheses that the left ventricles of non-failing human hearts exhibit transmural heterogeneity of cellular level contractile properties, and that heart failure produces transmural region-specific changes in contractile function.
Permeabilized samples were prepared from the sub-epicardial, mid-myocardial, and sub-endocardial regions of the left ventricular free wall of non-failing (n=6) and failing (n=10) human hearts. Power, an in vitro index of systolic function, was higher in non-failing mid-myocardial samples (0.59±0.06 μW mg−1) than in samples from the sub-epicardium (p=0.021) and the sub-endocardium (p=0.015). Non-failing mid-myocardial samples also produced more isometric force (14.3±1.33 kN m−2) than samples from the sub-epicardium (p=0.008) and the sub-endocardium (p=0.026). Heart failure reduced power (p=0.009) and force (p=0.042) but affected the mid-myocardium more than the other transmural regions. Fibrosis increased with heart failure (p=0.021) and mid-myocardial tissue from failing hearts contained more collagen than matched sub-epicardial (p<0.001) and sub-endocardial (p=0.043) samples. Power output was correlated with the relative content of actin and troponin I, and was also statistically linked to the relative content and phosphorylation of desmin and myosin light chain- 1. Non-failing human hearts exhibit transmural heterogeneity of contractile properties. In failing organs, region-specific fibrosis produces the greatest contractile deficits in the mid-myocardium. Targeting fibrosis and sarcomeric proteins in the mid-myocardium may be particularly effective therapies for heart failure.
Left ventricular function; Mechanics; Collagen; Sarcomere; Myofilament Protein
Based on our recent finding that cardiac myosin binding protein C (cMyBP-C) phosphorylation affects muscle contractility in a site-specific manner, we further studied the force per cross-bridge and the kinetic constants of the elementary steps in the six-state cross-bridge model in cMyBP-C mutated transgenic mice for better understanding of the influence of cMyBP-C phosphorylation on contractile functions. Papillary muscle fibres were dissected from cMyBP-C mutated mice of ADA (Ala273-Asp282-Ala302), DAD (Asp273-Ala282-Asp302), SAS (Ser273-Ala282-Ser302), and t/t (cMyBP-C null) genotypes, and the results were compared to transgenic mice expressing wide-type (WT) cMyBP-C. Sinusoidal analyses were performed with serial concentrations of ATP, phosphate (Pi), and ADP. Both t/t and DAD mutants significantly reduced active tension, force per cross-bridge, apparent rate constant (2πc), and the rate constant of cross-bridge detachment. In contrast to the weakened ATP binding and enhanced Pi and ADP release steps in t/t mice, DAD mice showed a decreased ADP release without affecting the ATP binding and the Pi release. ADA showed decreased ADP release, and slightly increased ATP binding and cross-bridge detachment steps, whereas SAS diminished the ATP binding step and accelerated the ADP release step. t/t has the broadest effects with changes in most elementary steps of the cross-bridge cycle, DAD mimics t/t to a large extent, and ADA and SAS predominantly affect the nucleotide binding steps. We conclude that the reduced tension production in DAD and t/t is the result of reduced force per cross-bridge, instead of the less number of strongly attached cross-bridges. We further conclude that cMyBP-C is an allosteric activator of myosin to increase cross-bridge force, and its phosphorylation status modulates the force, which is regulated by variety of protein kinases.
Top-down proteomics have recently started to gain attention as a novel
method to provide insight into the structure of proteins in their native state,
specifically the number and location of disulfide bridges. However, previous
techniques still relied on complex and time-consuming protein purification and
reduction reactions to yield useful information. In this issue of
Proteomics, Zhao et al. (High throughput
Screening of Disulfide-Containing Proteins in a Complex Mixture,
Proteomics 2013) devise a clever and rapid method for
high-throughput determination of disulfides in proteins via reduction by
tris(2-carboxyethyl)phosphine. Their work provides the foundation necessary to
undertake more complex experiments in biological samples.
Disulfide; Glutathionylation; Mass spectrometry; Post-translational Modification; Redox Proteomics; Redox Modifications
Mouse models of myocardial infarction (MI) are commonly used to explore the pathophysiological role of the monocytic response in myocardial injury and to develop translational strategies. However, no study thus far has examined the potential impact of inter-individual variability and sham surgical procedures on monocyte subset kinetics after experimental MI in mice. Our goal was to investigate determinants of systemic myeloid cell subset shifts in C57BL/6 mice following MI by developing a protocol for sequential extensive flow cytometry (FCM).
Methods and Results
Following cross-sectional multiplex FCM analysis we provide for the first time a detailed description of absolute quantities, relative subset composition, and biological variability of circulating classical, intermediate, and non-classical monocyte subsets in C57BL/6 mice. By using intra-individual longitudinal measurements after MI induction, a time course of classical and non-classical monocytosis was recorded. This approach disclosed a significant reduction of monocyte subset dispersion across all investigated time points following MI. We found that in the current invasive model of chronic MI the global pattern of systemic monocyte kinetics is mainly determined by a nonspecific inflammatory response to sham surgery and not by the extent of myocardial injury.
Application of sequential multiplexed FCM may help to reduce the impact of biological variability in C57BL/6 mice. Furthermore, the confounding influence of sham surgical procedures should always be considered when measuring monocyte subset kinetics in a murine model of MI.
High-myofilament Ca2+-sensitivity has been proposed as trigger of disease pathogenesis in familial hypertrophic cardiomyopathy (HCM) based on in vitro and transgenic mice studies. However, myofilament Ca2+-sensitivity depends on protein phosphorylation and muscle length, and at present, data in human are scarce.
To investigate whether high-myofilament Ca2+-sensitivity and perturbed length-dependent activation are characteristics for human HCM with mutations in thick- and thin-filament proteins.
Methods and Results
Cardiac samples from patients with HCM harboring mutations in genes encoding thick (MYH7, MYBPC3) and thin (TNNT2, TNNI3, TPM1) filament proteins were compared with sarcomere mutation-negative HCM and nonfailing donors. Cardiomyocyte force measurements showed higher myofilament Ca2+-sensitivity in all HCM samples and low phosphorylation of protein kinase A (PKA)-targets compared with donors. After exogenous PKA treatment, myofilament Ca2+-sensitivity was either similar (MYBPC3mut, TPM1mut, sarcomere mutation-negative HCM), higher (MYH7mut, TNNT2mut), or even significantly lower (TNNI3mut) compared with donors. Length-dependent activation was significantly smaller in all HCM than in donor samples. PKA treatment increased phosphorylation of PKA-targets in HCM myocardium and normalized length-dependent activation to donor values in sarcomere mutation-negative HCM and HCM with truncating MYBPC3 mutations, but not in HCM with missense mutations. Replacement of mutant by wild-type troponin in TNNT2mut and TNNI3mut corrected length-dependent activation to donor values.
High-myofilament Ca2+-sensitivity is a common characteristic of human HCM and partly reflects hypophosphorylation of PKA-targets compared with donors. Length-dependent sarcomere activation is perturbed by missense mutations, possibly via post-translational modifications other than PKA-hypophosphorylation or altered protein–protein interactions, and represents a common pathomechanism in HCM.
calcium; cardiomyopathy; contractility; hypertrophy; myocardium
Cardiac myosin binding protein C (cMyBP-C) regulates cross-bridge cycling kinetics and thereby fine-tunes the rate of cardiac muscle contraction and relaxation. Its effects on cardiac kinetics are modified by phosphorylation. Three phosphorylation sites (Ser275, Ser284, Ser304) have been identified in vivo, all located in the cardiac-specific M-domain of cMyBP-C. However recent work has shown that up to four phosphate groups are present in human cMyBP-C.
To identify and characterize additional phosphorylation sites in human cMyBP-C.
Methods and results
Cardiac MyBP-C was semi-purified from human heart tissue. Tandem mass-spectrometry analysis identified a novel phosphorylation site on serine 133 in the proline-alanine (Pro-Ala) rich linker sequence between the C0 and C1 domains of cMyBP-C. Unlike the known sites, Ser133 was not a target of protein kinase A. In silico kinase prediction revealed glycogen synthase kinase 3β (GSK3β) as the most likely kinase to phosphorylate Ser133. In vitro incubation of the C0C2 fragment of cMyBP-C with GSK3β showed phosphorylation on Ser133. In addition, GSK3β phosphorylated Ser304, although the degree of phosphorylation was less compared to PKA-induced phosphorylation at Ser304. GSK3β treatment of single membrane-permeabilized human cardiomyocytes significantly enhanced the maximal rate of tension redevelopment.
GSK3β phosphorylates cMyBP-C on a novel site, which is positioned in the Pro-Ala rich region and increases kinetics of force development, suggesting a non-canonical role for GSK3β at the sarcomere level. Phosphorylation of Ser133 in the linker domain of cMyBP-C may be a novel mechanism to regulate sarcomere kinetics.
Cardiac myosin binding protein-C; phosphorylation; GSK3β; heart failure; contractile proteins; hypertrophy/remodeling; myocardial contractility
Background: Myocardial infarction (MI) leads to proteolytic cleavage of cMyBP-C (hC0C1f) and decreased contractility.
Results: hC0C1f can incorporate into the human cardiac sarcomere, depressing force generation and increasing tension cost.
Conclusion: Interaction between hC0C1f and both actin and α-tropomyosin causes disruption of intact cMyBP-C function.
Significance: Proteolytic cleavage of cMyBP-C is sufficient to cause contractile dysfunction following MI.
Myocardial infarction (MI) is associated with depressed cardiac contractile function and progression to heart failure. Cardiac myosin-binding protein C, a cardiac-specific myofilament protein, is proteolyzed post-MI in humans, which results in an N-terminal fragment, C0-C1f. The presence of C0-C1f in cultured cardiomyocytes results in decreased Ca2+ transients and cell shortening, abnormalities sufficient for the induction of heart failure in a mouse model. However, the underlying mechanisms remain unclear. Here, we investigate the association between C0-C1f and altered contractility in human cardiac myofilaments in vitro. To accomplish this, we generated recombinant human C0-C1f (hC0C1f) and incorporated it into permeabilized human left ventricular myocardium. Mechanical properties were studied at short (2 μm) and long (2.3 μm) sarcomere length (SL). Our data demonstrate that the presence of hC0C1f in the sarcomere had the greatest effect at short, but not long, SL, decreasing maximal force and myofilament Ca2+ sensitivity. Moreover, hC0C1f led to increased cooperative activation, cross-bridge cycling kinetics, and tension cost, with greater effects at short SL. We further established that the effects of hC0C1f occur through direct interaction with actin and α-tropomyosin. Our data demonstrate that the presence of hC0C1f in the sarcomere is sufficient to induce depressed myofilament function and Ca2+ sensitivity in otherwise healthy human donor myocardium. Decreased cardiac function post-MI may result, in part, from the ability of hC0C1f to bind actin and α-tropomyosin, suggesting that cleaved C0-C1f could act as a poison polypeptide and disrupt the interaction of native cardiac myosin-binding protein C with the thin filament.
Contractile Protein; Heart Failure; Myocardial Infarction; Protein Degradation; Protein-Protein Interactions
Following activation by G-protein-coupled receptor agonists, protein kinase C (PKC) modulates cardiac myocyte function by phosphorylation of intracellular targets including myofilament proteins cardiac troponin I (cTnI) and cardiac myosin binding protein C (cMyBP-C). Since PKC phosphorylation has been shown to decrease myofibril ATPase activity, we hypothesized that PKC phosphorylation of cTnI and cMyBP-C will lower myocyte power output and, in addition, attenuate the elevation in power in response to protein kinase A (PKA)-mediated phosphorylation. We compared isometric force and power generating capacity of rat skinned cardiac myocytes before and after treatment with the catalytic subunit of PKC. PKC increased phosphorylation levels of cMyBP-C and cTnI and decreased both maximal Ca2+ activated force and Ca2+ sensitivity of force. Moreover, during submaximal Ca2+ activations PKC decreased power output by 62 %, which arose from both the fall in force and slower loaded shortening velocities since depressed power persisted even when force levels were matched before and after PKC. In addition, PKC blunted the phosphorylation of cTnI by PKA, reduced PKA-induced spontaneous oscillatory contractions, and diminished PKA-mediated elevations in myocyte power. To test whether altered thin filament function plays an essential role in these contractile changes we investigated the effects of chronic cTnI pseudo-phosphorylation on myofilament function using myocyte preparations from transgenic animals in which either only PKA phosphorylation sites (Ser-23/Ser-24) (PP) or both PKA and PKC phosphorylation sites (Ser-23/Ser-24/Ser-43/Ser-45/T-144) (All-P) were replaced with aspartic acid. Cardiac myocytes from All-P transgenic mice exhibited reductions in maximal force, Ca2+ sensitivity of force, and power. Similarly diminished power generating capacity was observed in hearts from All-P mice as determined by in situ pressure–volume measurements. These results imply that PKC-mediated phosphorylation of cTnI plays a dominant role in depressing contractility, and, thus, increased PKC isozyme activity may contribute to maladaptive behavior exhibited during the progression to heart failure.
PKC; PKA; Cardiac myocyte; Cardiac troponin I; Power output
Gravin, an A-kinase anchoring protein, targets protein kinase A (PKA), protein kinase C (PKC), calcineurin and other signaling molecules to the beta2-adrenergic receptor (β2-AR). Gravin mediates desensitization/resensitization of the receptor by facilitating its phosphorylation by PKA and PKC. The role of gravin in β-AR mediated regulation of cardiac function is unclear. The purpose of this study was to determine the effect of acute β-AR stimulation on cardiac contractility in mice lacking functional gravin. Using echocardiographic analysis, we observed that contractility parameters such as left ventricular fractional shortening and ejection fraction were increased in gravin mutant (gravin-t/t) animals lacking functional protein compared to wild-type (WT) animals both at baseline and following acute isoproterenol (ISO) administration. In isolated gravin-t/t cardiomyocytes, we observed increased cell shortening fraction and decreased intracellular Ca2+ in response to 1 µmol/L ISO stimulation. These physiological responses occurred in the presence of decreased β2-AR phosphorylation in gravin-t/t hearts, where PKA-dependent β2-AR phosphorylation has been shown to lead to receptor desensitization. cAMP production, PKA activity and phosphorylation of phospholamban and troponin I was comparable in WT and gravin-t/t hearts both with and without ISO stimulation. However, cardiac myosin binding protein C (cMyBPC) phosphorylation site at position 273 was significantly increased in gravin-t/t versus WT hearts, in the absence of ISO. Additionally, the cardioprotective heat shock protein 20 (Hsp20) was significantly more phosphorylated in gravin-t/t versus WT hearts, in response to ISO. Our results suggest that disruption of gravin’s scaffold mediated signaling is able to increase baseline cardiac function as well as to augment contractility in response to acute β-AR stimulation by decreasing β2-AR phosphorylation and thus attenuating receptor desensitization and perhaps by altering PKA localization to increase the phosphorylation of cMyBPC and the nonclassical PKA substrate Hsp20.
Myosin binding protein-C (MyBP-C) exists in three major isoforms: slow skeletal, fast skeletal, and cardiac. While cardiac MyBP-C (cMyBP-C) expression is restricted to the heart in the adult, it is transiently expressed in neonatal stages of some skeletal muscles. However, it is unclear whether this expression is necessary for the proper development and function of skeletal muscle. Our aim was to determine whether the absence of cMyBP-C alters the structure, function, or MyBP-C isoform expression in adult skeletal muscle using a cMyBP-C null mouse model (cMyBP-C(t/t)). Slow MyBP-C was expressed in both slow and fast skeletal muscles, whereas fast MyBP-C was mostly restricted to fast skeletal muscles. Expression of these isoforms was unaffected in skeletal muscle from cMyBP-C(t/t) mice. Slow and fast skeletal muscles in cMyBP-C(t/t) mice showed no histological or ultrastructural changes in comparison to the wild-type control. In addition, slow muscle twitch, tetanus tension, and susceptibility to injury were all similar to the wild-type controls. Interestingly, fMyBP-C expression was significantly increased in the cMyBP-C(t/t) hearts undergoing severe dilated cardiomyopathy, though this does not seem to prevent dysfunction. Additionally, expression of both slow and fast isoforms was increased in myopathic skeletal muscles. Our data demonstrate that i) MyBP-C isoforms are differentially regulated in both cardiac and skeletal muscles, ii) cMyBP-C is dispensable for the development of skeletal muscle with no functional or structural consequences in the adult myocyte, and iii) skeletal isoforms can transcomplement in the heart in the absence of cMyBP-C.
Inflammation plays a critical role in adverse cardiac remodeling and heart failure. Therefore, approaches geared towards inhibiting inflammation may provide therapeutic benefits. We tested the hypothesis that genetic deletion of interleukin-10 (IL10), a potent anti-inflammatory cytokine, exacerbates pressure-overload induced adverse cardiac remodeling and hypertrophy and that IL10 therapy inhibits this pathology.
Methods and Results
Cardiac hypertrophy was induced in Wild-type (WT) and IL10-knockout (KO) mice by isoproterenol (ISO) infusion. ISO-induced left ventricular (LV) dysfunction and hypertrophic remodeling, including fibrosis and fetal gene expression, were further exaggerated in KO mice compared to WT. Systemic recombinant mouse IL10 administration markedly improved LV function and not only inhibited but also reversed ISO-induced cardiac remodeling. Intriguingly, very similar cardio-protective response of IL10 was found in transverse aortic constriction (TAC)-induced hypertrophy and heart failure model. In neonatal rat ventricular myocytes (NRCM) and H9c2 myoblasts, ISO activated NFκB while it inhibited STAT3 phosphorylation. Interestingly, IL10 suppressed ISO-induced NFκB activation and attenuated STAT3 inhibition. Moreover, pharmacological and genetic inhibition of STAT3 reversed the protective effects of IL10 while ectopic expression of constitutively active STAT3 mimicked the IL10 responses on the ISO effects, confirming that IL10 mediated inhibition of NFκB is STAT3 dependent.
Taken together our studies suggest IL10 treatment as a potential therapeutic approach to limit the progression of pressure overload-induced adverse cardiac remodeling.
heart failure; hypertrophy; interleukins; myocardium; signal transduction
Elevated protein kinase C βII (PKCβII) expression develops during heart failure and yet the role of this isoform in modulating contractile function remains controversial. The present study examines the impact of agonist-induced PKCβII activation on contractile function in adult cardiac myocytes. Diminished contractile function develops in response to low dose phenylephrine (PHE, 100 nM) in controls, while function is preserved in response to PHE in PKCβII-expressing myocytes. PHE also caused PKCβII translocation and a punctate distribution pattern in myocytes expressing this isoform. The preserved contractile function and translocation responses to PHE are blocked by the inhibitor, LY379196 (30 nM) in PKCβII-expressing myocytes. Further analysis showed downstream protein kinase D (PKD) phosphorylation and phosphatase activation are associated with the LY379196-sensitive contractile response. PHE also triggered a complex pattern of end-target phosphorylation in PKCβII-expressing myocytes. These patterns are consistent with bifurcated activation of downstream signaling activity by PKCβII.
Earlier studies have shown that cardiac myosin binding protein-C (cMyBP-C) is easily releasable into the circulation following myocardial infarction (MI) in animal models and patients. However, since its release kinetics has not been clearly demonstrated, no parameters are available to judge its efficacy as a bona fide biomarker of MI in patients with MI. To make this assessment, plasma levels of cMyBP-C and six known biomarkers of MI were determined by sandwich enzyme-linked immunosorbent assay in patients with MI who had before and after Percutaneous Transcoronary Angioplasty (PTCA), as well as healthy controls. Compared to healthy controls (22.3 ± 2.4 ng/mL (n=54)), plasma levels of cMyBP-C were significantly increased in patients with MI (105.1 ± 8.8 ng/mL (n=65), P<0.001). Out of 65 patients, 24 had very high levels of plasma cMyBP-C (116.5 ± 13.3 ng/mL), indicating high probability of MI. Importantly, cMyBP-C levels were significantly decreased in patients (n=40) at 12 hours post-PTCA (41.2 ± 9.3 ng/mL, P<0.001), compared to the patients with MI. Receiver operating characteristic analysis revealed that a plasma cMyBP-C reading of 68.1 ng/mL provided a sensitivity of 66.2% and a specificity of 100%. Also, myoglobin, carbonic anhydrase and creatine kinase-MB levels were significantly increased in MI patients who also had higher cMyBP-C levels. In contrast, levels of cardiac troponin I, glycogen phosphorylase and heart-type fatty acid binding protein were not significantly changed in the samples, indicating the importance of evaluating the differences in release kinetics of these biomarkers in the context of accurate diagnosis. Our findings suggest that circulating cMyBP-C is a sensitive and cardiac-specific biomarker with potential utility for the accurate diagnosis of MI.
Acute coronary syndrome; cardiac biomarker; cardiac myosin binding protein-C; contractile protein; cMyBP-C; myocardial infarction