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Protein kinase G I α (PKGIα) inhibits cardiac remodeling, and this effect requires the PKGIα leucine zipper (LZ) binding domain. However, PKGIα LZ-dependent cardiac substrates remain poorly understood. Clinical trials of PKGI activating drugs have been limited to date by hypotension arising from vascular PKGI activation. Therefore, we explored downstream PKGIα substrates in the heart which may inhibit remodeling, yet circumvent the hypotensive effects of systemic PKGI activation. A screen for PKGIα LZ-interacting proteins identified: 1)cardiac myosin binding protein-C (cMyBP-C) and 2) mixed lineage kinase 3 (MLK3). cMyBP-C is a cardiac myocyte protein known to inhibit remodeling when phosphorylated. Co-precipitations with cGMP-conjugated beads confirmed the PKGIα-cMyBP-C interaction. Purified PKGIα phosphorylated cMyBP-C in vitro at Ser-273, Ser-282, and Ser-302. cGMP induced cMyBP-C phosphorylation at these sites in COS cells transfected with WT PKGIα, but not in cells transfected with either LZ mutant PKGIα or kinase-inactive PKGIα. In hearts of 9 month old PKGIα Leucine Zipper mutant mice, which have LV hypertrophy (LVH) and diastolic dysfunction, we observed decreased phosphorylated cMyBP-C as well as decreased total cMyBP-C, compared with WT littermate hearts. We next tested the effect of MLK3, which interacts with PKGIα in the heart, on remodeling in vivo. We performed 7 day Transaortic Constriction (TAC) on MLK3 KO mice and WT littermates (n=5 shams, 8 TAC per genotype). MLK3 KO TAC mice had increased LVH (LV mass/tibia length 71.1 ± 2.7 g/cm KO TAC vs 62.1 ± 2.7 WT TAC; p These studies reveal 2 novel PKGIα anti-remodeling substrates, and they support that exploring PKGIα substrates in the heart may identify novel therapeutic targets to inhibit cardiac remodeling but avoid excessive PKGI induced vasodilation.

Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is necessary for the regulation of sarcomeric structure and myocardial contractility in vivo. Different from skeletal isoforms, cMyBP-C has M-domain phosphorylation motifs in which three serines, Ser273, 282 and 302, are known to be substrates for several kinases. Phosphorylation of these M-domain sites regulates the interaction of cMyBP-C with thick and thin myofilaments during muscle contraction. While protein kinase C (PKC) is known to phosphorylate Ser273 and Ser302, the precise functional consequences of PKC-mediated cMyBP-C phosphorylation are not fully characterized. Therefore, the objective of this study was to determine how PKC-mediated phosphorylation at Ser273/302 might control the function of cMyBP-C in the context of thick and thin myofilament regulation and sarcomere contraction. Using recombinant proteins and yeast-2-hybrid (Y2H) clones expressing Ser273-Ser282-Ser302 (cMyBP-CWT), Ala273-Asp282-Ala302 (cMyBP-CADA) or Asp273-Ala282-A...

Rationale Cardiac myosin binding protein-C (cMyBP-C) is a trans-filament protein that has been shown to regulate cardiac function via its amino terminal (N′) region. In vitro studies have suggested the importance of the first 271 N′-residues of cMyBP-C (C0-C1f region) in slowing actin filament sliding over myosin to regulate cross-bridge cycling kinetics within the cardiac sarcomere. However, the role and necessity of the C0-C1f region of cMyBP-C in regulating contractile and cardiac function in vivo have not been elucidated. Hypothesis The N′-C0-C1f region of cMyBP-C is critical for proper cardiac function in vivo . Methods and Results Transgenic mice with approximately 95% expression of a mutant truncated cMyBP-C missing the N′-C0-C1f region (cMyBP-C 110 kDa ), compared to endogenous cMyBP-C, were generated and characterized at 3-months of age. cMyBP-C 110 kDa hearts had significantly elevated heart weight/body weight ratio, fibrosis, nuclear area and collagen content compared to hearts from non-transgenic (NTG) littermates. Electron microscopic analysis revealed normal sarcomere structure in cMyBP-C 110 kDa hearts but with apparently weaker cMyBP-C stripes. Furthermore, the ability of cMyBP-C to slow actin-filament sliding within the C-zone of native thick filaments isolated from NTG hearts was lost on thick filaments from cMyBP-C 110 kDa hearts. Short axis M-mode echocardiography revealed a significant increase in left ventricular (LV) internal diameter during diastole in cMyBP-C 110 kDa hearts. Importantly, cMyBP-C 110 kDa hearts displayed a significant reduction in fractional shortening compared to hearts from NTG mice. We further observed a decrease in the thickness of the LV interventricular septum and free wall during systole in cMyBP-C 110 kDa hearts. Strain analysis using images acquired from ECG-Gated Kilohertz Visualization identified a significant deficit in global longitudinal strain in cMyBP-C 110 kDa hearts compared to NTG hearts. Consistent with cardiac hypertrophy, we observed a significant increase in the expression of the hypertrophic genes MYH7 and NPPA by real-time PCR analysis. As expected, the expression levels of the MYBPC3 gene were significantly elevated in cMyBP-C 110 kDa hearts compared to NTG hearts. Surprisingly, our Western blot analyses revealed no significant difference in total cMyBP-C levels between NTG and cMyBP-C 110 kDa heart homogenates. However, intriguingly, we observed a significant elevation in cMyBP-C phosphorylation at Ser-273, Ser-282, and Ser-302, sites important for cMyBP-C9s regulation of actomyosin interaction, in cMyBP-C 110 kDa heart homogenates compared to those from NTG mice. Conclusion The N′-C0-C1f region of cMyBP-C is essential for maintaining normal cardiac morphology and function in vivo and loss of this region promotes contractile dysfunction both at the molecular and tissue level.

Background— Hypertrophic cardiomyopathy (HCM) is a common genetic disorder caused mainly by mutations in sarcomeric proteins and is characterized by maladaptive myocardial hypertrophy, diastolic heart failure, increased myofilament Ca 2+ sensitivity, and high susceptibility to sudden death. We tested the following hypothesis: correction of the increased myofilament sensitivity can delay or prevent the development of the HCM phenotype. Methods and Results— We used an HCM mouse model with an E180G mutation in α-tropomyosin (Tm180) that demonstrates increased myofilament Ca 2+ sensitivity, severe hypertrophy, and diastolic dysfunction. To test our hypothesis, we reduced myofilament Ca 2+ sensitivity in Tm180 mice by generating a double transgenic mouse line. We crossed Tm180 mice with mice expressing a pseudophosphorylated cardiac troponin I (S23D and S24D; TnI-PP). TnI-PP mice demonstrated a reduced myofilament Ca 2+ sensitivity compared with wild-type mice. The development of pathological hypertrophy did not occur in mice expressing both Tm180 and TnI-PP. Left ventricle performance was improved in double transgenic compared with their Tm180 littermates, which express wild-type cardiac troponin I. Hearts of double transgenic mice demonstrated no changes in expression of phospholamban and sarcoplasmic reticulum Ca 2+ ATPase, increased levels of phospholamban and troponin T phosphorylation, and reduced phosphorylation of TnI compared with Tm180 mice. Moreover, expression of TnI-PP in Tm180 hearts inhibited modifications in the activity of extracellular signal-regulated kinase and zinc finger-containing transcription factor GATA in Tm180 hearts. Conclusions— Our data strongly indicate that reduction of myofilament sensitivity to Ca 2+ and associated correction of abnormal relaxation can delay or prevent development of HCM and should be considered as a therapeutic target for HCM.

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±0.06μWmg(-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±1.33kNm(-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 (p0.001) 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.

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 [Ca(2+)]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, PKCe. 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.

AimsMouse 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 resultsFollowing 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.ConclusionsApplication 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.

Cardiomyocyte apoptosis contributes toward the loss of muscle mass in myocardial pathologies. Previous reports have implicated type I cAMP-dependent protein kinase (PKA) and p90 ribosomal S6 kinase (RSK) in cardiomyocyte apoptosis. However, the precise mechanisms and the isoform of RSK involved in this process remain undefined. Using adult rat ventricular myocytes and mouse-derived cardiac HL-1 cardiomyocytes, we demonstrate that hypoxia/reoxygenation (H/R)-induced apoptosis is accompanied by a decrease in the type I PKA regulatory subunit (PKARIα) and activation of RSK1. As previously described by us for other cell types, in cardiomyocytes, inactive RSK1 also interacts with PKARIα, whereas the active RSK1 interacts with the catalytic subunit of PKA. Additionally, small interfering (siRNA)-mediated silencing of PKARIα or disrupting the RSK1/PKARIα interactions with a small, cell-permeable peptide activates RSK1 and recapitulates the H/R-induced apoptosis. Inhibition of RSK1 or siRNA-mediated silencing of RSK1 attenuates H/R-induced apoptosis, demonstrating the role of RSK1 in cardiomyocyte apoptosis. Furthermore, silencing of RSK1 decreases the H/R-induced phosphorylation of sodium–hydrogen exchanger 1 (NHE1), and inhibition of NHE1 with 5′-N-ethyl-N-isopropyl-amiloride blocks H/R induced apoptosis, indicating the involvement of NHE1 in apoptosis. Overall, our findings demonstrate that H/R-mediated decrease in PKARIα protein levels leads to activation of RSK1, which via phosphorylation of NHE1 induces cardiomyocyte apoptosis.

Hypertrophic cardiomyopathy (HCM) is the most common form of inherited cardiac disease and the leading cause of sudden cardiac death in young people. HCM is caused by mutations in genes encoding contractile proteins. Cardiac myosin binding protein-C (cMyBP-C) is a thick filament contractile protein that regulates sarcomere organization and cardiac contractility. About 200 different mutations in the cMyBP-C gene (MYBPC3) have thus far been reported as causing HCM. Among them, a 25 base pair deletion in the branch point of intron 32 of MYBPC3 is widespread, particularly affecting people of South Asian descent, with 4% of this population carrying the mutation. This polymorphic mutation results in skipping of exon 33 and a reading frame shift, which, in turn, replaces the last 65 amino acids of the C-terminal C10 domain of cMyBP-C with a novel sequence of 58 residues (cMyBP-C(C10mut)). Carriers of the 25 base pair deletion mutation are at increased risk of developing cardiomyopathy and heart failure. Because of the high prevalence of this mutation in certain populations, genetic screening of at-risk groups might be beneficial. Scientifically, the functional consequences of C-terminal mutations and the precise mechanisms leading to HCM should be defined using induced pluripotent stem cells and engineered heart tissue in vitro or mouse models in vivo. Most importantly, therapeutic strategies that include pharmacology, gene repair, and gene therapy should be developed to prevent the adverse clinical effects of cMyBP-C(C10mut). This review article aims to examine the effects of cMyBP-C(C10mut) on cardiac function, emphasizing the need for the development of genetic testing and expanded therapeutic strategies.

Cardiac myosin binding protein-C (cMyBP-C) is a cardiac-specific, thick-filament regulatory protein that is differentially phosphorylated at Ser(273), Ser(282), and Ser(302) by various kinases and modulates contraction. In this study, phosphorylation-site-specific effects of cMyBP-C on myocardial contractility and cross-bridge kinetics were studied by sinusoidal analysis in papillary and trabecular muscle fibers isolated from t/t (cMyBP-C-null) mice and in their counterparts in which cMyBP-C contains the ADA (Ala(273)-Asp(282)-Ala(302)), DAD (Asp(273)-Ala(282)-Asp(302)), and SAS (Ser(273)-Ala(282)-Ser(302)) mutations; the results were compared to those from mice expressing the wild-type (WT) transgene on the t/t background. Under standard activating conditions, DAD fibers showed significant decreases in tension (~50%), stiffness, the fast apparent rate constant 2πc, and its magnitude C, as well as its magnitude H, but an increase in the medium rate constant 2πb, with respect to WT. The t/t fibers showed a smaller drop in stiffness and a significant decrease in 2πc that can be explained by isoform shift of myosin heavy chain. In the pCa-tension study using the 8 mM phosphate (Pi) solution, there was hardly any difference in Ca(2+) sensitivity (pCa50) and cooperativity (nH) between the mutant and WT samples. However, in the solutions without Pi, DAD showed increased nH and slightly decreased pCa50. We infer from these observations that the nonphosphorylatable residue 282 combined with phosphomimetic residues Asp(273) and/or Asp(302) (in DAD) is detrimental to cardiomyocytes by lowering isometric tension and altering cross-bridge kinetics with decreased 2πc and increased 2πb. In contrast, a single change of residue 282 to nonphosphorylatable Ala (SAS), or to phosphomimetic Asps together with the changes of residues 273 and 302 to nonphosphorylatable Ala (ADA) causes minute changes in fiber mechanics.

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time. Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Background: Recently we have shown that IL-10, an anti-inflammatory cytokine, markedly inhibited the pressure overload-induced cardiac fibrosis, however, antifibrotic mechanisms of IL-10 are largely unknown. In most of organs, including heart, extracellular matrix (ECM) remodeling is primarily mediated by excessive proliferation of activated fibroblasts and myofibroblasts. Here we hypothesized that IL-10 inhibits stress-induced homing, proliferation and differentiation of nonresident bone marrow-derived fibroblast progenitor cells and therefore, attenuates cardiac remodeling and improves of heart function. Methods and Results: Cardiac hypertrophy was induced in Wild-type (WT) and IL-10-knockout (KO) mice by transverse aortic constriction (TAC). TAC-induced left ventricular (LV) dysfunction and fibrosis were further exaggerated in KO mice compared to WT. TAC significantly increased TGF-β, collagen Iα and IIIα genes expression. Systemic recombinant mouse IL-10 administration markedly improved LV function, inhibited TAC-induced cardiac fibrosis and fibrosis associated genes expression. To identify the role of fibroblast progenitor cells (FPCs), we measured the mobilization of FPCs (Prominin1 positive cells) from bone marrow to heart by FACs. Exacerbated mobilization of FPCs in peripheral blood and heart in IL-10 KO mice were found 3 and 7 days after aortic constriction. Bone marrow transplantation experiments were performed where WT-GFP positive marrow was transplanted in BM depleted IL-10 KO mice. TAC-induced mobilization was significantly reduced in WT-transplanted marrow as compare to TAC-IL-10 KO mice. To identify the role IL-10 on TGFβ-induced endothelial cells trans-differentiation to myofibroblasts, we treated aortic endothelial cells with IL-10 and TGFβ2 for 96 hrs. Both Immunocytochemistry and Western blot analysis results suggested that TGF-β2-induced EndMT was significantly inhibited by IL-10 treatment. To understand the mechanisms, we found that TGF-β2-induced Notch1 signaling was reduced by IL-10. Conclusion: Taken together our observations suggest that the anti-fibrotic effects of IL-10 treatment are mediated by reduced proliferation and differentiation of non-resident myofibroblasts.

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.

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.

Rationale: High-myofilament Ca 2+ sensitivity has been proposed as a trigger of disease pathogenesis in familial hypertrophic cardiomyopathy (HCM) on the basis of in vitro and transgenic mice studies. However, myofilament Ca 2+ sensitivity depends on protein phosphorylation and muscle length, and at present, data in humans are scarce. Objective: To investigate whether high myofilament Ca 2+ 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 Ca 2+ sensitivity in all HCM samples and low phosphorylation of protein kinase A (PKA) targets compared with donors. After exogenous PKA treatment, myofilament Ca 2+ sensitivity was similar ( MYBPC3 mut , TPM1 mut , sarcomere mutation-negative HCM), higher ( MYH7 mut , TNNT2 mut ), or even significantly lower ( TNNI3 mut ) 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 TNNT2 mut and TNNI3 mut corrected length-dependent activation to donor values. Conclusions: High-myofilament Ca 2+ 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 posttranslational modifications other than PKA hypophosphorylation or altered protein–protein interactions, and represents a common pathomechanism in HCM.

Hearts in volume overload (VO) undergo progressive ventricular hypertrophy resulting in chronic heart failure that is unresponsive to β-adrenergic agonists. This study compared left ventricular (LV) and isolated cardiomyocyte contractility and β-adrenergic responsiveness in rats with end-stage VO heart failure (HF). Adult male Sprague-Dawley rats were studied 21 weeks after aortocaval fistula (ACF) or sham surgery. Echocardiography revealed decreased fractional shortening accompanied by increased LV chamber diameter and decreased eccentric dilatation index at end-stage ACF compared to sham. Hemodynamic measurements showed a decrease in the slope of end-systolic pressure-volume relationship, indicating systolic dysfunction. Isolated LV myocytes from ACF exhibited decreased peak sarcomere shortening and kinetics. Both Ca2+ transient amplitude and kinetics were increased in ACF myocytes, with no change under the integrated Ca2+ curves relating to contraction and relaxation phases. Increases in ryanodine receptor and phospholamban phosphorylation, along with a decrease in SERCA2 levels, were observed in ACF. These changes were associated with decreased expression of β-myosin heavy chain, cardiac troponin I and cardiac myosin binding protein-C. In vivo inotropic responses to β-adrenergic stimulation were attenuated in ACF. Interestingly, ACF myocytes exhibited a similar peak shortening to those of sham in response to a β-adrenergic agonist. The protein expression of the gap junction protein connexin-43 was decreased, although its phosphorylation at Ser-368 increased. These changes were associated with alterations in Src and ZO-1. In summary, these data suggest that the disconnect in β-adrenergic responsiveness between in vivo and in vitro conditions may be associated with altered myofilament Ca2+ sensitivity and connexin-43 degradation.

Neuronal nitric oxide synthase (NOS1 or nNOS) exerts negative inotropic and positive lusitropic effects through Ca(2+) handling processes in cardiac myocytes from healthy hearts. However, underlying mechanisms of NOS1 in diseased hearts remain unclear. The present study aims to investigate this question in angiotensin II (Ang II)-induced hypertensive rat hearts (HP). Our results showed that the systolic function of left ventricle (LV) was reduced and diastolic function was unaltered (echocardiographic assessment) in HP compared to those in shams. In isolated LV myocytes, contraction was unchanged but peak [Ca(2+)]i transient was increased in HP. Concomitantly, relaxation and time constant of [Ca(2+)]i decay (tau) were faster and the phosphorylated fraction of phospholamban (PLN-Ser(16)/PLN) was greater. NOS1 protein expression and activity were increased in LV myocyte homogenates from HP. Surprisingly, inhibition of NOS1 did not affect contraction but reduced peak [Ca(2+)]i transient; prevented faster relaxation without affecting the tau of [Ca(2+)]i transient or PLN-Ser(16)/PLN in HP, suggesting myofilament Ca(2+) desensitization by NOS1. Indeed, relaxation phase of the sarcomere length-[Ca(2+)]i relationship of LV myocytes shifted to the right and increased [Ca(2+)]i for 50% of sarcomere shortening (EC50) in HP. Phosphorylations of cardiac myosin binding protein-C (cMyBP-C(282) and cMyBP-C(273)) were increased and cardiac troponin I (cTnI(23/24)) was reduced in HP. Importantly, NOS1 or PKG inhibition reduced cMyBP-C(273) and cTnI(23/24) and reversed myofilament Ca(2+) sensitivity. These results reveal that NOS1 is up-regulated in LV myocytes from HP and exerts positive lusitropic effect by modulating myofilament Ca(2+) sensitivity through phosphorylation of key regulators in sarcomere.

Rationale: Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is essential for normal heart function. We recently demonstrated a direct correlation between cMyBP-C dephosphorylation and its degradation during ischemia-reperfusion (I-R) injury. Strikingly, cMyBP-C phosphorylation protects the heart from I-R injury. However, the mechanism of cMyBP-C-mediated cardioprotection is unknown. Objective: To determine if preventing cMyBP-C proteolysis and cleavage confers cardioprotection during I-R injury. Methods and Results: cMyBP-C is a substrate for calpains and dephosphorylation makes cMyBP-C more susceptible to proteolysis. In patients with heart failure, such proteolysis leads to the cleavage and release of the predominant N'-fragment (40 kDa) in association with increased calpain activity. MS/MS analysis identified a cardiac-specific phosphorylation motif responsible for the release of the 40 kDa fragment, 272-TSLAGAGRR-280, which we term the calpain-targeted site (CTS). Next, we utilized cardiac-specific transgenic mice expressing cMyBP-C[[Unable to Display Character: △]]CTS in which CTS motif was ablated and bred into cMyBP-C null (t/t) mice. We hypothesized that ablation of the CTS motif would confer resistance to calpain-mediated proteolysis and thus protect the heart from I-R injury. An animal that transgenically expressed the normal cardiac isoform, cMyBP-CWT, served as a control. Histological, electron microscopic and immunofluorescence analyses confirm that cMyBP-C[[Unable to Display Character: ∆]]CTS incorporates normally into the cardiac sarcomere and results in complete rescue of the cMyBP-Ct/t phenotype, compared control groups. Moreover, echocardiography and in vivo hemodynamic studies revealed that cMyBP-C[[Unable to Display Character: ∆]]CTS:t/t mice have normal cardiac function, similar to control mice. Altogether, these data suggest that cMyBP-C[[Unable to Display Character: ∆]]CTS is benign. Compared to cMyBP-CWT, cMyBP-C[[Unable to Display Character: ∆]]CTS protein is protected from calpain-mediated degradation. Finally, we determined that ablation of the CTS reduced infarct size and preserved cMyBP-C stability and cardiac function during I-R injury. Conclusion: Our data suggest that preserving cMyBP-C stability by thwarting calpain-mediated proteolysis protects cardiac structure and function from I-R injury, as a supportive therapy.

Mutations of cardiac myosin binding protein-C (cMyBP-C) are inherited by an estimated 60 million people worldwide, and the protein is the target of several kinases. Recent evidence further suggests that cMyBP-C mutations alter Ca(2+) transients, leading to electrophysiological dysfunction. Thus, while the importance of studying this cardiac sarcomere protein is clear, preliminary data in the literature have raised many questions. Therefore, in this article, we propose to review the structure and function of cMyBP-C with particular respect to the role(s) in cardiac contractility and whether its release into the circulatory system is a potential biomarker of myocardial infarction. We also discuss future directions and experimental designs that may lead to expanding the role(s) of cMyBP-C in the heart. In conclusion, we suggest that cMyBP-C is a regulatory protein that could offer a broad clinical utility in maintaining normal cardiac function.

Normal hearts increase contractile force in response to mechanical stretch caused by increasing volume. Although this phenomenon has been extensively studied from myofilaments’ spacing perspective, possible coupling of mechanical-sensing signaling to modulation of myofilament function remains unknown. Cardiac myosin binding protein-C (MyBPC3) is a component of heart muscle thick filament. Phosphorylation of MyBPC3 releases its inhibition on cross-bridge cycling to increase cardiac contractility. Thus, we postulate that mechanical stretch of the myocardium causes phosphorylation of MyBPC3 to increase contractility. We tested this hypothesis by performing static stretch of 20% from baseline on cultured neonatal rat cardiac myocytes (NRCM) at durations of 2, 5, 15, 30, and 60 minutes. NRCM culture provides the advantage of cells living in an environment free of adrenergic stimulation to avoid catecholamine stimulation mediated phosphorylation of MyBPC3. Site-specific phospho-serine antibodies were used to detect phosphorylation of rat equivalent of S282 and S302 of mouse MyBPC3. We used MyBPC3 antibody made from a different species than site-specific phospho-serine antibodies to account for loading. S282P transiently peaked after 5 minutes of stretch, whereas S302P continued to increase with time through 60 minutes (see figure ). Consequently, our data show that mechanical stretch alone can cause phosphorylation of MyBPC3 as a mechanism that couples different signaling pathways to myofilament function.

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament assembly protein that stabilizes sarcomeric structure and regulates cardiac function; however, the profile of cMyBP-C degradation after myocardial infarction (MI) is unknown. We hypothesized that cMyBP-C is sensitive to proteolysis and is specifically increased in the bloodstream post-MI in rats and humans. Under these circumstances, elevated levels of degraded cMyBP-C could be used as a diagnostic tool to confirm MI. To test this hypothesis, we first established that cMyBP-C dephosphorylation is directly associated with increased degradation of this myofilament protein, leading to its release in vitro. Using neonatal rat ventricular cardiomyocytes in vitro, we were able to correlate the induction of hypoxic stress with increased cMyBP-C dephosphorylation, degradation, and the specific release of N′-fragments. Next, to define the proteolytic pattern of cMyBP-C post-MI, the left anterior descending coronary artery was ligated in adult male rats. Degradation of cMyBP-C was confirmed by a reduction in total cMyBP-C and the presence of degradation products in the infarct tissue. Phosphorylation levels of cMyBP-C were greatly reduced in ischemic areas of the MI heart compared to non-ischemic regions and sham control hearts. Post-MI plasma samples from these rats, as well as humans, were assayed for cMyBP-C and its fragments by sandwich ELISA and immunoprecipitation analyses. Results showed significantly elevated levels of cMyBP-C in the plasma of all post-MI samples. Overall, this study suggests that cMyBP-C is an easily releasable myofilament protein that is dephosphorylated, degraded and released into the circulation post-MI. The presence of elevated levels of cMyBP-C in the blood provides a promising novel biomarker able to accurately rule in MI, thus aiding in the further assessment of ischemic heart disease.

We investigated the influence of cardiac myosin binding protein-C (cMyBP-C) and its constitutively unphosphorylated status on the radial and longitudinal stiffnesses of the myofilament lattice in chemically skinned myocardial strips of the following mouse models: nontransgenic (NTG), effective null for cMyBP-C (t/t), wild-type cMyBP-C expressed into t/t (WT(t/t)), and constitutively unphosphorylated cMyBP-C (AllP-(t/t)). We found that the absence of cMyBP-C in the t/t and the unphosphorylated cMyBP-C in the AllP-(t/t) resulted in a compressible cardiac myofilament lattice induced by rigor not observed in the NTG and WT(t/t). These results suggest that the presence and phosphorylation of the N-terminus of cMyBP-C provides structural support and radial rigidity to the myofilament lattice. Examination of myofilament longitudinal stiffness under rigor conditions demonstrated a significant reduction in cross-bridge-dependent stiffness in the t/t compared with NTG controls, but not in the AllP-(t/t) compared with WT(t/t) controls. The absence of cMyBP-C in the t/t and the unphosphorylated cMyBP-C in the AllP-(t/t) both resulted in a shorter myosin cross-bridge lifetime when myosin isoform was controlled. These data collectively suggest that cMyBP-C provides radial rigidity to the myofilament lattice through the N-terminus, and that disruption of the phosphorylation of cMyBP-C is sufficient to abolish this structural role of the N-terminus and shorten cross-bridge lifetime. Although the presence of cMyBP-C also provides longitudinal rigidity, phosphorylation of the N-terminus is not necessary to maintain longitudinal rigidity of the lattice, in contrast to radial rigidity.

In myocardium, the 90-kDa ribosomal S6 kinase (RSK) is activated by diverse stimuli and regulates the sarcolemmal Na(+)/H(+) exchanger through direct phosphorylation. Only limited information is available on other cardiac RSK substrates and functions. We evaluated cardiac myosin-binding protein C (cMyBP-C), a sarcomeric regulatory phosphoprotein, as a potential RSK substrate. In rat ventricular myocytes, RSK activation by endothelin 1 (ET1) increased cMyBP-C phosphorylation at Ser(282), which was inhibited by the selective RSK inhibitor D1870. Neither ET1 nor D1870 affected the phosphorylation status of Ser(273) or Ser(302), cMyBP-C residues additionally targeted by cAMP-dependent protein kinase (PKA). Complementary genetic gain- and loss-of-function experiments, through the adenoviral expression of wild-type or kinase-inactive RSK isoforms, confirmed RSK-mediated phosphorylation of cMyBP-C at Ser(282). Kinase assays utilizing as substrate wild-type or mutated (S273A, S282A, S302A) recombinant cMyBP-C fragments revealed direct and selective Ser(282) phosphorylation by RSK. Immunolabeling with a Ser(P)(282) antibody and confocal fluorescence microscopy showed RSK-mediated phosphorylation of cMyBP-C across the C-zones of sarcomeric A-bands. In chemically permeabilized mouse ventricular muscles, active RSK again induced selective Ser(282) phosphorylation in cMyBP-C, accompanied by significant reduction in Ca(2+) sensitivity of force development and significant acceleration of cross-bridge cycle kinetics, independently of troponin I phosphorylation at Ser(22)/Ser(23). The magnitudes of these RSK-induced changes were comparable with those induced by PKA, which phosphorylated cMyBP-C additionally at Ser(273) and Ser(302). We conclude that Ser(282) in cMyBP-C is a novel cardiac RSK substrate and its selective phosphorylation appears to regulate cardiac myofilament function.

During the past 5 years there has been an increasing body of literature describing the roles cardiac myosin binding protein C (cMyBP-C) phosphorylation play in regulating cardiac function and heart failure. cMyBP-C is a sarcomeric thick filament protein that interacts with titin, myosin and actin to regulate sarcomeric assembly, structure and function. Elucidating the function of cMyBP-C is clinically important because mutations in this protein have been linked to cardiomyopathy in more than sixty million people worldwide. One function of cMyBP-C is to regulate cross-bridge formation through dynamic phosphorylation by protein kinase A, protein kinase C and Ca(2+)-calmodulin-activated kinase II, suggesting that cMyBP-C phosphorylation serves as a highly coordinated point of contractile regulation. Moreover, dephosphorylation of cMyBP-C, which accelerates its degradation, has been shown to associate with the development of heart failure in mouse models and in humans. Strikingly, cMyBP-C phosphorylation presents a potential target for therapeutic development as protection against ischemic-reperfusion injury, which has been demonstrated in mouse hearts. Also, emerging evidence suggests that cMyBP-C has the potential to be used as a biomarker for diagnosing myocardial infarction. Although many aspects of cMyBP-C phosphorylation and function remain poorly understood, cMyBP-C and its phosphorylation states have significant promise as a target for therapy and for providing a better understanding of the mechanics of heart function during health and disease. In this review we discuss the most recent findings with respect to cMyBP-C phosphorylation and function and determine potential future directions to better understand the functional role of cMyBP-C and phosphorylation in sarcomeric structure, myocardial contractility and cardioprotection.

Background— Cardiac myosin binding protein-C (cMyBP-C) phosphorylation modulates cardiac contractility. When expressed in cMyBP-C–null (cMyBP-C (t/t) ) hearts, a cMyBP-C phosphomimetic (cMyBP-C AllP+ ) rescued cardiac dysfunction and protected the hearts from ischemia/reperfusion injury. However, cMyBP-C function may be dependent on the myosin isoform type. Because these replacements were performed in the mouse heart, which contains predominantly α-myosin heavy chain (α-MyHC), the applicability of the data to humans, whose cardiomyocytes contain predominantly β-MyHC, is unclear. We determined the effect(s) of cMyBP-C phosphorylation in a β-MyHC transgenic mouse heart in which >80% of the α-MyHC was replaced by β-MyHC, which is the predominant myosin isoform in human cardiac muscle. Methods and Results— To determine the effects of cMyBP-C phosphorylation in a β-MyHC background, transgenic mice expressing normal cMyBP-C (cMyBP-C WT ), nonphosphorylatable cMyBP-C (cMyBP-C AllP − ), or cMyBP-C AllP+ were bred into the β-MyHC background (β). These mice were then crossed into the cMyBP-C (t/t) background to ensure the absence of endogenous cMyBP-C. cMyBP-C (t/t)/β and cMyBP-C AllP − :(t/t)/β mice died prematurely because of heart failure, confirming that cMyBP-C phosphorylation is essential in the β-MyHC background. cMyBP-C AllP+:(t/t)/β and cMyBP-C WT:(t/t)/β hearts showed no morbidity and mortality, and cMyBP-C AllP+:(t/t)/β hearts were significantly cardioprotected from ischemia/reperfusion injury. Conclusions— cMyBP-C phosphorylation is necessary for basal myocardial function in the β-MyHC background and can preserve function after ischemia/reperfusion injury. Our studies justify exploration of cMyBP-C phosphorylation as a therapeutic target in the human heart.

Mutations in myosin binding protein-C (MyBP-C) cause both cardiac and skeletal muscle diseases, such as hypertrophic cardiomyopathy and distal arthrogryposis. There are three isoforms of MyBP-C: slow-skeletal, fast-skeletal, and cardiac (ssMyBP-C, fsMyBP-C, and cMyBP-C, respectively). These isoforms reside within the sarcomere, the functional unit of muscle contraction at the molecular level. However, the function of the three major MyBP-C isoforms remains unclear. The present study is the first to focus on the least characterized isoform, fsMyBP-C, which is expressed in fast- and mixed-type skeletal muscles. To determine the necessity of fsMyBP-C for regulation of contraction in the sarcomere, we generated a conventional fast-skeletal MyBP-C knockout (FSKO) mouse model. We analyzed both structural changes and regulatory function of skeletal muscles from heterozygous (FSKO −/+ ) and homozygous (FSKO −/− ), compared to wild-type (WT) mice. Neither heterozygous nor homozygous FSKO mice exhibited changes in morbidity or mortality relative to WT mice. Molecular analyses revealed a complete knockout of fsMyBP-C in the FSKO −/− skeletal muscles compared to FSKO −/+ and WT mice. Histopathological analyses of both Extensor digitorum longus (EDL) and soleus muscles revealed no obvious abnormalities, such as fibrosis or calcification, in either heterozygous or homozygous FSKO mice. Though fiber structure is preserved, we demonstrated that EDL muscles from FSKO −/− mice increases Ca 2+ -sensitivity of force development, suggesting that fsMyBP-C regulates contraction at the molecular level by decreasing Ca 2+ -sensitivity. While others have previously proposed the role of cMyBP-C is to increase Ca 2+ -sensitivity to normalize a Ca 2+ gradient imbalance in the heart, we propose that the role of fsMyBP-C in skeletal muscles is to reduce Ca 2+ -sensitivity of the thin filaments in order to normalize the reversed Ca 2+ gradient imbalance. Despite opposite effects on Ca 2+ -sensitivity, MyBP-C share the same functional role in both cardiac and skeletal muscles. Thus, in addition to elucidating the role of fast-skeletal MyBP-C and its regulation of skeletal muscle contraction, the present study provides insight into the cardiac isoform and its regulation of cardiac contraction.

Cardiac myosin binding protein-C (cMyBP-C) is a thick-filament protein whose presence and phosphorylation by protein kinase A (PKA) regulates cross-bridge formation and kinetics in isolated myocardium. We tested the influence of cMyBP-C and its PKA-phosphorylation on contraction/relaxation kinetics in intact hearts and revealed its essential role in several classic properties of cardiac function.Comprehensive in situ cardiac pressure-volume analysis was performed in mice harboring a truncation mutation of cMyBP-C (cMyBP-C(t/t)) that resulted in nondetectable protein versus hearts re-expressing solely wild-type (cMyBP-C(WT:(t/t))) or mutated protein in which known PKA-phosphorylation sites were constitutively suppressed (cMyBP-C(AllP-:(t/t))). Hearts lacking cMyBP-C had faster early systolic activation, which then terminated prematurely, limiting ejection. Systole remained short at faster heart rates; thus, cMyBP-C(t/t) hearts displayed minimal rate-dependent decline in diastolic time and cardiac preload. Furthermore, prolongation of pressure relaxation by afterload was markedly blunted in cMyBP-C(t/t) hearts. All 3 properties were similarly restored to normal in cMyBP-C(WT:(t/t)) and cMyBP-C(AllP-:(t/t)) hearts, which supports independence of PKA-phosphorylation. However, the dependence of peak rate of pressure rise on preload was specifically depressed in cMyBP-C(AllP-:(t/t)) hearts, whereas cMyBP-C(t/t) and cMyBP-C(AllP-:(t/t)) hearts had similar blunted adrenergic and rate-dependent contractile reserve, which supports linkage of these behaviors to PKA-cMyBP-C modification.cMyBP-C is essential for major properties of cardiac function, including sustaining systole during ejection, the heart-rate dependence of the diastolic time period, and relaxation delay from increased arterial afterload. These are independent of its phosphorylation by PKA, which more specifically modulates early pressure rise rate and adrenergic/heart rate reserve.

Elucidating the relative roles of cardiac troponin I (cTnI) and phospholamban (PLN) in β-adrenergic–mediated hastening of cardiac relaxation has been challenging and controversial. To test the hypothesis that β-adrenergic phosphorylation of cTnI has a prominent role in accelerating cardiac myocyte relaxation performance we used transgenic (Tg) mice bearing near complete replacement of native cTnI with a β-adrenergic phospho-mimetic of cTnI whereby tandem serine codons 23/24 were converted to aspartic acids (cTnI S23/24D). Adult cardiac myocytes were isolated and contractility determined at physiological temperature under unloaded and loaded conditions using micro-carbon fibers. At baseline, cTnI S23/24D myocytes had significantly faster relaxation times relative to controls, and isoproterenol stimulation (Iso) had only a small effect to further speed relaxation in cTnI S23/24D myocytes (delta Iso: 7.2 ms) relative to the maximum Iso effect (31.2 ms) in control. The Ca 2+ transient decay rate was similarly accelerated by Iso in Tg and nontransgenic (Ntg) myocytes. Gene transfer of cTnI S23/24D to myocytes in primary culture showed comparable findings. Gene transfer of cTnI with both serines 23/24 converted to alanines (cTnI S23/24A), or gene transfer of slow skeletal TnI, both of which lack PKA phosphorylation sites, significantly blunted Iso-mediated enhanced relaxation compared with controls. Gene transfer of wild-type cTnI had no effect on relaxation. These findings support a key role of cTnI in myocyte relaxation and highlight a direct contribution of the myofilaments in modulating the dynamics of myocardial performance.

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, 13, 3256-3260) 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.

Purpose: Myosin binding protein C is an accessory protein of vertebrate striated muscle thick filaments. Many studies have aimed to elucidate both its structure and function since mutations in the cardiac isoform (cMyBP-C) gene (MYBPC3) are known to be a leading cause of familial hypertrophic cardiomyopathy (HCM) affecting millions of people worldwide. There is strong evidence that phosphorylation of N-terminus M-domain of cMyBP-C is essential for normal cardiac function and that their dephosphorylation is associated with contractile dysfunction and HCM. However, the details of the underlying molecular-level ultrastructure are still poorly understood. The aim of this work is to provide details of the ultrastructure of thick filaments in different states of cMyBP-C phosphorylation. Methods: Two transgenic mouse strains expressing a constitutive phosphorylation or a non-phosphorylatable cMyBP-C have been compared to a homozygous knockout mouse for cMyBP-C and a wild-type (WT) control. We have carried out detailed transmission electron microscopy (TEM) in intact cardiac muscles, and electron tomography (ET) combined with sub-tomographic averaging. Results: TEM shows that cardiomyocyte ultrastructure is relatively well maintained under the total deletion of cMyBP-C from the heart and due to the expression of constitutive phosphorylation of cMyBP-C. However, the constitutive expression of non-PKA-phosphorylatable cMyBP-C leads to both sarcomeric disarray and mitochondria disruption. Longitudinal views of the average 3D reconstructions employing ET illustrate the surface density features of thick filaments. In the WT, two projection masses can be identified originating from cMyBP-C. Both the absence of cMyBP-C and the presence of constitutive non-phosphorylatable cMyBP-C seem to be associated with poorer structural preservation of the thick filaments. Conclusions: This study shows that cMyBP-C constitutive phosphorylation effectively preserves the intracellular arrangement of cardiac myocytes. However, non-PKA-phosphorylatable cMyBP-C leads to a dramatic overall decrease of tissue organization which is much more deleterious than the total deletion of cMyBP-C. This finding provides a structural explanation for cardiac dysfunction in this sample. This study shows that chemically fixed samples can be used for ET to reveal 3D structural information of thick filaments. Results obtained by using this methodology are in good agreement with previous studies using isolated filament approaches. However, in order to obtain higher resolution, advanced methods of sample processing will be required.

We report a case of Noonan syndrome with compound mutations in a sarcomeric contractile protein gene and several novel mutations in mitochondrial genes. Our case forms the first report, which emphasizes the importance of mtDNA mutations in Noonan syndrome and extends the scope for mitochondrial related syndromes.

Myosin binding protein-C (MyBP-C) is a thick filament accessory protein of vertebrate striated muscle. It is essential for normal cardiac function, and mutations in MyBP-C cause cardiac and skeletal muscle disease. The 40 nm long molecule is composed of 10 or 11 immunoglobulin- or fibronectin-like domains and is located in up to 9 axial stripes 43 nm apart in each half of the A-band. To understand MyBP-C function it is important to know its structural organization in the sarcomere. Several models have been proposed, in which MyBP-C either wraps around the thick filament, or extends radially from or longitudinally along the filament (or some combination of these). To distinguish between these models, we have used immuno-EM of mouse cardiac myofibrils labeled with antibodies to different domains of MyBP-C to determine the relative axial positions of the domains. Myofibrils were obtained from mouse cardiac muscle by homogenizing chemically skinned hearts under rigor conditions. They were labeled with antibodies specific for the N-terminal domain, the middle of the molecule, and the C-terminal domain, and observed by negative stain EM. Labeled myofibrils showed nine stain-excluding stripes in each half of the A-band. The average distance of each stripe from the middle of the M-line was measured. All antibodies labeled axially within 10 nm of each other, at the same axial positions as the unlabeled stripes, suggesting that the bulk of MyBP-C runs radially or circumferentially. A small axial difference between the C-terminal and the central and N-terminal antigenic sites suggests that a short portion of the C-terminus runs longitudinally. Electron tomography of muscle sections (Luther et al., PNAS 2011) and 3D reconstruction of isolated thick filaments (Zoghbi et al., PNAS 2008) support the radial arrangement.

To understand the functional significance of phosphorylation that takes place in the M domain of cMyBP-C, chemically skinned papillary muscle fibers of transgenic mice were studied by sinusoidal length alterations and concomitant tension transients. Muscle fibers were maximally activated at pCa 4.55 in the solution that mimic physiological conditions (5 mM MgATP, 8 mM Pi, 200 mM total ionic strength with K-acetate) in myocytes. WT mice possess phosphorylation sites S273, S282, and S302. SAS is a single mutant S282A, and ADA and DAD are triple mutants S273A/S282D/S302A and S273D/S282A/S302D, respectively. D models for phosphorylation (phosphomimetic), and A models for non-phosphorylation (phosphor-ablation). Isometric tension and stiffness of DAD were respectively ∼0.5x of those of WT, but tension and stiffness of t/t (cMyBP-C null), ADA, and SAS were respectively similar to WT. The fast rate constant 2πc of DAD and t/t was ∼0.6x of WT, but that of ADA and SAS was similar to WT. The intermediate rate constant 2πb of DAD and SAS was ∼1.3x of WT, but that of ADA and t/t was similar to WT. These results demonstrate that cMyBP-C M domain phosphorylation affects the cross-bridge kinetics at ATP binding and phosphate release steps, indicating that phosphorylation affects myosin structure and its interaction with actin. However, pCa-tension and pCa-stiffness studies demonstrated that pCa50 (Ca2+ sensitivity) and nH (cooperativity) were respectively not different among mutants and WT groups, indicating that phosphorylation of cMyBP-C has a minimal effect on the regulatory system. The decreased amount of isometric tension only in DAD indicates that phosphorylation of S273 and S302 are most significant and they diminish the force generation capability, presumably owing to the extra electrostatic interaction of the M domain of cMyBP-C with actin thin filament, which may serve as a break.

Cardiac myosin binding protein-C (cMyBP-C) is a sarcomeric thick filament assembly protein with regulatory functions in the heart. The cMyBP-C protein differs from the skeletal isoform in that it has a small insertion near the carboxyl terminus that contains 3 phosphorylatable serines, Ser-273, −282 and −302. While the precise functional correlates of cMyBP-C phosphorylation remain obscure, we do know that cMyBP-C is targeted by multiple kinases, such as PKA, PKC, RSK, PKD, CaMKII and PKG, suggesting that it plays a vital role in cardiac signaling. We previously reported that cMyBP-C phosphorylation is essential for normal heart function and that Ser-282 phosphorylation is critical for the subsequent phosphorylation of Ser-302 and normal cardiac function. However, the role of Ser-282 cMyBP-C phosphorylation in cardiac function, particularly as it affects contractile properties and sarcomere organization, is unclear. Therefore, to better understand the mechanisms and significance of cMyBP-C phosphorylation, we established several transgenic mouse models to determine the necessity and sufficiency of Ser-282 phosphorylation for normal cardiac function. Our findings suggested that cMyBP-C phosphorylation at Ser-282 is essential for normal cardiac function and that dephosphorylation at this site accelerates cMyBP-C degradation and cleavage of a 40 kDa fragment. During MI, we showed that cMyBP-C is extensively fragmented when dephosphorylated and that such fragmentation correlates well with contractile dysfunction and heart failure. Meanwhile, we also established that the release of cMyBP-C in the blood post-MI could be a potential diagnostic biomarker for MI. Overall, these studies show that Ser-282 phosphorylation is a critical determinant of Ser-302 phosphorylation and that cMyBP-C dephosphorylation accelerates its degradation and release into the circulation. In conclusion, we provide strong evidence that cMyBP-C phosphorylation directly affects the heart's contractile properties, sarcomere organization and cardioprotection.

Phosphorylation sites in Cardiac MyBP-C have been predicted at Ser273,282 and 302 but studies in intact tissue have identified 5 phosphorylted sites and suggested up to 4.6molsPi/mol MyBP-C is present in human heart.We analysed MyBP-C phosphospecies in human heart myofibrils by phosphate affinity SDS-PAGE using a non-specific antibody raised against the MyBP-C peptide 2-14. We observed six bands corresponding to 0, 1P, 2P, 3P, 4P, 5P phospho-species. Control experiments with pure MyBP-C indicated that the antibody labelled all phosphospecies equally. The assigned phosphorylation levels were confirmed by staining western blots with PhosTools phosphoprotein stain. This separation permits direct quantitative determination of MyBP-C phosphospecies without need for calibration.In donor heart myofibrils the highly phosphorylated species predominated: 0, 7±3%: 1P, 1±1%: 2P, 23±7%: 3P, 41±2%: 4P, 20±8% (n=4) from which total phosphorylation of MyBP-C was calculated to be 3.4molsPi/mol. In failing heart unphosphorylated MyBP-C predominated (0, 48±4%: 1P, 4±4%: 2P, 27±5%: 1±1%: 3P, 17±4%: 4P, 4±2%, n=4) and calculated total phosphorylation was 1.5 molsPi/mol. Total phosphorylation in failing heart myofibrils was 44% of donor and in myectomy samples from HCM patients it was 29% of donor, compared with 45 and 40% respectively determined in previous assays.We conclude that MyBP-C is highly phosphorylated in vivo with significant phosphorylation of at least 5 sites and that phosphorylation is dynamic, being greatly reduced in pathological muscle. Initial tests using antibodies specific to Ser 273, 282 and 302 show distinct patterns on phosphate affinity SDS-PAGE indicating varying preferences for the highly phosphorylated species of MyBP-C in normal and pathological muscle.

Rationale: Cardiac myosin binding protein-C (cMyBP-C) has three phosphorylatable serines at N-terminus (Ser-273, Ser-282, and Ser-302) and phosphorylation states of these serines may alter the thick filament structure and function. Phosphorylation of these serines in cMyBP-C may also alter sarcomere length-dependent modulation of contractile force development (LDA), which would be expected to modulate the Frank-Starling Law of the Heart.Objective: To analyze length dependent activation in cMyBP-C for nonphosphorylated and phosphomimetic states of serines 273, 282, 302 and to study the contribution of cMyBP-C in regulating cardiac contraction at different sarcomere lengths.Methods & Results: We studied the force-Ca2+ relationship in skinned myocytes isolated from (a) nonphosphorylated alanine transgenic mice (cMyBP-CAAA), (b) phosphomimetic aspartic acid transgenic mice (cMyBP-CDDD), and normal cMyBP-CWT mice at 2.0 and 2.3 μm sarcomere length (SL).At short SL, Ca2+ sensitivity was similar in all groups. However, Ca2+ sensitivity was increased in the cMyBP-CAAA group compared to either the WT or the cMyBP-CDDD group. Similar results were obtained for maximum force development. As a consequence, LDA was blunted (∼40%) in cMyBP-CAAA myocardium. There were no differences in the level of cooperativety as indexed by the Hill coefficient in any group.Conclusion: Phosphorylated cMyBP-C has been shown to contribute to regulation of cardiac sarcomere function via modulation of the cMyBP-C-actin interaction as well as the disposition of the cross-bridges in relation to the thin filament. Moreover removal of cMyBP-C results in blunted LDA, and a cardiac dysfunction that can be prevented by cMyBP-CDDD but not cMyBP-CAAA. Our data showed that lack of cMyBP-C phosphorylation results in blunted LDA, similar to that found previously in the absence cMyBP-C. We conclude that cMyBP-C phosphorylation modulates myofilament length dependent activation, possibly via modulation of the cMyBP-C interaction with actin.

Cardiac myosin binding protein-C (cMyBP-C) is a modular protein involved in stabilizing interactions with the thick filament of the sarcomere. The N-terminus of cMyBP-C associates with actin and myosin S2 and the C-terminus interacts with titin and myosin rods. While no high-resolution structure of C-terminal cMyBP-C exists, disruption of this region is proposed to destabilize cMyBP-C and adversely affect cardiac structure and function. In particular, deletion of 25 base pairs (Δ25) in the gene encoding for cMyBP-C results in amino acid substitutions in the C10 domain of cMyBP-C (C10 Δ25) which may be associated with the development of hypertrophic and dilated cardiomyopathies by unknown molecular mechanisms. The prevalence of this mutation is approximately 1% of the world population, underscoring the necessity of determining its role(s) in the pathogenesis of cardiomyopathies. In this study, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies have been used to examine the conformation of wild-type (Wt) and mutant C-terminal domains of cMyBP-C. Comparison of near UV CD spectra revealed alterations in the packing of aromatic residues in C10 Δ25 suggesting it is less stably folded as compared to C10 Wt. C10 Δ25 exhibited less beta-sheet content than C10 Wt as evidenced by the estimation of secondary structure from CD data. NMR analyses of amide proton/nitrogen chemical shifts and line-widths were used to probe the conformation of C10 domains and to map residues of importance in protein-protein association onto cMyBP-C models. Taken together, these data suggest that the Δ25 mutation structurally modulates cMyBP-C sites involved in binding titin and myosin.

Rationale: Myocardial infarction (MI) is associated with depressed cardiac contractile function and progression to heart failure. Cardiac myosin binding protein-C (cMyBP-C), a cardiac-specific myofilament protein, is proteolyzed post-MI in humans and results in an N-terminal fragment, C0C1f. The presence of C0C1f in cultured adult cardiomyocytes results in decreased Ca2+ transients and cell shortening, in addition to the induction of heart failure in a mouse model. However, the underlying mechanisms remain unclear.Objective: To determine how C0C1f causes altered contractility in human cardiac myofilaments in vitro.Methods and Results: We generated recombinant human C0C1f (hC0C1f) and incorporated it into skinned human left ventricular myocytes. Mechanical properties were then studied at sarcomere lengths of 2.0 and 2.3 µm. Our data demonstrate that the presence of hC0C1f in the sarcomere decreased maximal force myofilament Ca2+ sensitivity, increased cooperative activation at short lengths and enhanced length-dependent activation. Furthermore, hC0C1f led to increased cross-bridge cycling kinetics and tension cost at both short and long sarcomere lengths. We further established that the detrimental effects of hC0C1f occur through direct interaction with the thin filament proteins actin and α-tropomyosin (α-TM).Conclusions: 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 myofilament preparations. Decreased cardiac function post-MI may result, in part, from the ability of hC0C1f to bind actin and α-TM, suggesting that cleaved C0C1f could act as a poison peptide and disrupt the interaction of native cMyBP-C with the thin filament.Keywords: Cross-bridge cycling kinetics; length-dependent activation; cMyBP-C; C0C1f protein.

Understanding how cellular level mechanical properties change as failing hearts remodel may help identify the protein modifications that produce contractile dysfunction associated with this condition. We therefore procured through wall left ventricular samples from patients undergoing heart transplants at the University of Kentucky and from organ donors who did not have heart failure. Multicellular chemically permeabilized preparations were subsequently obtained from sub epicardial, midwall and sub endocardial regions of these samples by mechanical homogenization and triton treatment. The mechanical properties of the preparations were assessed by attaching them between a force transducer and a motor and subjecting them to a standard series of mechanical assays. There was a 30% decrease in maximum power output (p-value = 0.01) and steady-state force (p-value=0.005) in patients with end-stage heart failure (n=8, total of 72 preparations) as compared to samples from nonfailing organ donors (n=4, total of 36 preparations). The phosphorylation of two cardiac sarcomeric proteins- myosin binding protein-C (cMyBP-C) and troponin-I (cTnI) which have been previously associated with contractile dysfunction were investigated using phospho specific antibodies. Western blot analysis of three major phosphorylated sites of cMyBP-C, PSer273, PSer282 and PSer302 were not significantly different between heart failure and non failing groups but cTnI phosphorylation at PSer22/23 decreased significantly in heart failure (p=0.01). This study suggests that the cellular level mechanical dysfunction seen in heart failure may be in part due to phosphorylation modifications.