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N-terminal fragment of cardiac myosin binding protein-C triggers pro-inflammatory responses in vitro.

ABSTRACT: Myocardial infarction (MI) leads to loss and degradation of contractile cardiac tissue followed by sterile inflammation of the myocardium through activation and recruitment of innate and adaptive cells of the immune system. Recently, it was shown that cardiac myosin binding protein-C (cMyBP-C), a protein of the cardiac sarcomere, is degraded following MI, releasing a predominant N-terminal 40-kDa fragment (C0C1f) into myocardial tissue and the systemic circulation. We hypothesized that early release of C0C1f contributes to the initiation of inflammation and plays a key role in recruitment and activation of immune cells. Therefore, we investigated the role of C0C1f on macrophage/monocyte activation using both mouse bone marrow-derived macrophages and human monocytes. Here we demonstrate that C0C1f leads to macrophage/monocyte activation in vitro. Furthermore, C0C1f induces strong upregulation of pro-inflammatory cytokines (interleukin-6 (IL-6), tumor necrosis factor ? (TNF?), and interleukin-1? (IL-1?)) in cultured murine macrophages and human monocytes, resulting in a pro-inflammatory phenotype. We identified the toll-like receptor 4 (TLR4), toll-like receptor 2 (TLR2), and Advanced Glycosylation End Product-Specific Receptor (RAGE) as potential receptors for C0C1f whose activation leads to mobilization of the NF?B signaling pathway, a central mediator of the pro-inflammatory signaling cascade. Thus, C0C1f appears to be a key player in the initiation of inflammatory processes and might also play an important role upon MI.

SUBMITTER: Lipps C

PROVIDER: S-EPMC5107329 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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N-terminal fragment of cardiac myosin binding protein-C triggers pro-inflammatory responses in vitro.

Lipps Christoph C Nguyen Jenine H JH Pyttel Lukas L Lynch Thomas L TL Liebetrau Christoph C Aleshcheva Ganna G Voss Sandra S Dörr Oliver O Nef Holger M HM Möllmann Helge H Hamm Christian W CW Sadayappan Sakthivel S Troidl Christian C

Journal of molecular and cellular cardiology 20160908

Myocardial infarction (MI) leads to loss and degradation of contractile cardiac tissue followed by sterile inflammation of the myocardium through activation and recruitment of innate and adaptive cells of the immune system. Recently, it was shown that cardiac myosin binding protein-C (cMyBP-C), a protein of the cardiac sarcomere, is degraded following MI, releasing a predominant N-terminal 40-kDa fragment (C0C1f) into myocardial tissue and the systemic circulation. We hypothesized that early rel ...[more]

PMID: 27616755

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Project description:Cardiac myosin binding protein-C (cMyBP-C) plays a role in sarcomeric structure and stability, as well as modulating heart muscle contraction. The 150 kDa full-length (FL) cMyBP-C has been shown to undergo proteolytic cleavage during ischemia-reperfusion injury, producing an N-terminal 40 kDa fragment (mass 29 kDa) that is predominantly associated with post-ischemic contractile dysfunction. Thus far, the pathogenic properties of such truncated cMyBP-C proteins have not been elucidated. In the present study, we hypothesized that the presence of these 40 kDa fragments is toxic to cardiomyocytes, compared to the 110 kDa C-terminal fragment and FL cMyBP-C. To test this hypothesis, we infected neonatal rat ventricular cardiomyocytes and adult rabbit ventricular cardiomyocytes with adenoviruses expressing the FL, 110 and 40 kDa fragments of cMyBP-C, and measured cytotoxicity, Ca(2+) transients, contractility, and protein-protein interactions. Here we show that expression of 40 kDa fragments in neonatal rat ventricular cardiomyocytes significantly increases LDH release and caspase 3 activity, significantly reduces cell viability, and impairs Ca(2+) handling. Adult cardiomyocytes expressing 40 kDa fragments exhibited similar impairment of Ca(2+) handling along with a significant reduction of sarcomere length shortening, relaxation velocity, and contraction velocity. Pull-down assays using recombinant proteins showed that the 40 kDa fragment binds significantly to sarcomeric actin, comparable to C0-C2 domains. In addition, we discovered several acetylation sites within the 40 kDa fragment that could potentially affect actomyosin function. Altogether, our data demonstrate that the 40 kDa cleavage fragments of cMyBP-C are toxic to cardiomyocytes and significantly impair contractility and Ca(2+) handling via inhibition of actomyosin function. By elucidating the deleterious effects of endogenously expressed cMyBP-C N-terminal fragments on sarcomere function, these data contribute to the understanding of contractile dysfunction following myocardial injury.

Project description:RationaleMutations in the gene encoding the sarcomeric protein cardiac myosin-binding protein C (cMyBP-C) are a leading cause of hypertrophic cardiomyopathy (HCM). Mouse models targeting cMyBP-C and use of recombinant proteins have been effective in studying its roles in contractile function and disease. Surprisingly, while the N-terminus of cMyBP-C is important to regulate myofilament binding and contains many HCM mutations, an incorrect sequence, lacking the N-terminal 8 amino acids has been used in many studies.ObjectivesTo determine the N-terminal cMyBP-C sequences in ventricles and investigate the roles of species-specific differences in cMyBP-C on myofilament binding.Methods and resultsWe determined cMyBP-C sequences in mouse and human by inspecting available sequence databases. N-terminal differences were confirmed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cosedimentation assays with actin or myosin were used to examine binding in mouse, human and chimeric fusion proteins of cMyBP-C. Time-resolved FRET (TR-FRET) with site-directed probes on cMyBP-C was employed to measure structural dynamics. LC-MS/MS supported the sequencing data that mouse cMyBP-C contains an eight-residue N-terminal extension (NTE) not found in human. Cosedimentation assays revealed that cardiac myosin binding was strongly influenced by the presence of the NTE, which reduced binding by 60%. 75% more human C0-C2 than mouse bound to myosin. Actin binding of mouse C0-C2 was not affected by the NTE. 50% more human C0-C2 than mouse bound to actin. TR-FRET indicates that the NTE did not significantly affect structural dynamics across domains C0 and C1.ConclusionsOur functional results are consistent with the idea that cardiac myosin binding of N-terminal cMyBP-C is reduced in the mouse protein due to the presence of the NTE, which is proposed to interfere with myosin regulatory light chain (RLC) binding. The NTE is a critical component of mouse cMyBP-C, and should be considered in extrapolation of studies to cMyBP-C and HCM mechanisms in human.

Project description:Cardiac surgery encompasses various stimuli that trigger pro-inflammatory mediators, reactive oxygen species and mobilization of leucocytes. The aim of this study was to evaluate the effect of xenon on the inflammatory response during cardiac surgery.This randomized trial enrolled 30 patients who underwent elective on-pump coronary-artery bypass grafting in balanced anaesthesia of either xenon or sevoflurane. For this secondary analysis, blood samples were drawn prior to the operation, intra-operatively and on the first post-operative day to measure the pro- and anti-inflammatory cytokines interleukin-6 (IL-6), interleukin-8/C-X-C motif ligand 8 (IL-8/CXCL8), and interleukin-10 (IL-10). Chemokines such as C-X-C motif ligand 12/ stromal cell-derived factor-1? (CXCL12/SDF-1?) and macrophage migration inhibitory factor (MIF) were measured to characterize xenon's perioperative inflammatory profile and its impact on migration of peripheral blood mononuclear cells (PBMC).Xenon enhanced the postoperative increase of IL-6 compared to sevoflurane (Xenon: 90.7 versus sevoflurane: 33.7 pg/ml; p = 0.035) and attenuated the increase of IL-10 (Xenon: 127.9 versus sevoflurane: 548.3 pg/ml; p = 0.028). Both groups demonstrated a comparable intraoperative increase of oxidative stress (intra-OP: p = 0.29; post-OP: p = 0.65). While both groups showed an intraoperative increase of the cardioprotective mediators MIF and CXCL12/SDF-1?, only MIF levels decreased in the xenon group on the first postoperative day (50.0 ng/ml compared to 23.3 ng/ml; p = 0.012), whereas it remained elevated after sevoflurane anaesthesia (58.3 ng/ml to 53.6 ng/ml). Effects of patients' serum on chemotactic migration of peripheral mononuclear blood cells taken from healthy volunteers indicated a tendency towards enhanced migration after sevoflurane anaesthesia (p = 0.07).Compared to sevoflurane, balanced xenon anaesthesia triggers pro-inflammatory effects and suppresses the anti-inflammatory response in cardiac surgery patients even though the clinical significance remains unknown.This clinical trial was approved by the European Medicines Agency (EudraCT-number: 2010-023942-63) and at ClinicalTrials.gov ( NCT01285271 ; first received: January 24, 2011).

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N-terminal fragment of cardiac myosin binding protein-C triggers pro-inflammatory responses in vitro.

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N-terminal fragment of cardiac myosin binding protein-C triggers pro-inflammatory responses in vitro.

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