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. 2016 Oct:99:47-56.
doi: 10.1016/j.yjmcc.2016.09.003. Epub 2016 Sep 8.

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

Christoph Lipps  1 , Jenine H Nguyen  1 , Lukas Pyttel  2 , Thomas L Lynch 4th  3 , Christoph Liebetrau  2 , Ganna Aleshcheva  2 , Sandra Voss  2 , Oliver Dörr  1 , Holger M Nef  1 , Helge Möllmann  2 , Christian W Hamm  4 , Sakthivel Sadayappan  3 , Christian Troidl  5
Affiliations

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

Christoph Lipps et al. J Mol Cell Cardiol. 2016 Oct.

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 α (TNFα), and interleukin-1β (IL-1β)) 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 NFκB 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.

Keywords: C0C1f; Cardiac myosin binding protein-C; Cell signaling/signal transduction; Inflammation; Ischemic biology - basic studies; Myocardial infarction.

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Conflict of interest statement

On behalf of all other authors, the corresponding author states that there is no conflict of interest

Figures

Figure 1
Figure 1. Initiation of inflammatory responses by MI biomarkers
Murine macrophages were treated with 500 ng/ml C0C1f, 500 ng/ml cTnI, or 1µg/ml LPS for 6 hours. Thereafter, mRNA was isolated and mRNA levels of TNFα, IL-6, IL-1β, VCAM1, and ICAM1 were measured by qRT-PCR. Mean ± SEM; Statistical analysis was performed using Kruskal-Wallis one-way ANOVA with Dunn’s post-hoc test, n.s. non-significant, ** p<0.005, *** p<0.0005, **** p<0.0001 (n=13 for cTnI, n=31 for C0C1f and n=4 for LPS). b–c) Murine macrophages were treated for the indicated lengths of time with 500 ng/ml C0C1f and for 6 h with C0-L or LPS. Western blot analysis was used for determination of protein levels of b) IL-1β and c) TNFα. Depicted is the mean ± SEM of n=3 individual experiments. Statistical analysis was performed using the Mann Whitney U test, comparing each sample individually with control (p>0.07). d) Structure of cardiac MyBP-C. Calpain-dependent cleavage takes place in the M-domain. Various N-terminal fragments were designed.
Figure 2
Figure 2. Validation of pro- and anti-inflammatory responses upon treatment with N-terminal cMyBP-C fragments
a–e) Murine macrophages were treated for 6 h with 500 ng/ml C0C2, C0C1f, C0C1, or C0-L fragments, or 1 µg/ml LPS. mRNA levels of TNFα, IL-6, IL-1β, VCAM1, and ICAM1 were measured by qRT-PCR. f) Murine macrophages were treated with C0C1f peptide for 3, 6, 9, 24, or 72 h. g–i) Murine macrophages were treated for 6 h with C0C2, C0C1f, C0C1, or C0-L fragment and LPS; mRNA levels of Arg1, IL-10, and TGFβ were measured by qRT-PCR. Values shown are mean ± SEM; statistical analysis was performed using Kruskal-Wallis one-way ANOVA with Dunn’s post-hoc test, * p< 0.05, ** p<0.005, *** p<0.0005, **** p<0.0001 (C0: n=4, C0C2 and C0-L: n=11; C0C1: n=18, C0C1f: n=21, LPS: n=6).
Figure 3
Figure 3. C0C1f induces pro-inflammatory responses in human monocytes
a–e) Human monocytes were isolated from buffy coats of healthy donors and were treated with C0-L, C0C1f, or LPS for 6 h. Thereafter, mRNA was isolated and levels of TNFα, IL-6, IL-1β, VCAM1, and ICAM1 transcripts were measured by qRT-PCR. Values shown are mean ± SEM; statistical analysis was performed using the one-way ANOVA with Tukey’s post-hoc test, n= 10, * p<0.05, ** p<0.005, *** p<0.0005 and **** p<0.0001. f) Human monocytes were treated with C0C1f peptide for the indicated time periods and levels of TNFα, IL-6, IL-1β, VCAM1, and ICAM1 were measured by qRT-PCR. (n=3 donors) g–i) Protein levels of pro-inflammatory cytokines were detected by western blot analysis (g, j) and quantified (h, i, k, l). Values shown are mean ± SEM; statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc test, * p<0.05, ** p<0.005
Figure 4
Figure 4. C0C1f induces pro-inflammatory cytokine expression via activation of the NFκB signaling pathway
Murine macrophages were treated with C0C1f, LPS, or C0-L for 6 h. The NFκB activation was inhibited by 24-h pre-treatment (18 h prior to co-treatment with peptides or LPS) with Bay11–7082. qRT-PCR was performed to determine the gene expression levels of NFκB (a) and the pro-inflammatory cytokines TNFα (b), IL-6 (c), IL-1β (d), and MCP1 (e) as well as adhesion/migration molecules VCAM1 (f) and ICAM1 (g). Values shown are mean ± SEM; statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc test, (Bay11–7082: n=9, LPS, C0C1f+ Bay11–7082 and C0-L: n=28, C0C1f: n=38, n.s. not significant, *** p<0.005, **** p <0.0001). Protein expression of IL-1β (h–i) and TNFα (h, j) with or without 24-h Bay 11–7082 treatment was analyzed by western blotting. Depicted are the quantified data for 6-h treatment with C0C1f, C0C1f + Bay11–7082, LPS, and C0-L (n=4 independent experiments) (i, j). Values shown are mean ± SEM; statistical analysis was performed using the Mann Whitney U test, comparing each sample individually with control, * p<0.05.
Figure 5
Figure 5. C0C1f elicits its response via TLR4, TLR2, and RAGE
Murine macrophages were treated with various inhibitors. IL-6 and IL-1β mRNA expression was analyzed by qRT-PCR. Cells were treated with a–b) CLI-095, a TLR4-specific inhibitor (n=4), c–d) FPS-ZM1, a RAGE inhibitor (n=6), or e–f) CU CPT22, a TLR2-specific inhibitor (n=6) for 16 h prior to the 6-h treatment with C0C1f. Values shown are mean ± SEM; statistical analysis was performed using the Mann-Whitney U test, * p<0.05, ** p<0.005.
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