PMC

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.

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).

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

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.

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.