The C0-C1f Region of Cardiac Myosin Binding Protein-C Induces Pro-Inflammatory Responses in Fibroblasts via TLR4 Signaling

Abstract

Myocardial injury is associated with inflammation and fibrosis. Cardiac myosin-binding protein-C (cMyBP-C) is cleaved by µ-calpain upon myocardial injury, releasing C0-C1f, an N-terminal peptide of cMyBP-C. Previously, we reported that the presence of C0-C1f is pathogenic within cardiac tissue and is able to activate macrophages. Fibroblasts also play a crucial role in cardiac remodeling arising from ischemic events, as they contribute to both inflammation and scar formation. To understand whether C0-C1f directly modulates fibroblast phenotype, we analyzed the impact of C0-C1f on a human fibroblast cell line in vitro by performing mRNA microarray screening, immunofluorescence staining, and quantitative real-time PCR. The underlying signaling pathways were investigated by KEGG analysis and determined more precisely by targeted inhibition of the potential signaling cascades in vitro. C0-C1f induced pro-inflammatory responses that might delay TGFβ-mediated myofibroblast conversion. TGFβ also counteracted C0-C1f-mediated fibroblast activation. Inhibition of TLR4 or NFκB as well as the delivery of miR-146 significantly reduced C0-C1f-mediated effects. In conclusion, C0-C1f induces inflammatory responses in human fibroblasts that are mediated via TRL4 signaling, which is decreased in the presence of TGFβ. Specific targeting of TLR4 signaling could be an innovative strategy to modulate C0-C1f-mediated inflammation.

Keywords: C0-C1f; MYBPC3; cMyBP-C; fibroblasts; inflammation; miRNA-146.

Figure 1

Conversion to myofibroblasts. huFib cells were treated with 10 ng/mL TGFβ, 500 ng/mL C0-C1f, a combination of C0-C1f and TGFβ, 500 ng/mL C0-Linker, or 1 µg/mL LPS. (A) Nuclei (blue) and α-SMA (red) were stained after 48 h of treatment under serum-free conditions. Scale bar = 50 µm (B) Quantification of α-SMA-positive cells, showing mean and SD (unstimulated n = 6, C0-C1f n = 3, TGFβ n = 5, TGFβ + C0-C1f n = 4, LPS n = 3, C0-Linker n = 3; n represents technical replicates). (C,D) Quantification of mRNA expression levels of ACTA2 and COL1A1 after 24 h treatment by qPCR. Data are displayed as individual data points and means; p-values above each condition refer to differences between means (horizontal lines) and 0 fold change (unstimulated controls) unless otherwise indicated. (ACTA2 n = 7, TGFβ + C0-C1f n = 5; COL1A1 n = 10; n represents biological replicates).

Figure 2

Gene expression profiling. huFib were stimulated with 500 ng/mL C0-C1f, 500 ng/mL C0-Linker for 24h. As controls (Ctrl) unstimulated huFib were harvested. Total mRNA was isolated and microarray analyses performed. (A) Statistical significances of the KEGG pathway analyses as −log₁₀(p). The 50 pathways with lowest p-values in C0-C1f vs. control were selected. The vertical dashed line indicates p = 0.01. Black dots: C0-C1f vs. control, open circles: C0-linker vs. control. (B,C) Volcano plots for C0-C1f vs. control and C0-linker vs. control, respectively. The 20 genes with the lowest p-values are annotated.

Figure 3

Activation of fibroblasts. huFib cells were treated with 1 µg/mL LPS, 500 ng/mL C0-Linker, or 500 ng/mL C0-C1f and/or 10 ng/mL TGFβ. (A) CXCL-1 (B) CCL-2 (C) MMP-9, and (D) ICAM-1 mRNA abundance was detected by qPCR after 24 h treatment. Data are displayed as individual data points and means; p-values above each condition refer to differences between means (horizontal lines) and 0 fold change (unstimulated controls) unless otherwise indicated. (n = 4; n represents biological replicates).

Figure 4

Time-dependent response of fibroblasts. huFib cells were treated with 500 ng/mL C0-C1f, 500 ng/mL C0-Linker or 1 µg/mL LPS for different time periods, and (A) CXCL-1, (B) CCL-2, and (C) MMP-9 mRNA abundance was quantified by qPCR. Data are displayed as mean ± SD (n = 4 biologically independent experiments). * above each condition refer to differences between means (data points) and 0 fold change (unstimulated controls). * p < 0.05 was considered statistically significant.

Figure 5

Effects of TLR4 and NFκB inhibition on C0C1f signaling. (A) huFib cells were pretreated for 18 h with CLI-095 (TLR4 inhibitor) and then stimulated with 500 ng/mL C0-C1f for 6 h. mRNA levels of CXCL-1, CCL-2, and MMP-9 were determined by qPCR and compared using an unpaired t-test. (B) huFib cells were pretreated for 18 h with Bay11-7085 (NFκB inhibitor) and stimulated with C0-C1f for 6 h. mRNA levels of CXCL-1, CCL-2, and MMP-9 were determined by qPCR. (C) huFib cells were transiently transfected with 25 pmol miR-146 or antimiR-146 oligos for 24 h prior to C0-C1f treatment for 6 h. CXCL-1, CCL-2, and MMP-9 mRNA were detected by qPCR. In all panels p-values above each condition refer to differences between means (horizontal lines) and 0 fold change (unstimulated controls) unless otherwise indicated. (A) CXCL1 n = 6; CCL2 and MMP9 n = 7; (B) CXCL-1 n = 4, CCL2 and MMP9 n = 5; (C) n = 8; n represents biological replicates.