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. 2022 Nov 23;23(23):14571.
doi: 10.3390/ijms232314571.

Endogenous Modulation of Extracellular Matrix Collagen during Scar Formation after Myocardial Infarction

David Schumacher  1   2   3 , Adelina Curaj  3   4 , Mareike Staudt  3 , Sakine Simsekyilmaz  3 , Isabella Kanzler  3 , Peter Boor  5   6   7 , Barbara Mara Klinkhammer  5 , Xiaofeng Li  3 , Octavian Bucur  8   9 , Adnan Kaabi  4 , Yichen Xu  4   10 , Huabo Zheng  4   10 , Pakhwan Nilcham  4 , Alexander Schuh  4 , Mihaela Rusu  4 , Elisa A Liehn  4   8   10   11
Affiliations

Endogenous Modulation of Extracellular Matrix Collagen during Scar Formation after Myocardial Infarction

David Schumacher et al. Int J Mol Sci. .

Abstract

Myocardial infarction is remains the leading cause of death in developed countries. Recent data show that the composition of the extracellular matrix might differ despite similar heart function and infarction sizes. Because collagen is the main component of the extracellular matrix, we hypothesized that changes in inflammatory cell recruitment influence the synthesis of different collagen subtypes in myofibroblasts, thus changing the composition of the scar. We found that neutrophils sustain the proliferation of fibroblasts, remodeling, differentiation, migration and inflammation, predominantly by IL-1 and PPARγ pathways (n = 3). They also significantly inhibit the mRNA expression of fibrillar collagen, maintaining a reduced stiffness in isolated myofibroblasts (n = 4-5). Reducing the neutrophil infiltration in CCR1-/- resulted in increased mRNA expression of collagen 11, moderate expression of collagen 19 and low expression of collagen 13 and 26 in the scar 4 weeks post infarction compared with other groups (n = 3). Mononuclear cells increased the synthesis of all collagen subtypes and upregulated the NF-kB, angiotensin II and PPARδ pathways (n = 3). They increased the synthesis of collagen subtypes 1, 3, 5, 16 and 23 but reduced the expression of collagens 5 and 16 (n = 3). CCR2-/- scar tissue showed higher levels of collagen 13 (n = 3), in association with a significant reduction in stiffness (n = 4-5). Upregulation of the inflammation-related genes in myofibroblasts mostly modulated the fibrillar collagen subtypes, with less effect on the FACIT, network-forming and globular subtypes (n = 3). The upregulation of proliferation and differentiation genes in myofibroblasts seemed to be associated only with the fibrillar collagen subtype, whereas angiogenesis-related genes are associated with fibrillar, network-forming and multiplexin subtypes. In conclusion, although we intend for our findings to deepen the understanding of the mechanism of healing after myocardial infarction and scar formation, the process of collagen synthesis is highly complex, and further intensive investigation is needed to put together all the missing puzzle pieces in this still incipient knowledge process.

Keywords: extracellular matrix; inflammation; myocardial infarction; remodeling; scar formation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Characterization the origin and collagen subtype expression of myofibroblasts after myocardial infarction. (A) Time course of alpha smooth muscle actin staining (red, upper panel; scale bar, 50 µm) and Sirius red staining (lower panel, scale bar 200 µm) after myocardial infarction. (B) Percentage of alpha smooth muscle actin-positive (myofibroblasts) cells differentiated from cardiac fibroblasts and blood mononuclear cells (PBMC). As determined by α-SMA staining, local cardiac fibroblasts have an increased potential to differentiate towards myofibroblasts under TGF-ß1 stimulation and under hypoxic conditions compared with blood cells (** p < 0.01, t-test, n = 3–7 mice). (C) GFP-positive bone marrow reconstitution of lethally irradiated mice reveals that local differentiated myofibroblasts constitute most of the cells responsible for extracellular matrix synthesis; GFP/SMA double-positive cells (gray columns) are not significant compared with the control (two-way ANOVA followed by Tukey’s post hoc test, n = 3 mice per group). (D) Selected representative images of GFP/SMA double-positive cells (upper panel, yellow) and α-SMA cells (lower panel, red) at the time points characteristic of increased presence of myofibroblasts (scale bar, 50 µm). (E) mRNA expression levels of different collagen subtypes at different time points after myocardial infarction (normalized to α-actin) (* p < 0.05, *** p < 0.001, one-way ANOVA for each collagen subtype followed by Tukey’s post hoc test, n = 3 mice per group).
Figure 2
Figure 2
Assessment of infarction size and functional parameters of the heart after myocardial infarction. (A) Infarction size measured after Gomori’s one-step trichrome staining in a serial section in wild-type and different knockout mice four weeks after myocardial infarction (* p < 0.05, *** p < 0.001, one-way ANOVA, one-way ANOVA followed by Tukey’s post hoc test comparing the groups, n = 4–5 mice). (B) Echocardiographic measurements of ejection fractions 4 weeks after myocardial infarction (* p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA followed by Tukey’s post hoc test comparing the groups, n = 4–5 mice). (C) Representative Gomori’s one-step trichrome stain images of cross-sectioned tissues from wild-type and knockout mice 4 weeks after myocardial infarction. (D) Representative images of scar structures in atomic force microscopy topological images of scar microstructures (50 µm/50 µm) and examples of areas (inserts) considered for analysis (pores were avoided). (E) Scar tissue stiffness of different knockout mice analyzed by liquid atomic-force microscopy (* p < 0.05 vs. wild type, one-way ANOVA followed by Tukey’s post hoc test, n = 4–5 mice). (F) mRNA expression levels of different collagen subtypes 4 weeks after myocardial infarction (normalized to α-actin) in the various mutant mice (* p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA followed by Tukey’s post hoc test for each collagen subtype, n = 3 mice per group).
Figure 3
Figure 3
The influence of immune cells on apoptosis and proliferation of myofibroblasts. (A) Proliferation of myofibroblasts under normoxic/hypoxic conditions in the presence of neutrophils (Ne) and mononuclear cells (Mo) (* p < 0.05 vs. control, § p < 0.05 vs. mononuclear cells, one-way ANOVA followed by Tukey’s post hoc test comparing the groups, n = 5–6; the experimental data for one run are presented; the experiment was repeated four times). (B) Representative images of proliferating myofibroblasts in the presence of neutrophils and mononuclear cells (phase light; scale bar, 100 µm). (C) Representative images of proliferation (Ki67 in bright green, upper panel) of myofibroblasts (lower panel, Ki67 in bright green; alpha-actin in red; scale bar, 50 µm). (D) Apoptosis of myofibroblasts under normoxic/hypoxic conditions in the presence of neutrophils and mononuclear cells (* p < 0.05 vs. control, § p < 0.05 vs. neutrophils, one-way ANOVA followed by Tukey’s post hoc test comparing the groups, n = 7; the experimental data for one run are presented, the experiment was repeated three times). (E) Representative images of apoptotic myofibroblasts in the presence of neutrophils and mononuclear cells (apoptotic cells in red; scale bar, 100 µm; DAPI blue counterstaining in inserts showing the similar distribution of the cells in the cultures; a correction of the brightness/contrast was applied equally to all images in the power point to improve visibility for the reader). (F) Representative images of apoptotic cells (TUNEL in bright green, upper panel) of myofibroblasts (lower panel, TUNEL in bright green; alpha-actin in red; scale bar, 50 µm). (G) mRNA expression of Bcl2 as a survival and antiapoptotic marker in myofibroblasts (* p < 0.05, ** p < 0.01, one-way ANOVA followed by Tukey’s post hoc test comparing the groups, n = 9; three samples from three runs in triplicate) under normoxia (N), hypoxia (H) in coculture with neutrophils (Ne) or mononuclear cells (Mo). (H) mRNA expression of Bax as ab apoptotic marker in myofibroblasts (* p < 0.05, ** p < 0.01, **** p < 0.0001, one-way ANOVA followed by Tukey’s post hoc test comparing the groups, n = 9; three samples from three runs in triplicate). (I) mRNA expression of p53 as an apoptotic marker in myofibroblasts (** p < 0.01, *** p < 0.001, one-way ANOVA followed by Tukey’s post hoc test comparing the groups, n = 9).
Figure 4
Figure 4
Assessment of mRNA expression of extracellular matrix proteins and biomechanics in mice four weeks after myocardial infarction. (A) qRT-PCR was performed for a semi-quantitative assessment of collagen subtypes in the absence/presence of different immune cells. Isolated myofibroblast were cultured under normoxic and hypoxic conditions and in coculture with neutrophils (+Ne) or mononuclear cells (+Mo). A heat map representing the level of mRNA expression was created in PowerPoint; significant values after ANOVA analysis of each protein are shown (* p < 0.05; ** p < 0.01; *** p < 0.001, one-way ANOVA followed by Tukey’s post hoc test, n = 3 mice). Significant differences between coincubation with mononuclear cells and neutrophils are shown separately (* p’ < 0.05; ** p’ < 0.01, one-way ANOVA followed by Tukey’s post hoc test, n = 3). (B) Myofibroblast stiffness in the absence and presence of neutrophil and mononuclear fractions, respectively (* p < 0.05, *** p < 0.001, one-way ANOVA followed by Tukey’s post hoc test, n = 3 mice). (C) Fiber thickness in myofibroblasts in the absence and presence of neutrophil and mononuclear fractions, respectively (** p < 0.01, *** p < 0.001, one-way ANOVA followed by Tukey’s post hoc test, n = 3). (D) Representative AFM images of myofibroblasts coincubated with immune cells under normoxia and hypoxia. (E) Myofibroblast stiffness after successive incubation steps with neutrophil followed by mononuclear fractions and with mononuclear followed by neutrophil fractions (*** p < 0.001, one-way ANOVA followed by Tukey’s post hoc test, n = 3 mice). (F) Fiber thickness in myofibroblasts after successive incubation steps with neutrophil followed by mononuclear fractions and with mononuclear followed by neutrophil fractions (** p < 0.01, one-way ANOVA followed by Tukey’s post hoc test, n = 3 mice).
Figure 5
Figure 5
The modulation of gene expression by immune cells. (A) qRT-PCR was performed for a semi-quantitative assessment of collagen subtypes in the absence/presence of different immune cells. Isolated myofibroblasts were cultured under normoxic (N) and hypoxic conditions (H) and in coculture with neutrophils (+Ne) or mononuclear cells (+Mo). A heat map representing the level of mRNA expression was created in PowerPoint; significant values after ANOVA analysis of each protein are shown (* p < 0.05; ** p < 0.005; *** p < 0.0005, one-way ANOVA followed by Tukey’s post hoc test, n = 3; the experimental data for one run are presented; the experiment was repeated five times). Significantly differences between coincubation with mononuclear cells and neutrophils are shown separately (* p’ < 0.05; ** p’ <0.01, one-way ANOVA followed by Tukey’s post hoc test, n = 3; the experimental data for one run are presented; the experiment was repeated five times). (B–H) Interactions between signaling pathways and collagen subtypes identified in the gene–protein interaction analysis.
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