Tenascin-C regulates recruitment of myofibroblasts during tissue repair after myocardial injury

Abstract

Tenascin-C (TN-C) is an extracellular matrix molecule that is expressed during wound healing in various tissues. Although not detectable in the normal adult heart, it is expressed under pathological conditions. Previously, using a rat model, we found that TN-C was expressed during the acute stage after myocardial infarction and that alpha-smooth muscle actin (alpha-SMA)-positive myofibroblasts appeared in TN-C-positive areas. In the present study, we examined whether TN-C controls the dynamics of myofibroblast recruitment and wound healing after electrical injury to the myocardium of TN-C knockout (TNKO) mice compared with wild-type (WT) mice. In TNKO mice, myocardial repair seemed to proceed normally, but the appearance of myofibroblasts was delayed. With cultured cardiac fibroblasts, TN-C significantly accelerated cell migration, alpha-SMA expression, and collagen gel contraction but did not affect proliferation. Using recombinant fragments of murine TN-C, the functional domain responsible for promoting migration of cardiac fibroblasts was mapped to the conserved fibronectin type III (FNIII)-like repeats and the fibrinogen (Fbg)-like domain. Furthermore, alternatively spliced FNIII and Fbg-like domains proved responsible for the up-regulation of alpha-SMA expression. These results indicate that TN-C promotes recruitment of myofibroblasts in the early stages of myocardial repair by stimulating cell migration and differentiation.

Figures

Figure 1

Diagram of mouse TN-C and its recombinant fragments. FL: FNIII repeats including both conserved (1 to 5, 6 to 9) and alternatively spliced repeats (A1, A2, A4, B, D). SV: Alternatively spliced FNIII repeats. SO: Conserved FNIII repeats. EGF: the EGF-like domain. Fbg: fibrinogen-like domain.

Figure 2

Tissue healing and TN-C expression after electrical injury of the myocardium at day 1 (A, D), day 2 (B, E), and day 3 (C, F, G). A–C: H&E staining; D, E: double immunolabeling for α-SMA (blue) and TN-C (brown). F and G: Immunolabeling with the antibody clone 4F10TT reacting with constitutive sites of TN-C (F), and 4C8MS that specifically recognizes an alternative spliced repeat of TN-C (G). On day 1, TN-C deposition is clearly detectable in the interstitial spaces of border zone myocardium, but only vascular walls are positive for α-SMA (D). E: On day 2, myofibroblasts are apparent in TN-C-positive areas. G: Note staining for large splice variants of TN-C. int, intact area; inj, injured area. Scale bar, 50 μm.

Figure 3

Comparison of the histopathology of myocardial repair in a WT (A) and TN-C knockout mouse (B) 5 days after electric injury. H&E staining. Scale bar, 50 μm.

Figure 4

Myofibroblasts in the border zones of necrotic cardiac tissue. A: Myofibroblasts labeled with anti-α-SMA antibody. On day 2, α-SMA-positive cells are evident (arrows) in the myocardial interstitium near the necrotic area in a WT mouse. Fewer cells are apparent in a TN-C knockout (TNKO) mouse. B: α-SMA-positive cells were counted in three fields of view under a ×40 objective and the average of each sample was calculated. On days 1 and 2, the myofibroblasts in WT mice were more frequent than in TNKO mice (P < 0.01), but there was no difference on day 3. The data are averages and SDs of results from five animals. Scale bar, 50 μm.

Figure 5

Effect of TN-C on proliferation of cardiac fibroblasts in vivo and in vitro. A: BrdU-labeled cells in the injured areas of WT and TNKO mice. BrdU-positive nuclei were counted in three fields of view of a ×40 objective and the average of each sample was calculated. The data are expressed as averages and SDs of results from five animals. Note the lack of significant differences between WT and TNKO. B: Cells isolated from TNKO mice were plated and grown on culture glass slides. After serum starvation for 24 hours, TN-C was added. Cells labeled with BrdU were visualized by immunocytochemistry and BrdU-positive and total number of nuclei (more than 500) were counted and percentage values generated. The data are averages and SDs from three independent experiments. Note the lack of any increase with TN-C treatment.

Figure 6

Effects of TN-C on migration of cardiac fibroblasts in a transmigration assay. Cells isolated from cardiac ventricles of either TNKO or WT mice were plated on the culture inserts, treated with TN-C (5 or 10 μg/ml) or without TN-C, and allowed to migrate for 8 hours. A: Cells migrating through the membrane were stained with 0.1% crystal violet. B: Migration of TN-C-null cells was significantly lower than that of WT-cells, and addition of TN-C significantly enhanced cell migration in a dose-dependent manner. Cells were counted in three fields of view of 1 mm2 in each insert, and the data are averages and SDs of results from three independent experiments.

Figure 7

Effects of TN-C on α-SMA expression in cardiac fibroblasts. Cells isolated from cardiac ventricles of either TNKO or WT mice were plated and grown on the culture glass slides. After 24-hour serum starvation, TN-C (0 or 20 μg/ml) was added to TN-C-null cells and incubated for another 24 hours. A: α-SMA expression was detected by indirect immunofluorescent staining with fluorescein isothiocyanate-conjugated secondary antibody, and all cells were counterstained with rhodamine-phalloidin. The α-SMA-positive ratio of TN-C-null cells was significantly lower than that of WT cells. B: Addition of TN-C significantly up-regulated α-SMA expression in a dose-dependent manner. The α-SMA-positive and total cells (more than 200) were counted to allow generation of percentage values. The data are averages and SDs of results from three independent experiments.

Figure 8

Effects of TN-C on collagen gel contraction by cardiac fibroblasts. Cardiac fibroblasts of either TNKO or WT mice were combined with collagen gel mixture and allowed to polymerize. After 24 hours medium with or without TN-C (10 μg/ml) was added, then the gels were detached from the dish. Percentages of the contraction were measured at 4, 8, 12, and 24 hours. Contraction by WT cells (closed circle) was significantly stronger than that by TN-C-null cells (open triangle) at each time point. Addition of TN-C (closed triangle) significantly increased the gel contraction of TNKO fibroblasts at 8, 12, and 24 hours. The data are expressed as averages and SDs of triplicate samples.

Figure 9

Determination of functional domains promoting cell migration using recombinant fragments of TN-C. TN-C-null cardiac fibroblasts were plated on culture inserts and treated with TN-C (10 μg/ml) or TN-C fragments (10 μg/ml), and allowed to migrate for 8 hours. The cells were counted in three fields of view of 1 mm2 in each insert, and the data are averages and SDs of results from six independent experiments, relative to the control without TN-C. *P < 0.01.

Figure 10

Determination of functional domains up-regulating α-SMA expression using recombinant fragments of TN-C. TN-C-null cardiac fibroblasts were plated and grown on the culture glass slides. After 24-hour serum starvation, they were treated with TN-C (10 μg/ml) or TN-C fragments (10 μg/ml), and incubated for 24 hours. α-SMA expression was detected by immunofluorescence. α-SMA-positive cells and total number of cells (more than 200) were counted and the percentage values were generated. The data are averages and SDs of results from six independent experiments, relative to the control without TN-C. *P < 0.01, **P < 0.05.