Temporal spatial expression and function of non-muscle myosin II isoforms IIA and IIB in scar remodeling

Abstract

Scar contracture is believed to be caused by the cell contractility during the remodeling phase of wound healing. Cell contractility is mediated by non-muscle myosin II (NMMII) and actin, but the temporal-spatial expression profile of NMMII isoforms A and B (IIA and IIB) during the remodeling phase and the role of NMMII in scar fibroblast tissue remodeling are unknown. Human scar tissue immunostained for IIA and IIB showed that both isoforms were highly expressed in scar tissue throughout the remodeling phase of repair and expression levels returned to normal after the remodeling phase. Human scar tissue immunostained for β-, γ- and α-smooth muscle actin showed that all isoforms were consistently expressed throughout the remodeling phase of repair. The β- and γ-smooth muscle actin were widely expressed throughout the dermis, but α-smooth muscle actin was only locally expressed within the dermis. In vitro, fibroblasts explanted from scar tissue were shown to express more IIA than fibroblasts explanted from normal tissue and scar fibroblasts contracted collagen lattices to a greater extent than normal fibroblasts. Blebbistatin was used to demonstrate the function of NMMII in collagen lattice contraction. In normal tissue, fibroblasts are stress-shielded from external tensile stress by the extracellular matrix. After dermal injury and during remodeling, fibroblasts are exposed to a matrix of increased stiffness. The effect of matrix stiffness on IIA and IIB expression was examined. IIA expression was greater in fibroblasts cultured in collagen lattices with increasing stiffness, and in fibroblasts cultured on glass slides compared with polyacrylamide gels with stiffness of 1 kPa. In conclusion, NMMII and actin isoform expression changes coordinately with the remodeling phase of repair, and NMMII is increased as matrix stiffness increases. As NMMII expression increases, so does the fibroblast contractility.

Figure 1

Non-muscle myosin isoforms IIA and IIB, and α-smooth muscle-, β- and γ-actin expression in the remodeling phase of repair (a). There is a robust protein expression of non-muscle myosin isoforms IIA and IIB during the remodeling period (6 to 18 weeks) compared with early (2 weeks) and late scars (27 weeks) at low-power views (2 × objective). α-smooth muscle actin expression is focally expressed in the dermis throughout the remodeling phase of repair. There is a robust, widespread protein expression of β- and γ-actin throughout the remodeling phase of repair. Negative controls are rabbit and mouse non-specific immunogobulin for non-muscle myosin and actin isoforms, respectively. Scale bar = 1 mm.

Figure 2

Representative histological sections of non-muscle myosin isoforms IIA and IIB in scar and matched normal human skin. High-power objectives (100×) of human scar samples with normal skin tissue stained for IIA (a, b) and IIB (c, d). There is a significant difference in IIA and IIB protein localization within mesenchymal cells in scar compared with normal skin tissue. Scar mesenchymal cells are oval with rounded nuclei and normal skin cells are spindle shaped with coma-shaped nuclei. Non-muscle myosin is more robust and widely distributed in the cytoplasm in scar fibroblasts (black arrows) compared with peri-nuclear localization in normal skin cells (arrowheads). Scale bar = 10 μm.

Figure 3

Non-muscle myosin IIA and IIB protein level in explanted human scar and normal skin. Flow cytometry data show a representative increase in IIA (a) and IIB (b) protein level in an explanted normal and scar fibroblasts from the same patient. This increase is seen in all seven scar and normal pairs of human explanted fibroblasts (c). * denotes patient sample with representative histograms shown.

Figure 4

Scar fibroblasts contract collagen matrices faster than normal skin fibroblasts in free-floating (FF)-FPCL. Explanted fibroblasts were placed into FF-collagen lattices with lattice area measured after 6 h. Under basal condition (0.5% BSA in DMEM), scar fibroblasts display significantly greater contraction than normal fibroblasts in all seven sample pairs (a). Under stimulated condition (10% FBS in DMEM), means percent lattice contraction are greater in scars in all samples, but only three pairs with statistical significant difference in means (b). Inhibition of NMMII using 10 μM blebbistatin decreases overall contraction in all samples, but scar fibroblasts continue to have greater contraction than normal fibroblasts (b). * denotes statistical significance (P-value≤0.05).

Figure 5

Extracellular matrix mechanical tension upregulates non-muscle myosin isoform IIA. Three different normal human dermal fibroblast cell lines were seeded in stressed collagen type I matrix at increasing mechanical tension using increasing collagen concentration—0.64, 1.28, 2.56 and 4.83 mg/ml. Lattices were left anchored for 4 days to maximize isometric tension development. Western data show that mechanical strain causes an increase in IIA protein expression, but not IIB (a). Representative western blots IIA and IIB, and GAPDH are shown. Normal dermal fibroblasts cultured on glass slides or polyacrylamide gels with stiffness of 1kPa, for 48 h. Fibroblasts were stained for IIA and IIB and analyzed by flow cytometry. Flow cytometric data shows that IIA expression was increased when fibroblasts were cultured on glass (dashed lines) compared with polyacrylamide gels (solid lines, b).