Blebbistatin inhibits contraction and accelerates migration in mouse hepatic stellate cells

Abstract

Background and purpose: Blebbistatin, an inhibitor of myosin-II-specific ATPase, has been used to inhibit contraction of invertebrate and mammalian muscle preparations containing non-muscle myosin. Activated hepatic stellate cells have contractile properties and play an important role in the pathophysiology of liver fibrosis and portal hypertension. Therefore, hepatic stellate cells are considered as therapeutic target cells. In the present study, we studied the effect of blebbistatin during the transition of mouse hepatic stellate cells into contractile myofibroblasts.

Experimental approach: Effects of blebbistatin on cell morphology were evaluated by phase contrast microscopy. Cell stress fibres and focal adhesions were investigated by dual immunofluorescence staining and visualized using fluorescence microscopy. Contractile force generation was examined by silicone wrinkle formation assays and collagen gel contraction assays. Intracellular Ca(2+) release in response to endothelin-1 was measured by using Fluo-4. Cell migration was measured by wound healing experiments.

Key results: In culture-activated hepatic stellate cells, blebbistatin was found to change both cell morphology and function. In the presence of blebbistatin, stellate cells became smaller, acquired a dendritic morphology and had less myosin IIA-containing stress fibres and vinculin-containing focal adhesions. Moreover, blebbistatin impaired silicone wrinkle formation, reduced collagen gel contraction and blocked endothelin-1-induced intracellular Ca(2+) release. Finally, it promoted wound-induced cell migration.

Conclusions and implications: By inhibiting myosin II ATPase, blebbistatin has profound effects on the morphology and function of activated hepatic stellate cells. Our data suggest that myosin II could be a therapeutic target in the treatment of liver fibrosis and portal hypertension.

Figure 1

Effects of blebbistatin on mouse hepatic stellate cells (HSCs). (A) Phase contrast images showing that blebbistatin induced morphological changes in HSCs. Cells at day 10 were treated with or without blebbistatin (0, 12.5, 25 and 50 µM, 2 h), and removal of blebbistatin (only 50 µM shown) leads to re-appearance of normal activated HSC morphology. The scale bar represents 100 µm. (B) Western blots showing that blebbistatin did not influence protein expression. Cells at day 11 were treated with blebbistatin at different concentrations and for different periods as indicated in the figure. β-Actin was used as loading control.

Figure 2

Blebbistatin dissociates myosin IIA –α-smooth muscle actin (α-SMA)-containing stress fibres. Cells at day 11 were treated with or without blebbistatin (50 µM) for 2 h, and then fixed in acetone/ethanol (1:3) at −20^°C for 10 min. Dual immunofluoresence staining was performed by using anti-myosin IIA (1:400) and anti-α-SMA (1:1000) antibodies. Inserts represent higher-magnification images of areas enclosed in rectangles. The scale bar represents 50 µm.

Figure 3

Blebbistatin inhibits silicone wrinkle formation, both in the presence and in the absence of the potent constrictor ET-1. (A) Mouse hepatic stellate cells (HSCs) were cultured on silicone substrate. At day 7 wrinkle formation was assessed before and after treatment with blebbistatin or ET-1. Inserts represent higher magnification images of areas marked by arrow-points in the original images. The scale bar represents 50 µm. (B) Blebbistatin was withdrawn at day 8 (no ET-1 treatment) and cell culture continued in normal medium for 4 days. Wrinkle formation was visualized at day 12. Inserts represent higher-magnification images of areas enclosed in original images. The scale bar represents 100 µm.

Figure 4

Blebbistatin blocks collagen gel contraction by activated hepatic stellate cells (HSCs). (A) Morphology of 14-day-cultured HSCs on collagen gel, 24 h after stimulation with 2 × 10⁻⁸ M ET-1 in the absence or presence of 50 µM blebbistatin. Inserts show collagen fibril formation (Ctrl) or the absence of collagen fibril formation (BLEB). (B) Hydrated collagen lattices, photographed 24 h after treatment. Ctrl, control medium; BLEB + ET-1, ET-1 (2 × 10⁻⁸ M) after 20 min pre-treated with blebbistatin (50 µM); ET-1, treated with ET-1 (2 × 10⁻⁸ M). Photographs represent one of three independent experiments. (C) Mean % collagen gel area compared with original collagen gel area after 24 h stimulation by ET-1 (2 × 10⁻⁸ M) with or without blebbistatin (50 µM) pre-treatment. Original gel area was normalized to 100%. Each bar represents mean and error bars represent standard deviation. Data from three independent experiments. *P < 0.01, relative to medium only, **P < 0.001, relative to ET-1.

Figure 5

Blebbistatin blocks ET-1-induced intracellular Ca²⁺ release. Original fluorescence images of [Ca²⁺]_i measurements following addition of ET-1 (4 × 10⁻⁸ M) in hepatic stellate cells (HSCs) in the absence (A) or presence (B) of blebbistatin (50 µM, 2 h). Representative graph showing fluorescence intensity of [Ca²⁺]_i in response to ET-1 (C). Arrows in (B) and (C) (BLEB) point at a positively responding cell with morphology similar to that of control cells.

Figure 6

Wound healing assay showing that blebbistatin promotes wound-induced hepatic stellate cell (HSC) migration. (A) At day 4 after seeding, a ‘wound’ was made and cells were treated with blebbistatin (concentration: 0, 12.5, 25, 50 µM) at day 5. Photographs were taken at the start and 24 h after scratching. The scale bar represents 50 µm. (B) A ‘wound’ was made at day 10 and cells were treated with blebbistatin (0, 12.5, 25, 50 µM) at day 11. Photographs were taken at the start and 24 h after scratching. The scale bar represents 50 µm. (C) Effects of different concentrations of blebbistatin on wound-induced mouse HSC migration 24 h after treatment. Graphs show the number of cells in the wound (lower panel) and the % of area covered by migrated cells. Each bar represents means ± SD. Data from three independent experiments. *P < 0.05, **P < 0.001 compared with control (0 µM), the observed effect is not statistically significant for the different concentrations of blebbistatin. (D) Blebbistatin has no effect on HSC proliferation. Cell proliferation was measured by a WST-1 assay. HSCs were treated with (BLEB) or without (CONT) blebbistatin (25 µM), and in the presence or absence of platelet-derived growth factor (PDGF)-BB (20 ng·mL⁻¹).

Figure 7

Cell adhesion assay. (A) Blebbistatin has no significant effect on the adhesion of quiescent hepatic stellate cells (HSCs). Images show cell adhesion after crystal violet staining of cells at day 4, treated with different concentrations of blebbistatin (0, 12.5, 25, 50 µM). Graph showing absorbance analysis of three experiments. (B) Blebbistatin decreases cell adhesion in activated HSCs. Images show cell adhesion after crystal violet staining of cells at day 10, treated with blebbistatin at different concentrations (0, 12.5, 25, 50 µM). The graph shows the absorbance analysis of three experiments. Each bar represents mean ± SD. *P < 0.01, **P < 0.001 compared with control (0 µM). The observed effect was not statistically significant for the different concentrations of blebbistatin. (C) Vinculin-containing focal adhesions disappeared in blebbistatin-treated HSCs. Cells cultured at day 11 were treated with or without blebbistatin (50 µM) for 2 h and then fixed and stained with antibodies to vinculin. The scale bar represents 50 µm.