Mechanosensitive channel inhibition attenuates TGF132-induced actin cytoskeletal remodeling and reactivity in mouse optic nerve head astrocytes

Astrocytes within the optic nerve head undergo actin cytoskeletal rearrangement early in glaucoma, which coincides with astrocyte reactivity and extracellular matrix (ECM) deposition. Elevated transforming growth factor beta 2 (TGF132) levels within astrocytes have been described in glaucoma, and TGF13 signaling induces actin cytoskeletal remodeling and ECM deposition in many tissues. A key mechanism by which astrocytes sense and respond to external stimuli is via mechanosensitive ion channels. Here, we tested the hypothesis that inhibition of mechanosensitive channels will attenuate TGF132-mediated optic nerve head astrocyte actin cytoskeletal remodeling, reactivity, and ECM deposition. Primary optic nerve head astrocytes were isolated from C57BL/6J mice and cell purity was confirmed by immunostaining. Astrocytes were treated with vehicle control, TGF132 (5 ng/ml), GsMTx4 (a mechanosensitive channel inhibitor; 500 nM), or TGF132 (5 ng/ml) + GsMTx4 (500 nM) for 48 h. FITC-phalloidin staining was used to assess the formation of f-actin stress fibers and to quantify the presence of crosslinked actin networks (CLANs). Cell reactivity was determined by immunostaining and immunoblotting for GFAP. Levels of fibronectin and collagen IV deposition were also quantified. Primary optic nerve head astrocytes were positive for the astrocyte marker GFAP and negative for markers for microglia (F4/ 80) and oligodendrocytes (OSP1). Significantly increased %CLAN-positive cells were observed after 48-h treatment with TGF132 vs. control in a dose-dependent manner. Co-treatment with GsMTx4 significantly decreased %CLAN-positive cells vs. TGF132 treatment and the presence of f-actin stress fibers. TGF132 treatment significantly increased GFAP, fibronectin, and collagen IV levels, and GsMTx4 co-treatment ameliorated GFAP immunoreactivity. Our data suggest inhibition of mechanosensitive channel activity as a potential therapeutic strategy to modulate actin cytoskeletal remodeling within the optic nerve head in glaucoma.

Automated summary

In glaucoma, early rearrangement of actin cytoskeleton in astrocytes within the optic nerve head is associated with reactivity and ECM deposition. Inhibition of mechanosensitive channels may attenuate TGF132-mediated actin cytoskeletal remodeling in these astrocytes, suggesting a potential therapeutic strategy for glaucoma.