The role of mechanosensitive channels in cellular/metabolic response of optic nerve head (ONH) astrocytes to tension in a 3D hydrogel system

Abstract

Purpose : Glaucoma causes progressive retinal ganglion cell (RGC) loss and coincides with increased tension on the ONH. In early glaucoma, ONH astrocytes remodel to support RGCs through the lactate shuttle. To do this, astrocytes must retain great metabolic flexibility in response to strain. However, with chronic insult, the ONH undergoes reactive gliosis and metabolic insufficiency. In response to tension, astrocytes activate mechanosensitive channels like Piezo1 to amplify gliotic responses. Here, we use a 3D hydrogel system to investigate whether glaucomatous tensile strains alter mouse ONH astrocyte (MONHA) cellular/metabolic function through Piezo1.

Methods : MONHA-encapsulated hydrogels (N=3) were subjected to 0 or 5% tension +/- mechano-inhibitor 500nM GsMTx4 for 12-24h using the FlexCell System. Microregional strains were mapped by particle tracking velocimetry (PVC), and cell viability quantified via MTS assay. Astrocyte morphology was measured by Sholl analysis (N=10 cells/group), and nuclear morphology (N=350/group) analyzed via confocal microscopy. mRNA of reactivity marker C3, Piezo1, and lactate transporter MCT4 and extracellular lactate were measured. Mitochondrial superoxide content was evaluated by MitoSox stain in MONHAs treated with control, Piezo1 agonist 10 uM Yoda1, 500 nM GsMTx4 or co-treatment for 2h. Cellular metabolism was analyzed with Agilent Cell Mito Stress Seahorse Test.

Results : PVC analysis confirmed overall strain profile. Astrocyte viability was unchanged post stretch. Global hydrogel stiffness did not change with strain but softened with GsMTx4. Tension reduced process complexity and degree of branching (p<0.005) and increased nuclear volume (p<0.0001) with GsMTx4 rescuing these features. Strained MONHAs altered C3/MCT4 mRNA levels (p<0.0001) and increased lactate release (p<0.005). Yoda1 treatment increased MitoSox signal intensity (p<0.005), cellular respiration (p<0.05), extracellular acidification and ATP production (p<0.0001). Co-treatment with GsMTx4 restored mitochondrial superoxide level to control with no effect on cellular respiration readouts.

Conclusions : Our data implicate mechanosensitive channels such as Piezo1 in MONHA response to glaucomatous tensile strain. Future studies will investigate the role of Piezo1 in strain-induced Ca²⁺ and metabolic dynamics via cell-specific knockdown and live-cell imaging.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.