Abstract
Purpose :
The trabecular meshwork (TM) controls intraocular pressure by modulating the outflow of aqueous humor from the anterior chamber. A key homeostatic feature of this process rests on the capacity of TM cells for the sensing, transduction and adaptation to biomechanical stressors such as shear flow, tensile stretch, swelling and/or compression as they regulate cells’ contractility and outflow. We characterized the expression, functional properties and interactions of two mechanotransducing ion channels and evaluated their roles in TM calcium homeostasis and cytoskeletal remodeling.
Methods :
Transcript and protein expression was assessed in primary human TM cells with PCR, Western blots and immunocytochemistry. Channel activation was tested with patch-clamp, high-speed pressure clamp, optical imaging and biaxial cell stretch. Cytoskeletal and focal adhesion proteins were studied with phalloidin and antibody labeling, and fluorescent constructs.
Results :
TM cells expressed mRNAs that encode mechanosensitive TRPV4 and PIEZO1, but not PIEZO2 channels. Expression of TRPV4 and MYOC but not Piezo1 transcripts was upregulated in cells isolated from POAG donors. The selective PIEZO1 agonist Yoda-1 and TRPV4 agonist GSK1016790A induced a robust elevation of intracellular Ca2+ ([Ca2+]i), an effect that was abolished by Ruthenium red, removal of extracellular Ca2+, and isoform-specific short hairpin RNA (shRNA). Pressure stimulation induced single channel openings that exhibited the activation properties of PIEZO1 and were reduced by GsMTx4 and in cells expressing PIEZO1 shRNA, but not in control cells transfected with scrambled (Sc) shRNA. PIEZO1 and TRPV4 blockers differentially affected the expression of focal contacts in quiescent vs. stretched TM cells.
Conclusions :
We demonstrate that human TM cells strongly express at least two calcium-permeable mechanosensitive channels from different families. Both respond to pressure stimuli, with channel activation resulting in large [Ca2+]i elevations. However, TRPV4 and PIEZO1 appear to perform different functions in the sensing of ‘resting’ and applied membrane tension. Our data therefore suggest that trabecular physiology and outflow might be subject to parallel modulation from mechanotransducers with different pressure sensitivities, activation properties and cytoskeletal coupling.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.