Chronic activation of mechanosensitive channels in human trabecular meshwork cells involves dynamic interactions between TRPV4 and TRPM4-mediated signals

Abstract

Purpose : Calcium ions are believed to regulate the hydraulic conductivity of the conventional outflow pathway in part by modulating the contractility of the trabecular meshwork (TM). Mechanical stress facilitates TM contractility by elevating [Ca²⁺]_TM through stretch-activated TRPV4 channels but how this affects the dynamics of Ca homeostasis in the presence of chronic mechanoactivation is not known.

Methods : Immortalized (juxtacanalicular) and primary (corneoscleral) human TM cells were stimulated with biaxial strain (1-12%), pressure clamp or the selective TRPV4 agonist GSK1016790A (GSK101). mRNA and protein levels were measured with qRT-PCR and Western blots and spatial expression determined with antibody labeling. TM and HEK293 cells were transfected with GFP probes, scrambled (Sc) and Trpv4- or Trpm4-specific shRNAs and exposed to agonists/antagonists of intracellular Ca²⁺ signaling pathways. [Ca²⁺]_TM and transmembrane currents were measured with optical imaging and patch clamp, respectively.

Results : Mechanical stretch and TRPV4 activation elevated [Ca²⁺]_i and induced upregulation of actin stress fibers. The [Ca²⁺]_i increases induced by TRPV4 stimulation were associated with transient peaks and sustained plateaus, with superposed Ca²⁺ transients with periodicity of ~30 –120 sec. The frequency and amplitude of the Ca²⁺ fluctuations were suppressed by nonselective blockers of TRP channels, TRPM4 antagonists and Trpv4/Trpm4-specific shRNA. Ca²⁺ oscillations were observed in the presence of SERCA blockade. RT-PCR and Western blots showed prominent expression of Trpm4 gene and protein in TM cells, as well as Trpm4:GFP localization to membrane and cytosolic puncta. The TRPV4-dependent oscillatory calcium phenotype was reproduced in HEK293 cells transfected with Trpv4 and Trpm4.

Conclusions : These results show that TM calcium homeostasis is highly plastic, with amplitude- and time-dependence of Ca²⁺ fluctuations that depend on steady-state [Ca²⁺]_i and history of activation of the TM- intrinsic mechanosensitive channels. The functional link between TRPV4 and TRPM4-dependent calcium fluctuations is novel. We hypothesize that TRPV4-TRPM4 interactions contribute to the well-known pressure-dependent periodicity of fluctuations in IOP.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.