Emergent Temporal Signaling in Human Trabecular Meshwork Cells: Role of TRPV4-TRPM4 Interactions

Abstract

Trabecular meshwork (TM) cells are phagocytic cells that employ mechanotransduction to actively regulate intraocular pressure. Similar to macrophages, they express scavenger receptors and participate in antigen presentation within the immunosuppressive milieu of the anterior eye. Changes in pressure deform and compress the TM, altering their control of aqueous humor outflow but it is not known whether transducer activation shapes temporal signaling. The present study combines electrophysiology, histochemistry and functional imaging with gene silencing and heterologous expression to gain insight into Ca2+ signaling downstream from TRPV4 (Transient Receptor Potential Vanilloid 4), a stretch-activated polymodal cation channel. Human TM cells respond to the TRPV4 agonist GSK1016790A with fluctuations in intracellular Ca2+ concentration ([Ca2+]i) and an increase in [Na+]i. [Ca2+]i oscillations coincided with monovalent cation current that was suppressed by BAPTA, Ruthenium Red and the TRPM4 (Transient Receptor Potential Melastatin 4) channel inhibitor 9-phenanthrol. TM cells expressed TRPM4 mRNA, protein at the expected 130-150 kDa and showed punctate TRPM4 immunoreactivity at the membrane surface. Genetic silencing of TRPM4 antagonized TRPV4-evoked oscillatory signaling whereas TRPV4 and TRPM4 co-expression in HEK-293 cells reconstituted the oscillations. Membrane potential recordings suggested that TRPM4-dependent oscillations require release of Ca2+ from internal stores. 9-phenanthrol did not affect the outflow facility in mouse eyes and eyes from animals lacking TRPM4 had normal intraocular pressure. Collectively, our results show that TRPV4 activity initiates dynamic calcium signaling in TM cells by stimulating TRPM4 channels and intracellular Ca2+ release. It is possible that TRPV4-TRPM4 interactions downstream from the tensile and compressive impact of intraocular pressure contribute to homeostatic regulation and pathological remodeling within the conventional outflow pathway.

Osmosensing in trabecular meshwork cells

“…At the resting potential (−40 mV (Yarishkin, Phuong et al 2018, Yarishkin, Phuong et al 2022)) I Swell was inward and slow, taking min to reach the peak amplitude of –329 pA ± 45 pA (N =18). 285 mOsm saline evoked currents with correspondingly reduced amplitude.…”

“…2) (Jo, Ryskamp et al 2015) and (ii) swelling is augmented in cells that overexpress TRPV4 (Barile, Mola et al 2023), AQPs may not be required to facilitate cell swelling in response TRPV4-mediated Ca 2+ influx. NKCC1 transports water (Zeuthen and MacAulay 2002) and may modulate outflow resistance (Al-Aswad, Gong et al 1999) whereas TRPM4 shows strong expression in mouse and human TM and mediates 9-phenanthrol-sensitive Na + influx in GSK101-stimulated pTM cells (Yarishkin, Phuong et al 2022). Cell swelling may be inhibited by TRPM4 ablation and inhibition (Stokum, Kwon et al 2018).…”

“…De-identified postmortem eyes from 5 male and female donors aged 60 - 81 years with no history of glaucoma were procured from Utah Lions Eye Bank with written informed consent of the donors’ families and according to the standards set by the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report. Cells from juxtacanalicular and corneoscleral TM regions were isolated (Yarishkin, Phuong et al 2018, Lakk and Krizaj 2021, Yarishkin, Phuong et al 2022) and cultured, showing the typical spindle and hexagonal phenotypes (Stamer and Clark 2017). The cells were validated following the consensus recommendations (Keller, Bhattacharya et al 2018), including periodic profiling for MYOC, AQP1, MGP, ACTA2 expression and dexamethasone-dependent upregulation of MYOC (Lakk and Krizaj 2021, Yarishkin, Phuong et al 2022).…”

“…Cells from juxtacanalicular and corneoscleral TM regions were isolated (Yarishkin, Phuong et al 2018, Lakk and Krizaj 2021, Yarishkin, Phuong et al 2022) and cultured, showing the typical spindle and hexagonal phenotypes (Stamer and Clark 2017). The cells were validated following the consensus recommendations (Keller, Bhattacharya et al 2018), including periodic profiling for MYOC, AQP1, MGP, ACTA2 expression and dexamethasone-dependent upregulation of MYOC (Lakk and Krizaj 2021, Yarishkin, Phuong et al 2022). Passage 2–6 cells were seeded onto collagen I coverslips and grown in Trabecular Meshwork Cell Medium (ScienCell Research Laboratories, Carlsbad, CA) supplemented with 2% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin, at pH 7.4, 37°C and 5% CO 2 .…”

“…The presence of osmosensitive Ca 2+ channels and/or transporters has been predicted by swelling- and Ca 2+ -activated K + and Cl - currents in bovine, pig and huma preparations (Mitchell, Fleischhauer et al 2002, Soto, Comes et al 2004, Srinivas, Maertens et al 2004), but the source of the Ca 2+ signal has remained unknown. We focused on TRPV4 (Transient Receptor Potential Vanilloid Isoform 4) due to its prominent expression in rodent and human TM (Luo, Conwell et al 2014, Ryskamp, Frye et al 2016), roles ins ensing pressure, strain and body temperature (Ryskamp, Frye et al 2016, Yarishkin, Phuong et al 2021, Krizaj, Cordeiro et al 2023), role in TRPM4-mediated Na + influx (Yarishkin, Phuong et al 2022), AQP interactions (Jo, Ryskamp et al 2015, Mola, Sparaneo et al 2016) and sensitivity to hypotonic stimuli in other cell types (Liedtke and Friedman 2003, Hoffmann, Lambert et al 2009). TRPV4 is a tetrameric channel with the S5-S6 region forming a pore permeable to cations, and functional domains within the N- and C-termini regulating trafficking, protein-protein interactions and sensitivity to lipid and mechanical milieus.…”