Mechanotransduction and dynamic outflow regulation in trabecular meshwork requires Piezo1 channels

Abstract

<jats:title>Abstract</jats:title><jats:p>Mechanosensitivity of the trabecular meshwork (TM) is a key determinant of intraocular pressure (IOP) yet our understanding of the molecular mechanisms that subserve it remains in its infancy. Here, we show that mechanosensitive Piezo1 channels modulate the TM pressure response via calcium signaling and dynamics of the conventional outflow pathway. Pressure steps evoked fast, inactivating cation currents and calcium signals that were inhibited by Ruthenium Red, GsMTx4 and Piezo1 shRNA. Piezo1 expression was confirmed by transcript and protein analysis, and by visualizing Yoda1-mediated currents and [Ca<jats:sup>2+</jats:sup>]<jats:sub>i</jats:sub> elevations in primary human TM cells. Piezo1 activation was obligatory for transduction of physiological shear stress and was coupled to reorganization of F-actin cytoskeleton and focal adhesions. The importance of Piezo1 channels as pressure sensors was shown by the GsMTx4 -dependence of the pressure-evoked current and conventional outflow function. We also demonstrate that Piezo1 collaborates with the stretch-activated TRPV4 channel, which mediated slow, delayed currents to pressure steps. Collectively, these results suggest that TM mechanosensitivity utilizes kinetically, regulatory and functionally distinct pressure transducers to inform the cells about force-sensing contexts. Piezo1-dependent control of shear flow sensing, calcium homeostasis, cytoskeletal dynamics and pressure-dependent outflow suggests a novel potential therapeutic target for treating glaucoma.</jats:p><jats:sec><jats:title>Significance Statement</jats:title><jats:p>Trabecular meshwork (TM) is a highly mechanosensitive tissue in the eye that regulates intraocular pressure through the control of aqueous humor drainage. Its dysfunction underlies the progression of glaucoma but neither the mechanisms through which TM cells sense pressure nor their role in aqueous humor outflow are understood at the molecular level. We identified the Piezo1 channel as a key TM transducer of tensile stretch, shear flow and pressure. Its activation resulted in intracellular signals that altered organization of the cytoskeleton and cell-extracellular matrix contacts, and modulated the trabecular component of aqueous outflow whereas another channel, TRPV4, mediated a delayed mechanoresponse. These findings provide a new mechanistic framework for trabecular mechanotransduction and its role in the regulation of fast fluctuations in ocular pressure, as well as chronic remodeling of TM architecture that epitomizes glaucoma.</jats:p></jats:sec>

Membrane cholesterol regulates TRPV4 function, cytoskeletal expression, and the cellular response to tension

“…The phenotype was periodically validated by profiling for markers, including Aqp1, Timp3, Myoc, MGP, Acta2 (α-smooth muscle actin, αSMA) and dexamethasone-induced upregulation of myocilin expression. These data are shown in our previous characterizations of the cell line (21, 24, 90).…”

“…Patients with high levels of serum cholesterol have increased likelihood for elevated IOP, with POAG linked to several genes involved in cholesterol metabolism (e.g., ABCA1, CAV1, ARHGEF12 , and DGKG ) (17, 18)). IOP is dynamically regulated by the trabecular meshwork (TM), a multilayered tissue composed of smooth-muscle-like cells with mechanosensitive and contractile properties (16, 19–21). TM stiffness and the phospholipid composition are altered in mice and humans with glaucoma (22, 23) and it would therefore be useful to obtain insight into how the biomechanical milieu impact TM lipids and whether changes in membrane cholesterol affect mechanotransduction.…”

“…Patients with high levels of serum cholesterol have increased likelihood for elevated intraocular pressure (IOP), with glaucoma linked to multiple genes involved in cholesterol metabolism (17, 18). IOP is dynamically regulated by the trabecular meshwork (TM), a multilayered tissue composed of smooth-muscle-like cells with mechanosensitive and contractile functions that control the outflow of aqueous humor from the anterior eye (16, 19–21). In response to mechanical stress and glaucoma, TM cells upregulate cytoskeleton and secretion of ECM, with attendant increases in cell contractility and stiffness producing a suppression of aqueous outflow and elevation in IOP (22, 23, 36, 37).…”

“…We recently identified the mechanosensitive Transient Receptor Potential Vanilloid 4 (TRPV4) channel as a principal regulator of mechanically induced signaling in mouse and human TM (20, 24, 25). This ubiquitous nonselective cation channel transduces membrane strain, shear flow, swelling, temperature and polyunsaturated fatty acids into calcium signals (21, 24, 26, 27). Its sequence appears to have co-evolved with enzymes linked to cholesterol biosynthesis pathways (28) and studies in endothelial cells suggested that TRPV4 may be trafficked to cholesterol- and caveolin1 (Cav-1)-enriched rafts (29) to modulate vascular flow (30).…”

Membrane cholesterol regulates TRPV4 function, cytoskeletal expression, and the cellular response to tension

“…Patients with high serum cholesterol levels have an increased likelihood for elevated intraocular pressure (IOP), with glaucoma linked to multiple genes involved in cholesterol metabolism ( 17 , 18 ). A principal regulator of IOP is the trabecular meshwork (TM), a multilayered tissue composed of smooth muscle-like cells with mechanosensitive and contractile functions ( 16 , 19 , 20 , 21 ). In response to mechanical stress and glaucoma, TM cells upregulate actomyosin cytoskeleton, ECM secretion, and the size/number of focal cell-ECM contacts, thereby increasing cell contractility and tissue resistance to the outflow of aqueous humor ( 22 , 23 , 26 , 27 ).…”