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Andrew Jo, Sarah Redmon, Anthony Iuso, Tünde Molnar, Tam Thi Thanh Phuong, David Krizaj; TRPV4 and TRPC1 channels regulate strain sensitivity in retinal neurons and glia. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4209.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal ganglion cells (RGCs) and glia are immersed within a mechanically sensitive environment in which they must constantly cope with and adapt to pressure and osmotic stress. The purpose of this project was to (i) use different stretch paradigms to identify the molecular mechanism that underlies the mechanosensitive properties of mouse RGCs and Muller glial cells (MCs) and (ii) to characterize the relationship between membrane strain, TRP (transient receptor potential) channel activation and [Ca]2+homeostasis in RGC and MCs.
Dissociated or cultured mouse retinal cells were loaded with calcium indicator dyes and stimulated with defined magnitudes and time periods of applied cyclic uniaxial or biaxial substrate strain. RGCs were identified based on immunopanning or by expression of the transgenic Thy1:YFP marker. Calcium concentration [Ca2+]i was assessed with high-resolution optical imaging. Response selectivity was determined with selective TRP channel antagonists, agonists and TRPV4-/- and/or TRPC1-/- cells. Reactive gliosis was determined with GFAP immunolabeling.
MCs and a subset of RGCs (57 ± 14 %) responded to cyclical uniaxial or biaxial stretch (0.5 – 15%) with [Ca2+]i elevations. For example, 10% uniaxial stretch elevated [Ca2+]i in Thy1:RGC responders and in Muller glial cells by 89.4 ± 9.7% (n=21) and 72 ± 5.6% fluorescence ratio increase, respectively. Stretch evoked [Ca2+]i responses were reduced in KO cells and following the application of the selective TRPV4 antagonist HC 067047 whereas, capsazepine, an antagonist of TRPV1 channels had no inhibitory effect. The glial response to strain was reduced by inhibitors of the phospholipase A2 signaling pathway and by genetic elimination of TRPC1 channels. Further consistent with potential mechanosensitive function of glial TRPC1 was the observation of attenuated reactive gliotic response to elevation of intraocular pressure in a TRPC1-/- mouse model of glaucoma.
We applied novel mechanobiological paradigms to the retina to demonstrate that stretch- evoked [Ca2+]i signaling in RGCs and Muller cells is mediated by TRPV4 and TRPC1, but not TRPV1, channels. These findings identify possible mechanisms whereby retinal cells sense and transduce intraocular pressure and ECM strain at the optic nerve head, and may thus have implications for the understanding and treatment of blinding diseases such as ischemia and glaucoma.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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