Mitochondrial Defects and Dysfunction in Calcium Regulation in Glaucomatous Trabecular Meshwork Cells

Abstract

Purpose: : Disruption in intracellular calcium ion (Ca²⁺) homeostasis has important consequences for health. Persistent Ca²⁺ overload induces mitochondrial permeability transition pore (MPTP) opening prompting mitochondrial release of calcium (mCICR) and ROS into the cytosol which, in turn, compromises mitochondrial function. Here, we examined intracellular Ca²⁺ levels and mitochondrial vulnerability to Ca²⁺ stress in trabecular meshwork (TM) of individuals with primary open angle glaucoma (POAG).

Methods: : Primary cultures of TM cells from POAG (GTM) and age-matched, non-diseased (NTM) individuals obtained by standard surgical trabeculectomy, from postmortem donors eyes, were treated with the following calcium regulators: mitochondrial respiratory chain I inhibitor, rotenone (ROT); mitochondrial permeability transition pore (MPTP) inhibitor, cyclosporine A (CsA) and aristolochic acid (ArA); Ca²⁺ chelators, BAPTA/AM, or EDTA; mitochondrial Ca²⁺ uniporter inhibitor, ruthenium red (RR); Ca²⁺/Na⁺ exchanger inhibitor, trifluoperazine; and the inositol 1,4,5-triphosphate receptor type 3 (IP3R) inhibitors, 2-aminoethoxydiphenyl borane (2-APB) or Xestospongin C (Xe-C). Ca²⁺ concentrations in the cytoplasm ([Ca²⁺]_c) and mitochondria ([Ca²⁺]_m) were determined by confocal microscopy and flow cytometry using the fluorescent Ca²⁺ indicators, fluo-3/AM and rhod-2/AM, respectively. Mitochondrial membrane potential (ΔΨm) was examined using the fluorescent probe tetramethylrhodamine ethyl ester (TMRE). We also studied the expression of cyclophilin D, a protein that induces MPTP opening.

Results: : There was increased [Ca²⁺]_c, [Ca²⁺]_m, mCICR, MPTP opening and expression of cyclophilin D, and decreased ΔΨm in POAG TM cells compared to controls. ROT artificially exacerbated these conditions in GTM cells. Chelation of [Ca²⁺]_c and inhibition of IP3R and MPTP opening suppressed mitochondrial dysfunction and reduced the additional effects of ROT in GTM cells.

Conclusions: : We propose that POAG TM cells have defective mitochondrial function, which causes them to be abnormally vulnerable to Ca2+ stress. The dysfunction in calcium regulation by these cells may contribute to the failure of this tissue to control IOP. Pharmacological inhibitors of IP3R, MPTP opening, and cyclophilin D could have clinical implications for primary open angle glaucoma.

Keywords: calcium • mitochondria • degenerations/dystrophies