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

PURPOSE. Disruption in intracellular calcium ion (Ca2+) homeostasis has major effects on health. Persistent Ca2+ overload induces mitochondrial permeability transition pore ( MPTP) opening, which prompts mitochondrial release of calcium (mCICR) and reactive oxygen species (ROS) into the cytosol which, in turn, compromises mitochondrial function. This study was conducted to examine 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, nondiseased (NTM) eyes, obtained from postmortem donors eyes by standard surgical trabeculectomy, were treated with the following calcium regulators: the mitochondrial respiratory chain I inhibitor rotenone ( ROT); the mitochondrial permeability transition pore ( MPTP) inhibitors cyclosporine (Cys) and aristolochic acid (ArA); the Ca2+ chelators BAPTA/AM or EDTA; the mitochondrial Ca2+ uniporter inhibitor ruthenium red (RR); the Ca2+/Na+ exchanger inhibitor trifluoperazine; and the inositol 1,4,5-triphosphate receptor type 3 (IP3R) inhibitors 2-aminoethoxydiphenyl borane (2-APB) and xestospongin C (Xe-C). Ca2+ concentrations in the cytoplasm ([Ca2+](c)) and mitochondria ([Ca2+](m)) were determined by confocal microscopy and flow cytometry with the fluorescent Ca2+ indicators fluo-3/AM and rhod-2/AM, respectively. Mitochondrial membrane potential (Delta Psi m) was examined with the fluorescent probe tetramethylrhodamine ethyl ester ( TMRE). The expression of cyclophilin D, a protein that induces MPTP opening was also measured. RESULTS. There was increased [Ca2+](c), [Ca2+](m), mCICR, MPTP opening, and expression of cyclophilin D and decreased Delta Psi m in POAG TM cells compared with control cells. 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. 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. Pharmacologic inhibitors of IP3R, MPTP opening, and cyclophilin D could have clinical implications for primary open-angle glaucoma. (Invest Ophthalmol Vis Sci. 2008;49: 4912-4922) DOI:10.1167/iovs.08-2192