Morphological and functional evaluation of mitochondria in a Fuchs’ endothelial corneal dystrophy cell model

Abstract

Purpose: The pathogenesis of Fuchs’ endothelial corneal dystrophy (FECD) has yet to be fully elucidated, though the involvement of mitochondrial damage and endoplasmic reticulum stress have been suggested. We recently established an immortalized cellular model of FECD-patient human corneal endothelial cells (HCECs) (iFECD) and normal donor-cornea HCECs (iHCED) (article currently in revision). The purpose of this present study was to investigate the involvement of mitochondrial dysfunction in FECD by use of these cellular models.

Methods: To elucidate morphological change of mitochondria, iFECD and iHCEC were cultured on Transwell^® permeable supports (Corning Inc.) and assessed by transmission electron microscopy (TEM). Mitochondrial membrane potential (MMP) was evaluated by fluorescence microscopy following fluorescent probe JC-1 dye staining. The release of cytochrome c from mitochondria to the cytoplasm was evaluated by Western blotting. To evaluate the involvement of mitochondria dysfunction in apoptosis, MMP was evaluated by JC-1 dye, and apoptosis-related proteins such as caspase 9, caspase 3, and PARP were evaluated by western blotting in iHCECs stimulated by staurosporine that was used to induce mitochondrial damage.

Results: TEM showed that mitochondria were dilated in iFECD, though the morphology of mitochondria was normal in iHCEC. Staining with JC-1 dye showed that MMP was lower in iFECD than in iHCEC. Flow cytometry showed that the percentage of depolarized cells of iFECD was 41.7%, while that of iHCEC was 15.5% (p<0.05). Western blotting showed a higher level of cytochrome c leakage to the cytoplasm from mitochondrial fraction in iFECD than in iHCEC. In addition, western blotting revealed that caspase 9, caspase 3, and PARP were cleaved after treatment with staurosporine.

Conclusions: The findings of this study indicate that mitochondria dysfunction is involved in the pathogenesis of FECD, yet further studies are required to elucidate if mitochondria dysfunction might be a potent therapeutic target.