September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Analysis of the OXPHOS complex in the corneal endothelium suggests impaired mitochondrial respiration in Fuchs Endothelial Corneal Dystrophy
Author Affiliations & Notes
  • Adna Halilovic
    Ophthalmology, Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, United States
    Mass Eye and Ear Infirmary, Boston, Massachusetts, United States
  • Ula V Jurkunas
    Ophthalmology, Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, United States
    Mass Eye and Ear Infirmary, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Adna Halilovic, None; Ula Jurkunas, None
  • Footnotes
    Support  NEI/NIH R01 EY020581 and Alcon Research Institute Young Investigator Grant
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 5285. doi:
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      Adna Halilovic, Ula V Jurkunas; Analysis of the OXPHOS complex in the corneal endothelium suggests impaired mitochondrial respiration in Fuchs Endothelial Corneal Dystrophy. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5285.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Fuchs Endothelial Corneal Dystrophy (FECD) is characterized by progressive loss of functional corneal endothelium, a highly metabolic tissue with abundant mitochondria. We have previously demonstrated an increased DNA damage, decreased ATP levels, and mitochondrial fragmentation in FECD cells. In this study, we aim to further delineate the aberrant metabolic processes in mitochondrial respiration that can provide an insight on the underlying pathogenesis of FECD.

Methods : Oxidative phosphorylation (OXPHOS) complexes (CI-CV) were assessed by western blotting in normal and FECD ex vivo human specimens and immortalized normal and FECD corneal endothelial cells (HCEnC and FECDi, respectively). Mitochondrial respiration was analyzed with Seahorse Bioscience XF24 using the Mito Stress Test kit (which includes OXPHOS complex inhibitors: Oligomycin, FCCP, Rotenone, and Antimycin A) in HCEnC and FECDi cells. Baseline OCR and drug injection was measured 3 times for 3 min each separated by a 2 min wait and 2 min mix and 3 min for measurement.

Results : The protein expression of complex I and V were decreased in FECD compared to normal endothelium. Analysis of the OXPHOS complex in HCEnC and FECDi suggested a decrease in the protein expression of complex I and V concordant with FECD specimen, whereas, complex II, II, and IV did not show a differential expression. Using the Seahorse Mito Stress kit, basal respiration and mitochondrial respiration in FECDi cells was 3.3 and 2.8-fold higher compared to HCEnC cells suggesting that FECDi cells consume and require a greater amount of oxygen for homeostasis and metabolic function. Moreover, FECDi cells revealed 3.6-fold increase in proton leakage compared to HCEnC. Spare capacity was lower by 55% in FECDi compared to HCEnC suggesting that the FECDi cells uncoupled by FCCP were not able to respond by increasing maximal respiration.

Conclusions : Mitochondria in FECDi cells revealed a higher level of basal respiration resulting in a lower spare capacity. Since FECD endothelium has greater mitochondrial DNA damage, decrease in mitochondrially-encoded transcripts may manifest in a decline of OXPHOS complex synthesis. The resulting respiratory inefficiency may contribute to the elevated glycolytic activity (Warburg effect) and provide another venue in the study of FECD pathogenesis.

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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