Abstract
Purpose :
To characterize differences in mitochondrial fission, fusion, and mitophagy related proteins and mitochondrial morphology between diabetic and control corneal endothelial cells (CECs).
Methods :
Human CECs isolated from cornea donors with and without type II diabetes mellitus were characterized using a multidisciplinary approach including ultra high pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) for proteomic screening, semi-quantitative western blotting of select dynamics related proteins, and transmission electron microscopy (TEM). Healthy donor endothelium-Descemet membrane (EDM) tissue punches were also treated with reagents known to impact mitochondrial fission-fusion dynamics (MSICI1 and dynasore) and assayed for extracellular flux analysis of mitochondrial respiration parameters.
Results :
The relative abundance of dynamin 1 like peptides (DRP, involved in fission) were reduced in diabetic CECs compared to controls. Phospho-dynamin 1 like 637 (pDRP1-637, inhibits fission) was found to be significantly increased in diabetic samples (P<0.05) and PTEN induced putative kinase 1 (PINK1, involved in mitophagy) was found to be significantly reduced in diabetic CECs (P<0.05). Mitochondria in diabetic samples displayed elongated morphologies, disrupted cristae, and greater numbers of inclusion bodies compared to controls. Healthy EDM tissues treated with fission inhibiting reagents (MSICI1 and dynasore) displayed the lowest mitochondrial spare respiratory and maximal respiratory capacities (P<0.05 each), recapitulating results established previously in diabetic tissues.
Conclusions :
Overall, our studies show that type II diabetes mellitus alters mitochondrial dynamics in CECs and shifts the balance away from fission and proper mitophagy processes, resulting in dysfunctional mitochondria that build up within cells. These results support the distension, inclusion bodies, and dense packing of the mitochondria observed in TEM analyses, as well as the reduced spare respiratory capacity found in our previous functional assays of diabetic tissues and reproduced by blocking fission processes in normal tissue. A more complete understanding of the mechanisms involved in altering mitochondrial dynamics will provide better targets for interventions designed to protect CEC health, as well as other cell types affected by diabetes.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.