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K. Trudeau, A. Molina, S. Roy; High Glucose Alters Mitochondrial Morphology and Membrane Potential Heterogeneity in Retinal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):33.
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© ARVO (1962-2015); The Authors (2016-present)
Healthy mitochondria within a cell appear long and tubular-shaped and display little variation in membrane potential. The effect of high glucose (HG) on mitochondrial morphology and membrane potential heterogeneity in retinal endothelial cells was investigated.
Rat retinal endothelial cells (RRECs) grown in normal (5mM) or HG (30mM) medium for 5 days were double-stained with MitoTracker Green FM (MTG, 125nM) and tetramethylrhodamine-ethyl-ester-perchlorate (TMRE, 8nM) and imaged using confocal microscopy. MTG stains mitochondria in a membrane potential independent manner whereas TMRE stains mitochondria in a membrane potential sensitive manner. Images were analyzed for mitochondria shape change in N and HG cells using Form Factor (FF) and Aspect Ratio (AR) values of the mitochondria. FF value of 1 corresponds to a circular, un-branched mitochondrion, and higher FF values indicate a longer, more-branched mitochondrion. AR of 1 corresponds to a circular mitochondrion, and higher AR values indicate more elliptical mitochondrion. The images were also analyzed for membrane potential of individual mitochondrion using deviation of fluorescence intensity (FI) values for the ratio of red (TMRE) to green (MTG) dye for several mitochondria within each cell. FI values reflect dye concentration distributions over the mitochondrial membranes. The standard deviation of membrane potential for mitochondria in a cell was used to give a value for membrane potential heterogeneity.
RRECs grown in HG medium exhibited significant fragmentation of mitochondria compared to RRECs grown in normal medium (FF for HG: 1.377 compared to 2.248 in normal, p=0.0002, AR for HG: 1.793 compared to 2.094 in normal, p=0.0055). Simultaneously, the RRECs grown in HG showed greater heterogeneity of mitochondrial membrane potential compared to normal RRECs (FI deviation for HG: 354.5 compared to 182.9 in normal, p=0.0001).
Our findings indicate that HG induces mitochondrial fragmentation and alters membrane potential heterogeneity in retinal endothelial cells. This could play a role in mitochondrial dysfunction and subsequent apoptosis associated with retinal endothelial cells in diabetic retinopathy.
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