Abstract
Purpose :
Mitochondrial damage precedes histopathological abnormalities in diabetic retinopathy. Recently mitochondria and associated membranes were shown to contain sphingolipids and enzymes of sphingolipid pathway. Activation of acid sphingomyelinase (ASM) in diabetes increases ceramide production in retinal endothelial (REC) and retinal pigment epithelial (RPE) cells. Ceramide was shown to directly bind and suppress activity of complex III (CmpIII). Current methodology precludes direct studies of individual respiratory complexes needed for precise localization of the ceramide-induced mitochondrial damage in diabetic REC and RPE cells.
Methods :
CmpIII and IV in intact mitochondria were electrochemically manipulated in a novel microfluidic respirometer using natural and artificial redox mediators to selectively supply electrons to CmpIV and withdraw electrons from CmpIII. Mitochondria were isolated from control and STZ-induced diabetic mouse retina and from REC and RPE cells from control and diabetic human donor retinas, or ARPE-19 cells treated with 5.5 and 25 mM glucose.
Results :
Addition of oxidized CytC altered respiration rate (RO2) in mitochondria from high glucose-treated ARPE-19 cells, but not in control cells, and ASM inhibitor desipramine reversed this effect. TMPD- and CytC-mediated electrochemistry was used to determine changes in activities of the upstream and downstream complexes. Exogenous CytC alone did not affect RO2 in intact control mitochondria, but TMPD yielded concertation-dependent stimulation of RO2 by both TMPD and CytC under negative potentials in the absence of mitochondrial substrates (pyruvate etc.), approaching rates observed with TMPD/ascorbate. TMPD is capable of modulating RO2 in the presence of substrates, stimulating it under reducing potential and inhibiting under oxidizing potential. We demonstrate competition between the electrode/TMPD and CmpIV for electrons derived from substrates. Oxidizing electric current in anaerobic intact mitochondria can be used to assess activities of upstream complexes without respiration since the entire electron flux is diverted to the electrode.
Conclusions :
Increase in ASM activity and ceramide production under hyperglycemic conditions leads to change in mitochondria respiration. Electrochemistry was used as a sensitive probe to measure independent activities of CmpIII and IV and to assess specific inhibition of CmpIII by ceramide.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.