Tandem microfluidic respirometry with bio-mimetic electrochemistry in whole cells and intact mitochondria from diabetic retinopathy models
Nathan Frantz1, Yan Levitsky1,2,3, Sandra S.Hammer2, Artem Muchnik1,2, David Pegouske1,2, Julia V.Busik1, Denis A. Proshlyakov2
1Chemistry, 2Physiology, 3COM Michigan State University, E. Lansing, MI, USA
Increased acid sphingomyelinase (ASM) activity and ceramide production critically contribute to the pathogenesis of sight threatening complication of diabetes, diabetic retinopathy (DR). Recently mitochondria and mitochondria-associated membranes were shown to contain sphingolipids and enzymes of sphingolipid pathway, including ASM. Ceramide was shown to suppress activity of Complex III, however existing methodology precludes direct studies on electron flow through ETC or precise localization of the ceramide-induced damage in DR pathogenesis. To circumvent this, we developed microfluidic respirometry that allows measurements on as few as 200-300 whole cells with >100x linear dynamic range and constant steady-state rate of O2 consumption (RO2) to <50 mM O2. Sustained, bio-mimetic respiration in whole mitochondria was achieved in both aerobic and anaerobic conditions using microfluidic electrochemistry with direct and quantitative measure of electron flux. Selective supply of electrons to Complex IV and withdrawal of electrons from Complex III using natural (CytC) and artificial (TMPD) redox metabolites allowed direct electrochemical manipulation of ETC Complexes in intact mitochondria and mitoplasts from DR cell culture and animal models. Exogenous CytC alone did not affect pyruvate/succinate-stimulated RO2 due to impermeability of the outer mitochondrial membrane, but concertation-dependent stimulation of RO2 was observed with TMPD and TMPD/CytC under negative potentials without mitochondrial substrates. Rates of electrochemically-stimulated RO2 exceeded intrinsic uncoupled substrate-driven RO2. TMPD stimulated RO2 under reducing potential and inhibited under oxidizing potential in the presence of substrates, permitting studies on Complexes III and IV independently of each other or the endogenous CytC. Furthermore, at zero RO2 under anaerobic conditions the entire electron flux from substrates is diverted to the electrode. This approach was used to specifically assess inhibition of Complex III by ceramide and its role in overproduction of succinate and increased ROS generation in diabetic retina and retinal cells.