Mitochondrial respiration and ROS emission during β-oxidation in the heart: An experimental-computational study
Fig 6
Mitochondrial antioxidant defense protein levels and ROS emission in the absence or the presence of inhibitors of the GSH and Trx antioxidant systems in Sham and STZ hearts.
(A, B) Heart tissue from Sham or diabetic GPs was processed and the antioxidant proteins indicated were analyzed by Western Blot as described in Materials and Methods. Left panel shows representative Western Blot analysis of protein abundance. The bar plot on the right panel displays the statistical comparison between the results obtained with heart tissue from the two groups [n = 4, four experiments]. Protein was normalized to total protein abundance in a given lane based on Direct Blue 71 (DB71) staining of the membrane as described [16,21]. Key to symbols: GR, glutathione reductase; Gpx4, glutathione peroxidase 4; Trx2, thioredoxin 2; TrxR2, thioredoxin reductase 2; Nnt, nicotinamide nucleotide transhydrogenase; SOD2, superoxide dismutase 2; Prx3, peroxiredoxin 3. (C, D) Freshly isolated heart mitochondria (100μg mitochondrial protein) from Sham or STZ-treated GP were preincubated without or with 50nM auranofin (AF) plus 10μM 1-chloro-2,4 dinitrobenzene (DNCB) [13,16]. Monitoring of H2O2 was performed with an Amplex red assay during state 4 (with [A] 10μM PCoA/0.5mM malate/0.5mM L-carnitine or [B] 5mM each of G/M) and state 3 (+1 mM ADP) of mitochondrial respiration, using a wavelength scanning fluorometer (QuantaMaster; Photon Technology International, Inc.) [13,16] in the same assay medium utilized for high throughput measurements. The specific fluxes of H2O2 emission are shown for PCoA (C) and G/M (D) in the absence or the presence of AF + DNCB. (E, F) Depicted are the model simulated rates of ROS emission and O2 consumption upon inhibition of antioxidant defense activities, glutathione reductase (GR) and thioredoxin reductase (TrxR) to mimic the actions of the experimentally utilized inhibitors DNCB and auranofin, respectively (C, D). The plots display the steady state values of H2O2 emission (E) and VO2 (F) obtained at high (control: 100% GR and TrxR activities, empty bars) and low (15% and 22% of GR and TrxR activities, respectively, grey bars) scavenging capacity, under states 4 and 3 respiration as indicated. For comparative purpose, in panel E are shown the experimental values of Sham depicted in panel C, corresponding to the absence (filled squares) or presence (filled triangles) of inhibitors, under states 4 and 3 respiration as indicated. The simulations correspond to 40μM PCoA with 5x10-4mM and 0.1mM ADP in states 4 and 3, respectively, while the remaining parameters are described in S1 Text.