Figure 10. Isolated mitochondrial studies II — redox.

Mitochondrial redox: Freshly isolated mitochondria from mutants and respective littermate controls were assayed in parallel using a fluorometer. (A) NAD(P)H oxidation (autofluorescence: λ_exc: 340, λ_em: 450 nm) was examined in the presence of complex I substrates (5 mM glutamate/malate) during state 4 and state 3 respiration. The NAD(P)H signal was calibrated by adding KCN (2.5 mM) and 2,4-dinitrophenol (DNP, 20 μΜ) at the end of each experiment. MyHC mutants had higher levels of reduced NAD(P)H during state 4 and 3 respiration. TnT mutants had higher levels of reduced NAD(P)H at the end of state 3 respiration when compared with littermate controls, because of lower oxidation of NAD(P)H by complex I during the state 4 to state 3 transition. Data are presented as relative ratios: MyHC/control-M and TnT/control-Tm. n = 21 experiments from 7 mitochondrial preparations/14 mice in control-M/MyHC group; n = 15 experiments from 5 mitochondrial preparations/10 mice in control-T/TnT group. (B–D) ROS emission: Mitochondria were incubated in Amplex Red (10 μΜ) for assessment of H₂O₂ emission by fluorometry (λ_exc: 530 nm and λ_em: 590 nm). H₂O₂ emission was assessed during state 4 and state 3 respiration, in the presence of glutamate/malate or succinate with/without rotenone at 37°C. H₂O₂ (100 pM) was used for signal calibration at the end of each experiment. The 2 mutants demonstrated H₂O₂ emission similar to that of the respective controls. Inhibition of complex I by rotenone led to lower H₂O₂ emission during state 3 respiration (P = 0.01). n = 5 experiments from 5 mitochondrial preparations/10 mice in each group for control-M/MyHC, and n = 4 experiments from 4 mitochondrial preparations/8 mice in each group for control-T/TnT. (E and F) ROS scavenging: Isolated mitochondria were incubated at 37°C in the presence of Amplex Red to monitor H₂O₂ during state 4 and state 3 respiration, with glutamate/malate (5/5 mM) and inhibitors of thioredoxin (50 nM auranofin) and/or glutathione (10 μM dinitrochlorobenzene [DNCB]) systems. Lower ROS scavenging by the thioredoxin system during state 4 was observed in TnT mutants. No other differences in ROS scavenging were detected. In A–F, data are presented as mean ± SD. n = 5 experiments from 5 mitochondrial preparations/10 mice in each group for control-M/MyHC and control-T/TnT. *P < 0.05 and **P < 0.001, using 2-sided unpaired Student’s t test.