Carboxyatractyloside catr

ΔΨm of isolated mitochondria (0.5 mg for mouse liver and 0.25 mg for brain per 2 ml of medium) was estimated fluorimetrically with safranine O [9 (link)], acknowledging the considerations elaborated in [10 (link), 11 (link)] regarding inhibition of respiration as well as unspecific binding of safranine. Fluorescence was recorded in a Hitachi F-7000 spectrofluorimeter (Hitachi High Technologies, Maidenhead, UK) at a 5-Hz acquisition rate, using 495- and 585-nm excitation and emission wavelengths, respectively. Experiments were performed at 37 °C.
Reagents: Standard laboratory chemicals and 2-ketobutyrate (Cat. No.: K401, purity 97%) were from Sigma. SF 6847 and atpenin A5 were from Enzo Life Sciences (ELS AG, Lausen, Switzerland). Carboxyatractyloside (cATR) was from Merck (Merck KGaA, Darmstadt, Germany). Mitochondrial substrate stock solutions were dissolved in bi-distilled water and titrated to pH 7.0 with KOH. ADP was purchased as a K⁺ salt of the highest purity available (Merck) and titrated to pH 6.9.

Standard laboratory chemicals, enzyme substrates and itaconic acid were from
Sigma-Aldrich. SF 6847 was from Enzo Life Sciences (ELS AG, Lausen,
Switzerland). Carboxyatractyloside (cATR) was from Merck (Merck KGaA, Darmstadt,
Germany). KM4549SC (LY266500) was from Molport (SIA Molport, Riga, Latvia).
Mitochondrial substrate stock solutions were dissolved in bi-distilled water and
titrated to pH 7.0 with KOH. ADP was purchased as a K⁺ salt of
the highest purity available (Merck) and titrated to pH 6.9. TaqMan Gene
Expression Assays, XS, Suclg2 (AB, 4448892, FAM/MGB-NFQ) kit and Actb (AB,
4448489, VIC-MGB) kit were from Thermo Fisher Scientific. qPCR mix was qPCRBIO
SyGreen Mix Hi-Rox (PCR Biosystems).

H₂O₂ flux (ROS flux) in cardiac fibres was measured simultaneously with respirometry using an O2k-Fluorometer and the H₂O₂-sensitive probe Ampliflu™ Red (AmR) (Sigma-Aldrich) as described previously [27 (link), 28 (link)] with some modifications. First, 10 μM AmR, 1 U/ml horseradish peroxidase (HRP), and 5 U/ml superoxide dismutase (SOD) (all Sigma-Aldrich) were added to the chamber, and H₂O₂ detection was performed based on the conversion of AmR to fluorescent resorufin. Calibrations were performed by adding 0.1 μM H₂O₂ stepwise. The H₂O₂ flux was corrected to take into account the background (AmR slope before sample addition). The respiration protocol was similar to that described above, with the exception that after the assessment of FAO and pyruvate-linked respiration, succinate (10 mM, complex II (CII) substrate) (Sigma-Aldrich) was then added to reconstitute convergent FAO-CI-II-linked respiration. Then, an inhibitor of adenine nucleotide translocator, carboxyatractyloside (Catr) (Sigma-Aldrich), was added to determine the LEAK_Catr respiration. Titration with an uncoupler, carbonyl cyanide m-chlorophenyl hydrazine (0.5 μM steps to maximum oxygen consumption) (Sigma-Aldrich), was performed to determine the H₂O₂ production rate at the electron transfer system capacity state.