Mitochondrial Function in Endotoxemia

Mouse subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria were isolated from whole hearts by differential centrifugation at 24 h post-LPS, as previously described [35 (link), 36 (link)]. Mitochondrial respiration was assessed using a Clark-type electrode (Hansatech, UK) at room temperature after the addition of the substrates of complex I (malate and glutamate) or complex II (succinate and rotenone) to assess basal respiration (state 2). Maximal oxygen consumption (state 3) was activated by the addition of 200 µmol/L ADP. Oxygen consumption was expressed as nmols O2/min*CS. Hydrogen peroxide (H₂O₂) production was assessed from changes in fluorescence after the addition of respiration substrates in the presence of Amplex® Red (10 µmol/L) and horseradish peroxidase (5 U/mL). Changes in fluorescence over time were monitored at 590 nm using a multimode reader (iD3 SpectraMax, Molecular devices). H₂O₂ concentration was calculated using standard H₂O₂ curves and rates were expressed as pmol H₂O₂/min/mg protein.