Effect of LPI on mitochondrial ROS production in primary cardiomyocytes. Primary cardiomyocytes were preincubated for 24 hours in medium with LPI-containing serum from an iron-overloaded patient (A) or with the same serum depleted of LPI by addition of equimolar DFO (B). The final LPI in panel B was less than 0.5 μM and in panel A 2.0 μM. After washing, the cells were loaded with 50 μM DHR for 10 minutes at 37°C and washed, and ROS production was monitored by epifluorescence microscopy and recorded at 2-minute intervals. The base line fluorescence was established, and 50 μM H₂O₂ was added at 4 minutes (indicated by arrow). The montage depicts snapshots of phase contrast images (A: serum with LPI, and B: serum depleted of LPI) and 5 (of 17) epifluorescence images of the same time sequence before and after H₂O₂ addition, taken at 2, 10, 18, 26, and 34 minutes. (C) Scatter plots of A and B showing average fluorescence density of 3 to 5 cells in each field indicating treatment with 30% LPI-containing human serum (curve A; • or 30% human serum depleted of LPI (curve B; ○. The line graph ΔLPI (▪) is the difference between curve B and curve A. (D) Relative ROS production levels in cardiomyocytes (determined with DHR and fluorescence microscopy) after incubation overnight with FAC (100 μM) in growth medium containing 10% fetal calf serum and 10% horse serum or in growth medium containing 30% human LPI-containing serum (□). The growth media were supplemented with the indicated concentrations (10, 20, or 100 μM) of chelators either present during the 24-hour preincubation (before, ▨) or added only 20 minutes before ROS determination (after, ▪). The dotted line represents the basal level of ROS production (27 au, arbitrary units of fluorescence) obtained by addition of excess iron chelator (200 μM DFR).