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Fig. 6. Mitochondria buffer Ca²⁺ signals in atrial myocytes. (Aa) The experimental protocol. Cells were initially paced under control conditions. The stimulation was then halted and the cells incubated for 3 minutes with 20 µM antimycin + 20 µM oligomycin. After this period, the pacing was continued. The arrowheads indicate the timing of the depolarising pulses. The cell image (Ab) shows the position of the line that was used to generate the pseudo-linescan plots. (B) The response of the cell to electrical pacing under control conditions. The inset plots showing subsarcolemmal and central Ca²⁺ signals were obtained by sampling the linescan image along the regions shown by the red and black lines. (C) The reaction of the same cell following depolarisation of the mitochondrial membrane potential. The average cellular Ca²⁺ signal is shown as single black traces in Ba and Ca. The data are representative of 5 cells (from 3 hearts). (D) Ca²⁺ uptake into mitochondria. An image of a rhod-2-loaded atrial myocyte is depicted in Da. Ca²⁺ uptake into subsarcolemmal and central mitochondria was assessed by sampling the fluorescence changes within the regions indicated by the filled circles. The changes in mitochondrial Ca²⁺ are depicted by the correspondingly coloured traces for electrically evoked signals (Db) and a spontaneous Ca²⁺ wave (Dc), which occurred in the same cell. (Dd) Electrical stimulation only modestly increased rhod-2 fluorescence following application of 20 µM antimycin + 20 µM oligomycin for 3 minutes. The rhod-2 loading depicted in Da is representative of >20 cells from several independent experiments. The responses to electrical stimulation and generation of spontaneous Ca²⁺ waves are typical of six cells from three independent experiments.

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