Extended Data Figure 9.

Tracking DHE oxidation, NAD(P)H reduction state, and mitochondrial membrane potential in primary cardiomyocytes during in situ IR. a, Inhibition of mitochondrial complex I RET reduces DHE oxidation on reperfusion (n = 6; rotenone n =4). b,c Effect of manipulation of ischaemic succinate levels on NAD(P)H oxidation during early reperfusion (n = 3). Primary rat cardiomyocytes were subjected to 40 min ischaemia followed by reoxygenation and NAD(P)H reduction state was tracked throughout the experiment by measurement of NAD(P)H autofluorescence. Ischaemic buffer contained either no additions, 4 mM dimethyl malonate, or 4 mM dimethyl succinate. Average (b) and representative (c) traces from each condition are shown. The highlighted window in (c) indicates the period of the experiment expanded in detail in (b). d, Effect of inhibition of ischaemic succinate accumulation on mitochondrial membrane potential following late ischaemia (left panels) and early reperfusion (right panels). e,f, Primary rat cardiomyocytes were subjected to 40 min ischaemia and reoxygenation and mitochondrial membrane potential was tracked throughout the experiment by measurement of tetramethylrhoadmine (TMRM) fluorescence. Ischaemic buffer contained either no additions or 4 mM dimethyl malonate. (e), TMRM signal throughout the entire experiment. (f) TMRM signal during the transition from ischaemia to reoxygenation (n = 3). Data are shown as the mean ± s.e.m of at least three biological replicates. Replicates represent separate experiments on independent cell preparations.