Both K_ATP channel opening drugs and ischaemic preconditioning have been suggested to protect the ischaemic heart by acting on K_ATP channels in the inner mitochondrial membrane, partially uncoupling the proton gradient and dissipating mitochondrial membrane potential (▓Dgr│P) (O’Rourke, 2000). We have previously shown that pretreatment of isolated rat hearts with low doses of the mitochondrial uncoupler FCCP (optimal concentration 100 nM), which similarly dissipates ▓Dgr│P, exerts cardioprotective effects analogous to K_ATP channel openers. In isolated cells, 100 nM FCCP oxidises mitochondrial flavoproteins by 85 % (as assessed by autofluorescence). In this study we investigated whether FCCP pretreatment mediates its protective effects via a mitochondrial-derived oxidant stress or by depleting ATP and indirectly opening mitochondrial K_ATP channels.

Hearts (n = 6 per group) were isolated from anaesthetised (150 mg kg^-1 pentabarbitone I.P.) Wistar rats according to UK regulations. Hearts were Langendorff-perfused (37 °C, perfusion pressure 73 mmHg) for 20 min and contractile function assessed via a left ventricle balloon. Perfusion was switched to constant flow with Krebs (Control), FCCP (100 nM), FCCP+NAC (4 mM) or NAC alone, for 5 min prior to 25 min zero-flow global ischaemia and functional recovery assessed on reperfusion. Data are means ± S.E.M. and differences were assessed using one-way ANOVA followed by a Dunnett’s test.

FCCP pretreatment improved post-ischaemic functional recovery after 40 min reperfusion (58 ± 5 % vs. Control 33 ± 4 %: P < 0.05). However, NAC abolished this effect (14 ± 5 %: P < 0.05 vs. FCCP) and decreased recovery when compared with Control+NAC (30 ± 5 %: P < 0.05). NAC alone had no effect on recovery (cf. Control). Pretreatment of hearts with the K_ATP channel blockers 5-hydroxydecanoate (100 µM) or glibenclamide (1 µM) did not block the protection afforded by FCCP. To determine whether ATP synthesis was impaired by FCCP pretreatment, separate experiments using NMR spectroscopy were performed. Isolated rat hearts (n = 6 per group) were Langendorff perfused (37 °C) within an NMR spectrometer and [ATP] measured throughout the FCCP and Control protocols described above. ATP levels did not differ between the two groups during the 5 min pretreatment, nor did lactate production increase, indicating there was no switch to glycolytic metabolism.

In conclusion, in the isolated rat heart, partial mitochondrial uncoupling with low-dose FCCP may induce an oxidant stress which then activates unknown downstream effectors that in turn protect the heart against ischaemic injury. This cardioprotection is not mediated via the depletion of cellular ATP or mitochondrial K_ATP channel activation.Support from the British Heart Foundation is gratefully acknowledged.

O’Rourke, B. (2000). Circ. Res. 87, 845-855.