MITOCHONDRIAL NONSPECIFIC PORES REMAIN CLOSED DURING CARDIAC ISCHEMIA, BUT OPEN UPON REPERFUSION

1. The yield of mitochondria isolated from perfused hearts subjected t o 30 min ischaemia followed by 15 min reperfusion was significantly le ss than that for control hearts, and this was associated with a decrea se in the rates of ADP-stimulated respiration. 2. The presence of 0.2 mu M cyclosporin A (CsA) in the perfusion medium during ischaemia and reperfusion caused mitochondrial recovery to return to control values, but did not reverse the inhibition of respiration. 3. A technique has been devised to investigate whether the Ca2+-induced non-specific por e of the mitochondrial inner membrane opens during ischaemia and/or re perfusion of the isolated rat heart. The protocol involved loading the heart with 2-deoxy[H-3]glucose ([H-3]DOG), which will only enter mito chondria when the pore opens. Subsequent isolation of mitochondria dem onstrated that [H-3]DOG did not enter mitochondria during global isoth ermic ischaemia, but did enter during the reperfusion period. 4. The a mount of [3H]DOG that entered mitochondria increased with the time of ischaemia, and reached a maximal Value after 30-40 min of ischaemia. 5 . CsA at 0.2 mu M did not prevent [H-3]DOG becoming associated with th e mitochondria, but rather increased it; this was despite CsA having a protective effect on heart function similar to that shown previously [Griffiths and Halestrap (1993) J. Mol. Cell. Cardiol. 25, 1461-1469]. 6. The non-immunosuppressive CsA analogue [MeAla(6)]cyclosporin was s hown to have a similar K-i to CsA on purified mitochondrial peptidyl-p rolyl cis-trans-isomerase and mitochondrial pore opening, and also to have a similar protective effect against reperfusion injury. 7. Using isolated heart mitochondria, it was demonstrated that pore opening cou ld become CsA-insensitive under conditions of adenine nucleotide deple tion and high matrix [Ca2+] such as may occur during the initial phase of reperfusion. The apparent increase in mitochondrial [H-3]DOG in th e CsA-perfused hearts is explained by the ability of the drug to stabi lize pore closure and so decrease the loss of [H-3]DOG from the mitoch ondria during their preparation.