1. The yield of mitochondria isolated from perfused hearts subjected to 30 min ischaemia followed by 15 min reperfusion was significantly less than that for control hearts, and this was associated with a decrease in the rates of ADP-stimulated respiration. 2. The presence of 0.2 microM 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 Ca(2+)-induced non-specific pore of the mitochondrial inner membrane opens during ischaemia and/or reperfusion of the isolated rat heart. The protocol involved loading the heart with 2-deoxy[3H]glucose ([3H]DOG), which will only enter mitochondria when the pore opens. Subsequent isolation of mitochondria demonstrated that [3H]DOG did not enter mitochondria during global isothermic ischaemia, but did enter during the reperfusion period. 4. The amount 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 microM did not prevent [3H]DOG becoming associated with the 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 [MeAla6]cyclosporin was shown to have a similar Ki to CsA on purified mitochondrial peptidyl-prolyl 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 could become CsA-insensitive under conditions of adenine nucleotide depletion and high matrix [Ca2+] such as may occur during the initial phase of reperfusion. The apparent increase in mitochondrial [3H]DOG in the CsA-perfused hearts is explained by the ability of the drug to stabilize pore closure and so decrease the loss of [3H]DOG from the mitochondria during their preparation.

No abstract

Mitochondrial Ca(2+) transport was initially considered important only in buffering of cytosolic Ca(2+) by acting as a "sink" under conditions of Ca(2+) overload. The main regulator of ATP production was considered to be the relative concentrations of high energy phosphates. However, work by Denton and McCormack in the 1970s and 1980s showed that free intramitochondrial Ca(2+) ([Ca(2+)](m)) activated dehydrogenase enzymes in mitochondria, leading to increased NADH and hence ATP production. This leads them to propose a scheme, subsequently termed a "parallel activation model" whereby increases in energy demand, such as hormonal stimulation or increased workload in muscle, produced an increase in cytosolic [Ca(2+)] that was relayed by the mitochondrial Ca(2+) transporters into the matrix to give an increase in [Ca(2+)](m). This then stimulated energy production to meet the increased energy demand. With the development of methods for measuring [Ca(2+)](m) in living cells that proved [Ca(2+)](m) changed over a dynamic physiological range rather than simply soaking up excess cytosolic [Ca(2+)], this model has now gained widespread acceptance. However, work by ourselves and others using targeted probes to measure changes in both [Ca(2+)] and [ATP] in different cell compartments has revealed variations in the interrelationships between these two in different tissues, suggesting that metabolic regulation by Ca(2+) is finely tuned to the demands and function of the individual organ.

Opening of the mitochondrial permeability transition pore (MPTP) is thought to be a critical event in mediating the damage to hearts that accompanies their reperfusion following prolonged ischaemia. Protection from reperfusion injury occurs if the prolonged ischaemic period is preceded by short ischaemic periods followed by recovery. Here we investigate whether such ischaemic preconditioning (IPC) is accompanied by inhibition of MPTP opening. MPTP opening in Langendorff-perfused rat hearts was determined by perfusion with 2-deoxy[ We demonstrate that IPC inhibits initial MPTP opening in hearts reperfused after 30 min global ischaemia, and subsequently enhances pore closure as hearts recover. However, MPTP opening in mitochondria isolated from IPC hearts occurred more readily than control mitochondria, implying that MPTP inhibition by IPC in situ was secondary to other factors such as decreased calcium overload and oxidative stress. Hearts perfused with cyclosporin A or sanglifehrin A, powerful inhibitors of the MPTP, also recovered better from ischaemia than controls (improved haemodynamic function and less lactate dehydrogenase release). However, the mitochondrial DOG entrapment technique showed these agents to be less effective than IPC at preventing MPTP opening. Our data suggest that protection from reperfusion injury is better achieved by reducing factors that induce MPTP opening than by inhibiting the MPTP directly.

Stimulation of mitochondrial oxidative metabolism by Ca2+ is now generally recognised as important for the control of cellular ATP homeostasis. Here, we review the mechanisms through which Ca2+ regulates mitochondrial ATP synthesis. We focus on cardiac myocytes and pancreatic β-cells, where tight control of this process is likely to play an important role in the response to rapid changes in workload and to nutrient stimulation, respectively. We also describe a novel approach for imaging the Ca2+-dependent regulation of ATP levels dynamically in single cells.