Opening of the mitochondrial permeability transition pore causes depletionof mitochondrial and cytosolic NAD(+) and is a causative event in the death of myocytes in postischemic reperfusion of the heart

The opening of the mitochondrial permeability transition pore (PTP) has bee n suggested to play a key role in various forms of cell death, but direct e vidence in intact tissues is still lacking. We found that in the rat heart, 92% of NAD(+) glycohydrolase activity is associated with mitochondria. Thi s activity was not modified by the addition of Triton X-100, although it wa s abolished by mild treatment with the protease Nagarse, a condition that d id not affect the energy-linked properties of mitochondria. The addition of Ca2+ to isolated rat heart mitochondria resulted in a profound decrease in their NAD(+) content, which followed mitochondrial swelling. Cyclosporin A (CsA), a PTP inhibitor, completely prevented NAD(+) depletion but had no ef fect on the glycohydrolase activity. Thus, in isolated mitochondria PTP ope ning makes NAD(+) available for its enzymatic hydrolysis, Perfused rat hear ts subjected to global ischemia for 30 min displayed a 30% decrease in tiss ue NAD(+) content, which was not modified by extending the duration of isch emia, Reperfusion resulted in a more severe reduction of both total and mit ochondrial contents of NAD(+), which could be measured in the coronary effl uent together with lactate dehydrogenase. The addition of 0.2 muM CsA or of its analogue MeVal-4-Cs (which does not inhibit calcineurin) maintained hi gher NAD(+) contents, especially in mitochondria, and significantly protect ed the heart from reperfusion damage, as shown by the reduction in lactate dehydrogenase release. Thus, upon reperfusion after prolonged ischemia, PTP opening in the heart can be documented as a CsA-sensitive release of NAD(), which is then partly degraded by glycohydrolase and partly released when sarcolemmal integrity is compromised. These results demonstrate that PTP o pening is a causative event in reperfusion damage of the heart.