We investigated the role of pH,
reactive oxygen species (ROS), Ca2+, and the mitochondrial permeability transition (MPT) in pH-dependent
ischemia-reperfusion injury to adult rat myocytes. Myocytes were incubated in anoxic Krebs-Ringer-
HEPES buffer at pH 6.2 for 3 h to simulate
ischemia. To simulate reperfusion, myocytes were reoxygenated at pH 6.2 or 7.4 for 2 h. Some myocytes were treated with MPT blockers (
cyclosporin A and N-methyl-4-
isoleucine cyclosporin) and
antioxidants (
desferal, diphenylphenylene
diamine, and 2-mercaptopropionyl
glycine). Mitochondrial membrane potential, inner membrane permeabilization, and ROS formation were imaged with
tetramethylrhodamine methyl ester,
calcein, and chloromethyldichlorofluorescein diacetate, respectively. For Ca2+ imaging, myocytes were coloaded with
rhod-2 and
fluo-4 to evaluate mitochondrial and cytosolic Ca2+, respectively. After 10 min of reperfusion at pH 7.4,
calcein redistributed across the mitochondrial inner membrane, an event preceded by mitochondrial ROS formation and accompanied by hypercontracture, mitochondrial depolarization, and then cell death. Acidotic reperfusion,
antioxidants, and MPT blockers each prevented the MPT, depolarization, hypercontraction, and cell killing.
Antioxidants, but neither MPT blockers nor acidotic reperfusion, inhibited ROS formation after reperfusion. Furthermore, anoxic reperfusion at pH 7.4 prevented cell death. Both mitochondrial and cytosolic Ca2+ increased during
ischemia but recovered in the first minutes of reperfusion. Mitochondrial and cytosolic Ca2+ overloading again occurred late after reperfusion. This late Ca2+ overloading was blocked by MPT inhibition. Intramitochondrial Ca2+ chelation by cold loading/warm incubation of
BAPTA did not prevent cell death after reperfusion. In conclusion, mitochondrial ROS, together with normalization of pH, promote MPT onset and subsequent myocyte death after reperfusion. In contrast, Ca2+ overloading appears to be the consequence of bioenergetic failure after the MPT and is not
a factor promoting MPT onset.