Mitochondrial Ca2+ uptake during simulated ischemia does not affect permeability transition pore opening upon simulated reperfusion

Objective: Reenergization of ischemic cardiomyocytes may be associated with acute necrotic cell death due in part to cytosolic Ca2+ overload and opening of a permeability transition pore (PTP) in mitochondria. It has been suggested that Ca2+ overload during ischemia primes mitochondria for PTP opening during reperfusion. We investigated the ability of mitochondria to uptake Ca2+ during simulated ischemia (SI) and whether this uptake determines PTP opening and cell death upon simulated reperfusion (SR). Methods: Rat heart mitochondria were submitted to either hypoxia (anoxic chamber) or to SI (respiratory inhibition, substrate depletion and acidosis) and subsequent SR. Mitochondrial Ca2+ uptake was monitored using Ca2+ microelectrodes after exposure to different [Ca2+] up to 25 mu M during SI, and PTP opening was assessed by quantification of mitochondrial swelling (changes in absorbance rate at 540 nm) and calcein release. Mitochondrial Ca2+ uptake (Rhod-2 fluorescence) and cytosolic Ca2+ rise (Fura-2 ratio fluorescence) were further investigated in HL-I cardiac myocytes submitted to SI/SR, and the effect of reducing mitochondrial Ca2+ load (with 25 PM ruthenium red) or blocking PTP opening (with 0.5 mu M cyclosporin A) on the rate of cell death was investigated in adult cardiomyocytes exposed to SI/SR. Results: SI induced a progressive dissipation of mitochondrial membrane potential (TMRE fluorescence); however, prior to the completion of depolarization, high levels of Ca2+ uptake were observed in mitochondria. SR induced PTP opening but this phenomenon was not influenced by the magnitude of mitochondrial Ca2+ uptake during previous SI. Blockade of the mitochondrial Ca2+ uniporter during SI in cardiomyocytes allenuated mitochondrial Ca2+ uptake but increased cytosolic Ca2+ overload and cell death upon subsequent SR. Conclusion: Mitochondrial Ca2+ uptake during SI buffers cytosolic Ca2+ overload but its magnitude appears not to be an important determinant of PTP opening upon subsequent SR. (c) 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.