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 microM 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-1 cardiac myocytes submitted to SI/SR, and the effect of reducing mitochondrial Ca2+ load (with 25 microM ruthenium red) or blocking PTP opening (with 0.5 microM 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 attenuated mitochondrial Ca2+ uptake but increased cytosolic Ca2+ overload and cell death upon subsequent SR.


CONCLUSIONS
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.
Ruiz-Meana
Marisol
M
Laboratorio de Cardiología Experimental, Hospital Vall d'Hebron, Barcelona, Spain.
Garcia-Dorado
David
D
Miró-Casas
Elisabet
E
Abellán
Arancha
A
Soler-Soler
Jordi
J
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D016923:Cell Death; D002460:Cell Line; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D008856:Microscopy, Fluorescence; D008929:Mitochondria, Heart / Q000378:metabolism*; D008933:Mitochondrial Swelling; D017202:Myocardial Ischemia / Q000378:metabolism; D015428:Myocardial Reperfusion Injury / Q000378:metabolism*; D051381:Rats; D012430:Ruthenium Red / Q000494:pharmacology; D050994:Voltage-Dependent Anion Channels / Q000378:metabolism*
D002121:Calcium Channel Blockers; D050994:Voltage-Dependent Anion Channels; D012430:Ruthenium Red; D016572:Cyclosporine; D002118:Calcium