A subpopulation of mitochondria prevents cytosolic calcium overload in endothelial cells after cold ischemia/reperfusion

Background. Calcium represents a key mediator of cold ischemia/reperfusion (CIR) injury presumably by affecting mitochondrial function. In this study, we investigated cellular and mitochondrial changes of calcium homeostasis In sublethally damaged human endothelial cells. Methods, Changes in cellular and mitochondrial calcium concentrations were studied after cold ischemia in University of Wisconsin solution for 12 hr a nd reperfusion in ringer solution. Cytosolic-free calcium concentration ([C a2+](c)) and mitochondrial-free calcium content ([Ca2+](m)) were analyzed b y fura-2 and rhod-2 fluorescence, respectively. Pretreatment of cells with ruthenium red (RR) or a H+-ionophore was used to inhibit mitochondrial calc ium uptake. Mitochondrial membrane potential (Delta Psi (m)) was measured b y 5,5',6,6'-tetrachloro- 1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iod ide and 3,3'-dihexyloxacarbocyanine iodide fluorescence. Results. Twelve-hr cold ischemia did not induce apoptosis in endothelial ce lls. In such sublethally damaged cells, [Ca2+](c) rose from approximately 2 0 nmol/L after cold ischemia to approximately 120 nmol/L during reperfusion , Pretreatment with RR leads to an approximately 5-fold rise in [Ca2+](c). Image analysis revealed a significant increase of [Ca2+], in a subpopulatio n of mitochondria during reperfusion. This was not the case in RR-pretreate d cells. Delta Psi (m) decreased significantly during cold ischemia and was sustained during reperfusion, The loss of Delta Psi (m), can be related to a reduced portion of mitochondria exhibiting high Delta Psi (m). Conclusions, Our results suggest that cytosolic calcium influx during CIR i s buffered by a selective portion of mitochondria in human umbilical vein e ndothelial cells. These mitochondria protect cells against cytosolic calciu m overload and probably against subsequent cell injury.