Altered ATP-dependent mitochondrial Ca2+ uptake in cold ischemia is attenuated by ruthenium red

Background. Graft dysfunction as a result of preservation injury remains a major clinical problem in liver transplantation. This is related in part to accumulation of mitochondrial calcium (Ca2+), which has been linked to activation of proapoptotic factors. We hypothesized that cold ischemia increases mitochondrial Ca2+ uptake in a concentration dependent fashion and that ruthenium red (RR) will attenuate these changes by inhibiting the mitochondrial Ca2+ uniporter. Methods. Rat livers perfused with cold University of Wisconsin (UW) solution (4degreesC) with or without RR (10 muM) via the portal vein (n = 3 per group) were processed immediately (no ischemia) or after 24 h cold-storage (24 h cold ischemia). Mitochondria were separated by differential centrifugation, and adenosine triphosphate (ATP)-dependent Ca-45(2+) uptake was determined in the presence of ATP (5 mm), adenosine diphosphate (ADP), or adenosine 5'-beta,gamma-imidotriphosphate (AMP-PNP); variable concentrations of extramitochondrial Ca-45(2+) were used. All measurements were performed in triplicate. Student's t test with P < 0.05 was taken as significant. Results. Our data demonstrate the following: 1) ATP-dependent Ca-45(2+) uptake in mitochondria separated from livers following 24 h of cold ischemia in UW alone was higher than in mitochondria isolated from non-ischemic livers; the increased uptake was dependent on the concentration of Ca-45(2+) in the incubation buffer. 2) There was no difference in ATP-dependent Ca-45(2+) uptake between non-ischemic mitochondria and those separated from livers stored in UW-RR for 24 h. 3) Ca-45(2+) uptake in mitochondria from livers subjected to 24 h of cold ischemia in UW-RR was significantly lower compared to those from livers stored in UW alone when Ca-45(2+) concentrations were greater than 1 mum. Conclusion. 1) Cold ischemia affects mitochondrial Ca2+ handling, especially when it is challenged by high extramitochondrial Ca" concentrations. 2) The addition of RR in preservation solution attenuates the effects of cold ischemia on mitochondrial Ca2+ handling. 3) Inhibition of mitochondrial Ca2+ uniporter with RR protects mitochondria from Ca2+ overload at high Ca2+ concentrations. These findings may offer a potentially effective strategy for prevention of ischemia-reperfusion injury in liver transplantation. (C) 2003 Elsevier Inc. All rights reserved.