Altered ATP-dependent mitochondrial Ca 2+ uptake in cold ischemia is attenuated by ruthenium red

Abstract

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 (Ca 2+), which has been linked to activation of proapoptotic factors. We hypothesized that cold ischemia increases mitochondrial Ca 2+ uptake in a concentration dependent fashion and that ruthenium red (RR) will attenuate these changes by inhibiting the mitochondrial Ca 2+ uniporter. Methods. Rat livers perfused with cold University of Wisconsin (UW) solution (4°C) with or without RR (10 μ m) 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 45Ca 2+ uptake was determined in the presence of ATP (5 m m), adenosine diphosphate (ADP), or adenosine 5′-β,γ-imidotriphosphate (AMP-PNP); variable concentrations of extramitochondrial 45Ca 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 45Ca 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 45Ca 2+ in the incubation buffer. 2) There was no difference in ATP-dependent 45Ca 2+ uptake between nonischemic mitochondria and those separated from livers stored in UW-RR for 24 h. 3) 45Ca 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 45Ca 2+ concentrations were greater than 1 μm. Conclusion. 1) Cold ischemia affects mitochondrial Ca 2+ handling, especially when it is challenged by high extramitochondrial Ca 2+ concentrations. 2) The addition of RR in preservation solution attenuates the effects of cold ischemia on mitochondrial Ca 2+ handling. 3) Inhibition of mitochondrial Ca 2+ uniporter with RR protects mitochondria from Ca 2+ overload at high Ca 2+ concentrations. These findings may offer a potentially effective strategy for prevention of ischemia-reperfusion injury in liver transplantation.