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 (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 degrees C) with or without RR (10 microM) 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 (45)Ca(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 (45)Ca(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 (45)Ca(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 (45)Ca(2+) in the incubation buffer. 2) There was no difference in ATP-dependent (45)Ca(2+) uptake between nonischemic mitochondria and those separated from livers stored in UW-RR for 24 h. 3) (45)Ca(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 (45)Ca(2+) concentrations were greater than 1 microM.


CONCLUSIONS
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.
Belous
Andrey
A
Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee 37232-4753, USA.
Knox
Clayton
C
Nicoud
Ian B
IB
Pierce
Janene
J
Anderson
Christopher
C
Pinson
C Wright
CW
Chari
Ravi S
RS
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D003080:Cold Temperature; D007511:Ischemia / Q000378:metabolism*; D008099:Liver / Q000098:blood supply*; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D010477:Perfusion; D051381:Rats; D017207:Rats, Sprague-Dawley; D012430:Ruthenium Red / Q000494:pharmacology*; D012996:Solutions
D012996:Solutions; D012430:Ruthenium Red; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D002118:Calcium