Mitochondrial calcium uptake regulates cold preservation-induced Bax translocation and early reperfusion apoptosis.

Anderson CD, Belous A, Pierce J, Nicoud IB, Knox C, Wakata A, Pinson CW, Chari RS
Am J Transplant. 2004 4 (3): 352-62

PMID: 14961987 · DOI:10.1111/j.1600-6143.2004.00357.x

Mitochondrial calcium (mCa + 2) overload occurs during cold preservation and is an integral part of mitochondrial-dependent apoptotic pathways. We investigated the role of mCa + 2 overload in cell death following hypothermic storage using HepG2 cells stored in normoxic-hypothermic (4 degrees C) or hypoxic (< 0.1% O2)-hypothermic Belzer storage solution. Cells were stored for 6 h, with or without 10 microM ruthenium red (mCa + 2 uniporter inhibitor) followed by rewarming in oxygenated media at 37 degrees C. Cytoplasmic cytochrome c levels were studied by Western analysis and by fluorescent microscopy after transfection of cytochrome c-GFP expression plasmid. Immunofluorescence determined the intracellular, spatio-temporal distribution of Bax, and TUNEL staining was used to evaluate cell death after 180 min of rewarming. Caspase activation was evaluated using Western analysis and a caspase 3 activity assay. Bax translocation, cytochrome c release, and early rewarming cell death occurred following hypothermic storage and were exacerbated by hypoxia. Caspase 3 activation did not occur following hypothermic storage. Blockade of mCa + 2 uptake prevented Bax translocation, cytochrome c release, and early rewarming cell death. These studies demonstrate that mCa + 2 uptake during hypothermic storage, both hypoxic and normoxic, contributes to early rewarming apoptosis by triggering Bax translocation to mitochondria and cytochrome c release.

MeSH Terms (17)

Apoptosis bcl-2-Associated X Protein Calcium Caspase 3 Caspases Cryopreservation Cytochromes c Hepatoblastoma Humans Indicators and Reagents Mitochondria Protein Transport Proto-Oncogene Proteins Proto-Oncogene Proteins c-bcl-2 Reperfusion Injury Ruthenium Red Tumor Cells, Cultured

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