Early reperfusion apoptosis is initated by mitochondrial calcium uptake during cold preservation

Abstract

Introduction: Mitochondrial calcium (mCa+2) overload occurs during cold preservation. In other settings, mCa+2 overload has been linked to permeability transition pore formation and release of mitochondrial proapoptotic proteins such as cytochrome-c (cyt-c). We hypothesize that mCa+2 overload during cold ischemia and reperfusion (CIR) contributes to cellular injury by induction of apoptosis during the early reperfusion period. Methods: HepG2 cells were plated in UW with or without 10 μM ruthenium red (RR, a mitochondrial Ca+2 uniporter inhibitor) and subjected to cold ischemic storage for 6 hours. Cells were subsequently rewarmed with oxygenated media at 37°C to simulate reperfusion. To study the intracellular, spatio-temporal distribution of cyt-c, cells were plated on glass coverslips, transfected with a green fluorescence protein-tagged cyt-c cDNA construct (GFP-cyt-c), and then subjected to CIR as above. Cytoplasmic extraction (for western analysis) or cell fixation (for fluorescent microscopy) was performed at 0, 30, 60, 120, and 180 minutes following reperfusion. To evaluate early reperfusion apoptosis, TUNEL staining was performed following 180 min of rewarming. Controls were untreated HepG2 cells, and cold normoxic stored cells. Results: 1) Following cold ischemia, western analysis demonstrated an increase in cytoplasmic cyt-c (p = 0.01), which was significantly decreased by RR treatment (p = 0.05). 2) CIR resulted in release of GFP-cyt-c from mitochondria into the cytoplasm, and this intracellular redistribution was prevented by RR following cold ischemia and throughout rewarming. 3) RR decreased the fraction of apoptotic cells following 180 min of rewarming from 45% to 16% (p = 0.001). Conclusions: These data indicate that hypoxic cold storage leads to a cyt-c release from mitochondria which is attenuated by blockade of mCa+2 uptake. Prevention of mCa+2 overload by RR also caused a significant decrease in apoptosis during early reperfusion. Together, these results indicate that mCa+2 overload during cold storage is a precursor to cellular proapoptotic cascade activation following reperfusion.