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Resveratrol or 32°C hypothermia applied during reperfusion after cardiac ischemia reduces mitochondrial translocation of p66shc
Author(s) -
Yang Meiying,
Stowe David F.,
Heisner James S.,
Aldakkak Mohammed,
Camara Amadou K.S.
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.678.18
Subject(s) - chemistry , ischemia , cytosol , hypothermia , mitochondrion , reperfusion injury , reactive oxygen species , phosphorylation , western blot , pharmacology , biochemistry , medicine , biology , gene , enzyme
Reactive oxygen species (ROS) are important in ischemia (I) and reperfusion (RP) injury. Mitochondria are sources of ROS via electron leak from complexes I and III. P66 shc , a splice variant of ShcA adaptor protein family, enhances mROS by oxidizing reduced cyt c and reducing O 2 to H 2 O 2 . Mild hypothermia or resveratrol (RES) reduce ROS during IRP. Although p66 shc ablation protected against cardiac IR injury, it is unknown if and when p66 shc is activated during IRP, and if inhibiting p66 shc activation on RP protects against IRP injury. To test this, we isolated and perfused guinea pig hearts with KR buffer for 55 min (time control, TC), or 20 min or 35 min ischemia, or 35 min ischemia plus 20 min RP, with or without hypothermia or RES (10 μM). We isolated heart tissue total cell lysates, mitochondria, and cytosol and measured levels of p66 shc by Western blot (WB) with anti‐SHC antibody and phosphorylation of Serine 36 of p66 shc by immunoprecipitation with anti‐SHC followed by WB with p66‐ Ser 36 antibody. We found that after RP mitochondrial p66 shc increased two fold above TC while phosphorylation of Ser 36 increased at 20 min ischemia, but decreased at 35 min ischemia, and increased again at 20 min RP in total cell lysates. Our results demonstrate that p66 shc is activated by IRP, that activated p66 shc translocates from cytosol to mitochondria, and that RES or hypothermia applied only during RP reduces mitochondrial p66 shc to TC levels.