title: Inhibition of mitochondrial permeability transition pore opening by ischemic preconditioning is probably mediated by reduction of oxidative stress rather than mitochondrial protein phosphorylation.
abstract: Inhibition of mitochondrial permeability transition pore (MPTP) opening at reperfusion is critical for cardioprotection by ischemic preconditioning (IP). Some studies have implicated mitochondrial protein phosphorylation in this effect. Here we confirm that mitochondria rapidly isolated from preischemic control and IP hearts show no significant difference in calcium-mediated MPTP opening, whereas IP inhibits MPTP opening in mitochondria isolated from IP hearts following 30 minutes of global normothermic ischemia or 3 minutes of reperfusion. Analysis of protein phosphorylation in density-gradient purified mitochondria was performed using both 2D and 1D electrophoresis, with detection of phosphoproteins using Pro-Q Diamond or phospho-amino-specific antibodies. Several phosphoproteins were detected, including voltage-dependent anion channels isoforms 1 and 2, but none showed significant IP-mediated changes either before ischemia or during ischemia and reperfusion, and neither Western blotting nor 2D fluorescence difference gel electrophoresis detected translocation of protein kinase C (alpha, epsilon, or delta isoforms), glycogen synthase kinase 3beta, or Akt to the mitochondria following IP. In freeze-clamped hearts, changes in phosphorylation of GSK3beta, Akt, and AMP-activated protein kinase were detected following ischemia and reperfusion but no IP-mediated changes correlated with MPTP inhibition or cardioprotection. However, measurement of mitochondrial protein carbonylation, a surrogate marker for oxidative stress, suggested that a reduction in mitochondrial oxidative stress at the end of ischemia and during reperfusion may account for IP-mediated inhibition of MPTP. The signaling pathways mediating this effect and maintaining it during reperfusion are discussed.
authors: Department of Biochemistry and the Bristol Heart Institute, University of Bristol, Bristol BS8 1TD, United Kingdom.
authors: Samantha J
authors: SJ
authors: Clarke
authors: Igor
authors: I
authors: Khaliulin
authors: Manika
authors: M
authors: Das
authors: Joanne E
authors: JE
authors: Parker
authors: Kate J
authors: KJ
authors: Heesom
authors: Andrew P
authors: AP
authors: Halestrap
mesh_terms: D055372:AMP-Activated Protein Kinases; D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D019157:Ischemic Preconditioning, Myocardial; D008297:Male; D008929:Mitochondria, Heart / Q000201:enzymology / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000083162:Mitochondrial Permeability Transition Pore; D024101:Mitochondrial Proteins / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism; D015428:Myocardial Reperfusion Injury / Q000378:metabolism* / Q000517:prevention & control; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism*; D018384:Oxidative Stress; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D050050:Protein Carbonylation; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D040901:Proteomics / Q000379:methods; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors
chemical_list: D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D024101:Mitochondrial Proteins; D009097:Multienzyme Complexes; D010750:Phosphoproteins; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D011493:Protein Kinase C; D038362:Glycogen Synthase Kinase 3; D055372:AMP-Activated Protein Kinases