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 pre-ischemic 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 min global normothermic ischemia or 3 min 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. Nor did either Western blotting or 2-D fluorescence difference gel electrophoresis (DIGE) detect translocation of protein kinase C (α, ε or δ isoforms), glycogen synthase kinase 3β (GSK3β), or Akt to the mitochondria following IP. In freeze-clamped hearts changes in phosphorylation of GSK3β, Akt and AMP-activated protein kinase (AMPK) 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 might account for IP-mediated inhibition of MPTP. The signalling pathways mediating this effect and maintaining it during reperfusion are discussed.
Mitochondrial permeability transition; preconditioning; reperfusion injury; protein phosphorylation; oxidative stress
Urocortin (Ucn) protects hearts against ischemia and reperfusion injury whether given prior to ischemia or at reperfusion. Here we investigate the roles of protein kinase C, reactive oxygen species, and the mitochondrial permeability transition pore (MPTP) in mediating these effects. In Langendorff-perfused rat hearts, acute Ucn treatment improved hemodynamic recovery during reperfusion after 30 min global ischemia; this was accompanied by less necrosis (lactate dehydrogenase release) and MPTP opening (mitochondrial entrapment of [3H]-2-deoxyglucose). Ucn pre-treatment protected mitochondria against calcium-induced MPTP opening, but only if the mitochondria had been isolated from hearts after reperfusion. These mitochondria also exhibited less protein carbonylation, suggesting that Ucn decreases levels of oxidative stress. In isolated adult and neonatal rat cardiac myocytes, both acute (60 min) and chronic (16 hr) treatment with Ucn reduced cell death following simulated ischemia and re-oxygenation. This was accompanied by less MPTP opening as measured using tetramethylrhodamine methyl ester. The level of oxidative stress during reperfusion was reduced in cells which had been pre-treated with Ucn suggesting that this is the mechanism by which Ucn desensitizes the MPTP to reperfusion injury. Despite the fact that we could find no evidence that either PKCε or PKCα translocate to the mitochondria following acute Ucn treatment, inhibition of PKC with chelerythrine eliminated the effect of Ucn on oxidative stress. Our data suggests that acute Ucn treatment protects the heart by inhibiting MPTP opening. However, the mechanism appears to be indirect, involving a PKC-mediated reduction in oxidative stress.
reperfusion; mitochondria; oxygen radicals; peptide hormones
A prolonged period of ischaemia followed by reperfusion irreversibly damages the heart. Such reperfusion injury (RI) involves opening of the mitochondrial permeability transition pore (MPTP) under the conditions of calcium overload and oxidative stress that accompany reperfusion. Protection from MPTP opening and hence RI can be mediated by ischaemic preconditioning (IP) where the prolonged ischaemic period is preceded by one or more brief (2–5 min) cycles of ischaemia and reperfusion. Following a brief overview of the molecular characterisation and regulation of the MPTP, the proposed mechanisms by which IP reduces pore opening are reviewed including the potential roles for reactive oxygen species (ROS), protein kinase cascades, and mitochondrial potassium channels. It is proposed that IP-mediated inhibition of MPTP opening at reperfusion does not involve direct phosphorylation of mitochondrial proteins, but rather reflects diminished oxidative stress during prolonged ischaemia and reperfusion. This causes less oxidation of critical thiol groups on the MPTP that are known to sensitise pore opening to calcium. The mechanisms by which ROS levels are decreased in the IP hearts during prolonged ischaemia and reperfusion are not known, but appear to require activation of protein kinase Cε, either by receptor-mediated events or through transient increases in ROS during the IP protocol. Other signalling pathways may show cross-talk with this primary mechanism, but we suggest that a role for mitochondrial potassium channels is unlikely. The evidence for their activity in isolated mitochondria and cardiac myocytes is reviewed and the lack of specificity of the pharmacological agents used to implicate them in IP is noted. Some K+ channel openers uncouple mitochondria and others inhibit respiratory chain complexes, and their ability to produce ROS and precondition hearts is mimicked by bona fide uncouplers and respiratory chain inhibitors. IP may also provide continuing protection during reperfusion by preventing a cascade of MPTP-induced ROS production followed by further MPTP opening. This phase of protection may involve survival kinase pathways such as Akt and glycogen synthase kinase 3 (GSK3) either increasing ROS removal or reducing mitochondrial ROS production.
5HD, 5-hydroxydecanoate; AMPK, AMP activated protein kinase; ANT, Adenine nucleotide translocase; APD, Action potential duration; BCDH, branched chain 2-oxoacid dehydrogenase; CrK, creatine kinase; CsA, cyclosporin A; CyP, cyclophilin; Cx43, connexin43; GSK3, glycogen synthase kinase 3; IP, ischaemic preconditioning; KATP, ATP-dependent potassium channels; mitoKATP, mitochondrial ATP-dependent potassium channels; MCT1, monocarboxylate transporter 1; MPTP, mitochondrial permeability transition pore; PDH, pyruvate dehydrogenase; PDK1, phosphoinositide-dependent kinase 1; PI-3-kinase, phosphatidyl inositol 3 phosphate kinase; PKC, protein kinase C; PKG, cyclic GMP-dependent protein kinase; PPi, pyrophosphate; PPIase, peptidyl-prolyl cis-trans isomerase; PTEN, Phosphatase and TENsin homolog; ROS, reactive oxygen species; SfA, Sanglifehrin A; SUR, sulphohylurea receptor; VDAC, voltage activated anion channel; Mitochondrial permeability transition pore; Ischaemia; Reperfusion; ROS; Calcium; PKC; KATP channel