BackgroundThe mechanisms by which ischemic preconditioning (IP) inhibits mitochondrial permeability transition pore opening and, hence, ischemia–reperfusion injury remain unclear. Here we investigate whether and how mitochondria‐bound hexokinase 2 (mtHK2) may exert part of the cardioprotective effects of IP.Methods and ResultsControl and IP Langendorff‐perfused rat hearts were subject to ischemia and reperfusion with measurement of hemodynamic function and infarct size. Outer mitochondrial membrane (OMM) permeabilization after ischemia was determined by measuring rates of respiration and H2O2 production in the presence and absence of added cytochrome c in isolated mitochondria and permeabilized fibers. IP prevented OMM permeabilization during ischemia and reduced the loss of mtHK2, but not Bcl‐xL, observed in control ischemic hearts. By contrast, treatment of permeabilized fibers with glucose‐6‐phosphate at pH 6.3 induced mtHK2 loss without OMM permeabilization. However, metabolic pretreatments of the perfused heart chosen to modulate glucose‐6‐phosphate and intracellular pHi revealed a strong inverse correlation between end‐ischemic mtHK2 content and infarct size after reperfusion. Loss of mtHK2 was also associated with reduced rates of creatine phosphate generation during the early phase of reperfusion. This could be mimicked in permeabilized fibers after mtHK2 dissociation.ConclusionsWe propose that loss of mtHK2 during ischemia destabilizes mitochondrial contact sites, which, when accompanied by degradation of Bcl‐xL, induces OMM permeabilization and cytochrome c loss. This stimulates reactive oxygen species production and mitochondrial permeability transition pore opening on reperfusion, leading to infarction. Consequently, inhibition of mtHK2 loss during ischemia could be an important mechanism responsible for the cardioprotection mediated by IP and other pretreatments.

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 (␣, , or ␦ isoforms), glycogen synthase kinase 3␤, or Akt to the mitochondria following IP. In freeze-clamped hearts, changes in phosphorylation of GSK3␤, 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. Key Words: mitochondrial permeability transition Ⅲ preconditioning Ⅲ reperfusion injury Ⅲ protein phosphorylation Ⅲ oxidative stress A critical factor mediating reperfusion injury of the heart is the mitochondrial permeability transition pore (MPTP), the opening of which causes mitochondrial swelling with release of proapoptotic proteins and uncoupling of mitochondrial oxidative phosphorylation. The resulting ATP deprivation causes disruption of ionic homeostasis and contractile function and ultimately sarcolemma rupture and necrosis. 1 Inhibition of MPTP opening during reperfusion protects hearts from reperfusion injury. 1 Effective cardioprotection is also mediated by ischemic preconditioning (IP) before prolonged ischemia is initiated, 2 and this also involves inhibition of MPTP opening. [3][4][5][6] Extensive evidence points to protein kinase (PK)C playing a central role in IP, although controversy remains over which PKC isoform(s) are involved and their translocation to mitochondria. 7,8 The strongest evidence implicates PKC because PKC-knockout mice do not exhibit IP and transgenic mice with cardiac-specific overexpression of PKC or expression of a PKC activator are protected from reperfusion injury. 8 Several studies have reported PKC translocation to th...