Morphine has been shown to protect the myocardium against ischemia-reperfusion injury through inhibition of glycogen synthase kinase-3␤ (GSK-3␤). Given that GSK-3␤ is known to modulate the mitochondrial permeability transition pore (mPTP), we investigated the role of mPTP in the cardioprotective effect of morphine and the GSK-3␤ inhibitor SB216763 [SB; 3-(2,4-dichlorophenyl)-4(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione] during ischemia-reperfusion. Both morphine (0.3 mg/kg) and SB (0.6 mg/kg) reduced infarct size in a model of regional myocardial ischemia-reperfusion in rats (13 Ϯ 1 and 14 Ϯ 3% of the area at risk versus 33 Ϯ 4% in controls; p Ͻ 0.05). Morphine and SB protected the ischemic myocardium against Ca 2ϩ -induced mPTP opening as demonstrated by the increased capacity of mitochondria to retain Ca 2ϩ when they were isolated from the ischemic zone 10 min after the onset of reperfusion (59 Ϯ 8 and 66 Ϯ 3 versus 29.5 Ϯ 6 nmol Ca 2ϩ /mg ⅐ protein, respectively; p Ͻ 0.05). This was associated with a restoration of mitochondrial oxidative phosphorylation parameters. In isolated adult rat cardiomyocytes subjected to anoxiareoxygenation, morphine (2 M), SB (3 M), and the direct mPTP inhibitor cyclosporine A (3 M) delayed mPTP opening as assessed by the calcein loading Co 2ϩ -quenching technique. This was accompanied by an increase in cell survival as measured by nuclear staining with propidium iodide. These in vitro effects of morphine on inhibition of mPTP opening during anoxia-reoxygenation were suppressed by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin (0.1 M). These data indicate that the infarct-limiting effect of morphine and SB is linked by a cause-effect relationship, which leads to an increased mitochondrial resistance and inhibition of mPTP opening through the PI3-kinase pathway and subsequent inactivation of GSK-3␤.There are many investigations that suggest that the opening of mitochondrial permeability transition pore (mPTP) is involved in myocardial ischemia-reperfusion injury (Di Lisa and Bernardi, 2006;Halestrap, 2006;Javadov and Karmazyn, 2007) and that raising the threshold for mPTP opening is a relevant strategy to provide cardioprotection (Tissier et al., 2008).

Opening of the permeability transition pore (PTP) is a key event in ischemia-reperfusion injury and several ligands of the peripheral benzodiazepine receptor (PBR), a mitochondrial outer membrane protein possibly associated with PTP, have been demonstrated as potent cardioprotective agents. Here, we investigated the mechanisms by which the specific PBR ligand 4Ј-chlorodiazepam (CDZ) protected the myocardium against ischemia-reperfusion. In either global or regional models of myocardial ischemia-reperfusion in rats, CDZ reduced infarct size in a dose-dependent manner (e.g., 11 Ϯ 1% of the area at risk at 10 mg/kg versus 31 Ϯ 3% in control; p Ͻ 0.05) and to a similar extent as ischemic or diazoxide-induced preconditioning. CDZ (10 mg/kg) reduced apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling staining), restored mitochondrial recovery, improved oxidative phosphorylation parameters, and reduced mitochondrial membrane permeabilization with inhibition of cytochrome c and apoptosis-inducing factor releases. CDZ increased the resistance of mitochondria to Ca 2ϩ -induced PTP opening. All these cardioprotective effects of CDZ were associated with an improved stabilization of the association of Bcl-2 with the mitochondrial membrane and inhibition of the association of a cytosolic fragment of Bax, occurring during ischemia-reperfusion, with the outer mitochondrial membrane. In addition, the PTP opener atractyloside (20 M) and the Bcl-2 inhibitor ethyl-2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (HA14-1) (20 M) abrogated CDZ-induced reduction of infarct size. These results demonstrate that PBR occupancy by CDZ renders the heart more resistant to ischemia-reperfusion injury by limiting mitochondrial membrane permeabilization. This is due to a reorganization of the balance between pro-and antiapoptotic proteins of the Bcl-2 family proteins at the level of mitochondrial membranes.It is now well established that during myocardial ischemiareperfusion, pathological signals converge to the mitochondria and induce its membrane permeabilization, a phenomenon that ultimately leads to cell death (Gottlieb, 2003;Juhaszova et al., 2004;Lundberg and Szweda, 2004). Two mechanisms have been described to explain mitochondrial membrane permeabilization (Green, 2005). The first mechanism concerns permeabilization of the outer membrane without alteration of the inner membrane, which can be facilitated by the proapoptotic members of the Bcl-2 family proteins. The second mechanism involves both the inner and the outer membranes, and it corresponds to the opening of the permeability transition pore (PTP), a multiprotein structure whose opening leads to mitochondrial swelling and subsequent release of apoptogenic factors (Zoratti and Szabo, 1995).Through mitochondrial membrane permeabilization, ischemia-reperfusion (I/R) induces the release of cell death effectors and ultimately results in the loss of mitochondrial functions that are fundamental for cell survival. Therefore, every strat...

The presence of gene lesions in blood coagulation factor X (FX) was investigated in eight FX-deficient patients with severe bleeding symptoms, originating from five unrelated Algerian families (FX coagulant activity <1%, FX antigen ranging from 2% to 16%). A missense mutation (p.Phe31Ser) in the Gla domain was found in homozygous form for all patients but one, who is a compound heterozygote for the Phe31Ser mutation and for a non-sense mutation, Tyr130Term in EGF-2 domain. The haplotypes of FX alleles were determined by the following allelic variants located in the promoter: g.1323_1330delTTGTGA (A1/A2), g.1449T>C, g.1451C>A, upstream to exon 3: g.17257C>T and downstream to exon 3: g.17396A>C. The A1-C-A-T-C haplotype was found on each allele bearing the Phe31Ser mutation in the eight FX deficient patients contrasting with its low frequency (8%) in a control Algerian population (in which the Phe31Ser substitution was absent). The patients came from the same geographical area of Algeria (5/8 are certainly from Kabyle origin) and the haplotype analysis suggests a founder effect. Transient expression study reveals that, for the mutant FX-Phe31Ser, FX antigen level was 60% in conditioned media and 140% in cell lysates compared with the wild type FX. The partial retention and intracellular accumulation of the mutant FX might be due to impaired folding and/or conformational changes, and the discrepancies observed between the FX antigen level in COS-7 cell supernatant (60%) and in the patients plasma (2-16%) to an in vivo increased clearance of the secreted unstable FX mutant.