Abstract
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 4′-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 ± 1% of the area at risk at 10 mg/kg versus 31 ± 3% in control; p < 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 Ca2+-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 μM) and the Bcl-2 inhibitor ethyl-2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (HA14-1) (20 μM) 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.
Footnotes
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This work was supported by Fondation de France Grant 2004004775. F.N.O. was supported by the Ministère de la Recherche et de la Technologie.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.124255.
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ABBREVIATIONS: PTP, permeability transition pore; I/R, ischemia-reperfusion; PBR, peripheral benzodiazepine receptor; SSR180575, 7-chloro-N,N,5-trimethyl-4-oxo-3-phenyl-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide; Ro5-4864, 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepine-2-one; CDZ, 4′-chlorodiazepam; LVDP, left ventricular developed pressure; dP/dtmax, maximal first derivative of ventricular pressure over time; CF, coronary flow; IPC, ischemic preconditioning; PK-11195 (PK), 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinolinecarboxamide; CAO, coronary artery occlusion; AAR, area at risk; AIF, apoptosis-inducing factor; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; HA14-1, ethyl-2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl-4H-chromene-3-carboxylate.
- Received April 11, 2007.
- Accepted July 18, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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