Peripheral benzodiazepine receptor-induced myocardial protection is mediated by inhibition of mitochondrial membrane permeabilization.

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 microM) and the Bcl-2 inhibitor ethyl-2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (HA14-1) (20 microM) 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.