Opening of the mitochondrial permeability transition pore causes matrix expansion and outer membrane rupture in Fas-mediated hepatic apoptosis in mice

Although Fas stimulation has been reported to cause outer mitochondrial mem brane rupture in Jurkat cells, the mechanism of this effect is debated, and it is not known if outer membrane rupture also occurs in hepatocyte mitoch ondria, We studied the in vivo effects of Fas stimulation on ultrastructura l lesions and mitochondrial function in mice. Four hours after administrati on of an agonistic anti-Fas antibody (8 mu g/animal), caspase activity incr eased 5.4-fold. Nuclear DNA showed internucleosomal fragmentation, whereas supercoiled mitochondrial DNA was replaced by circular and linear forms. Mi tochondrial cytochrome c was partly released into the cytosol. Ultrastructu rally, mitochondrial lesions were observed in both apoptotic hepatocytes (w ith nuclear chromatin condensation/fragmentation) and nonapoptotic hepatocy tes (without nuclear changes). In nonapoptotic cells, outer mitochondrial m embrane rupture allowed herniation of the inner membrane and matrix through the outer membrane gap. In apoptotic hepatocytes, the matrix became electr on-lucent and no longer protruded through the outer membrane gap. Mitochond ria clustered around the nucleus, whereas rough endoplasmic reticulum ciste rnae became peripheral. In liver mitochondria isolated after Fas stimulatio n, the membrane potential decreased, whereas basal respiration increased. P retreatment with either z-VAD-fmk tan inhibitor of caspases) or cyclosporin A (a permeability transition inhibitor) totally or mostly prevented mitoch ondrial outer membrane rupture, membrane potential decrease, cytochrome c r elease, and apoptosis. In conclusion, in vivo Pas stimulation causes caspas e activation, mitochondrial permeability transition (decreasing the membran e potential and increasing basal respiration), mitochondrial matrix expansi on las shown by matrix herniation), outer mitochondrial membrane rupture, a nd cytochrome c release.