Reperfusion after myocardial ischemia is associated with a rapid influx of calcium, leading to activation of various enzymes including calpain. Isolated perfused adult rabbit hearts subjected to global ischemia and reperfusion were studied. Calpain or a calpain-like activity was activated within 15 min after reperfusion, and preconditioning suppressed calpain activation. In contrast, caspase activation was not detected although cytochrome c was released after ischemia and reperfusion. The pro-apoptotic BH3-only Bcl-2 family member, Bid, was cleaved during ischemia/reperfusion in the adult rabbit heart. Recombinant Bid was cleaved by calpain to a fragment that was able to mediate cytochrome c release. The calpain cleavage site was mapped to a region within Bid that is extremely susceptible to proteolysis. These findings suggest that there is cross-talk between apoptotic and necrotic pathways in myocardial ischemia/reperfusion injury.Reperfusion after ischemia is accompanied by a rapid influx of calcium. Prevention of calcium influx is widely recognized in order to protect the myocardium. Preconditioning, in which a brief period of ischemia and reperfusion confers protection against a longer episode of ischemia, preserves tissue viability through attenuation of ionic fluxes (notably protons and calcium), preservation of energy charge (less consumption of ATP), and a variety of less well characterized enzymatic alterations (1-5). We have previously shown that preconditioning attenuates cytoplasmic acidification and calcium overload (3,6). A variety of calcium-dependent enzymes are activated after calcium influx, including the cysteine protease, calpain. Calpain has been implicated as a mediator of contractile dysfunction during ischemic injury, in part because of proteolysis of structural proteins (7,8). In addition, calpain has been reported to cleave Bax to an 18-kDa fragment that mediates cytochrome c release (9, 10), and calpain cleaves Bcl-x L to convert it to a pro-apoptotic form (11).Apoptosis has been reported to occur after ischemia/reperfusion injury, indicated by DNA fragmentation and TUNEL 1 staining (12-16). Preconditioning has been reported to reduce apoptosis (17). Caspase activation in ischemic and reperfused rat heart has been reported using an antibody that recognizes a neoepitope in activated caspase-3 (18). Other studies examining apoptosis in neonatal cardiomyocytes have described cytochrome c release and caspase activation (19,20). In this study we report that calpain is activated shortly after ischemia/reperfusion and that preconditioning attenuates calpain activation.In addition, we demonstrate a connection between calpain and mitochondrial dysfunction mediated by Bid.
MATERIALS AND METHODSHeart Perfusion and Ischemia/Reperfusion Protocol-Male New Zealand White rabbits (2.0 -2.5 kg) were anesthetized and a midsternal thoracotomy was performed. The heart was rapidly excised and mounted onto a Langendorff heart perfusion apparatus using a protocol adapted from Tsuchida et al. (21). The heart w...
Objective To determine whether inhibition of p38 mitogen-activated protein kinase (p38MAPK) reduces the pathogenicity of anti-neutrophil cytoplasmic autoantibodies (ANCAs) in vitro and in vivo. Methods
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