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...

ABSTRACT:The cytosolic factor p47-phox, encoded by the NCF1 gene, is an essential component of the phagocyte NADPH-oxidase system. Upon activation of this multicomponent system, p47-phox translocates to the membrane and participates in the electron transfer from NADPH to molecular oxygen. A deficiency or absence of p47-phox is the most common autosomal form of chronic granulomatous disease (CGD). We have cloned and characterized the NCF1 gene from four bacteriophage clones, a P1 clone and genomic DNA from normal individuals. The gene is 15,236 base pairs long and includes 11 exons. It is 98.6% homologous in sequence to at least one pseudogene that maps to the same region of chromosome 7q11.23. Slightly more than half (50.37%) of the wild-type NCF1 gene consists of repetitive elements. In particular, the density of Alu sequences is high (1.4 Alu/kb); there are 21 Alu repeats interspersed through 10 introns. These findings are consistent with the observation that recombination events between the wild-type gene and its highly homologous pseudogenes account for the majority of potentially lethal mutations in p47-phox-deficient chronic granulomatous disease. Analysis of 1.96 kb of sequence 5Ј of the start of translation revealed a high homology (99.6%) between wild-type and pseudogene clones. Characterization of NCF1 establishes a foundation for detailed molecular analysis of p47-phox-deficient CGD patients as well as for the study of the regulation of the NCF1 gene and pseudogenes, both of which are present as full-length transcripts in normal individuals.

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