Calpain and mitochondria in ischemia/reperfusion injury.
Studies of ischemia/reperfusion (I/R) injury and preconditioning have shown that ion homeostasis, particularly calcium homeostasis, is critical to limiting tissue damage. However, the relationship between ion homeostasis and specific cell death pathways has not been investigated in the context of I/R. Previously we reported that calpain cleaved Bid in the absence of detectable caspase activation (1). In this study, we have shown that an inhibitor of the sodium/hydrogen exchanger prevented calpain activation after I/R. Calpain inhibitors prevented cleavage of Bid as well as the downstream indices of cell death, including DNA strand breaks, creatine kinase (CK) release, and infarction measured by triphenyl tetrazolium chloride (TTC) staining. In contrast, the broad spectrum caspase inhibitor IDN6734 was not protective in this model. To ascertain whether mitochondrial dysfunction downstream of these events was a required step, we utilized a peptide corresponding to residues 4-23 of Bcl-x(L) conjugated to the protein transduction domain of HIV TAT (TAT-BH4), which has been shown to protect mitochondria against Ca2+-induced deltaPsi(m) loss (2). TAT-BH4 attenuated CK release and loss of TTC staining, demonstrating the role of mitochondria and a pro-apoptotic Bcl-2 family member in the process leading to cell death. We propose the following pathway. (i) Reperfusion results in sodium influx followed by calcium accumulation. (ii) This leads to calpain activation, which in turn leads to Bid cleavage. (iii) Bid targets the mitochondria, causing dysfunction and release of pro-apoptotic factors, resulting in DNA fragmentation and death of the cell. Ischemia/reperfusion initiates a cell death pathway that is independent of caspases but requires calpain and mitochondrial dysfunction.
Chen
Min
M
Scripps Research Institute and The Burnham Institute, La Jolla, California 92037, USA.
Won
Dong-Jun
DJ
Krajewski
Stan
S
Gottlieb
Roberta A
RA
D000818:Animals; D051023:BH3 Interacting Domain Death Agonist Protein; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D004396:Coloring Agents / Q000494:pharmacology; D003402:Creatine Kinase / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D053938:DNA Fragmentation; D006321:Heart / Q000502:physiology; D008297:Male; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*; D008954:Models, Biological; D009336:Necrosis; D011485:Protein Binding; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury; D013778:Tetrazolium Salts / Q000494:pharmacology; D013997:Time Factors
D051023:BH3 Interacting Domain Death Agonist Protein; C494816:Bid protein, rat; D002352:Carrier Proteins; D004396:Coloring Agents; D013778:Tetrazolium Salts; C009591:triphenyltetrazolium; D004247:DNA; D003402:Creatine Kinase; D002154:Calpain; D002118:Calcium