Calpain and Mitochondria in Ischemia/Reperfusion Injury

Abstract

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 Ca 2؉ -induced ⌬⌿ 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.

Anthracyclines Induce Calpain-dependent Titin Proteolysis and Necrosis in Cardiomyocytes

“…Whereas caspase-3 has been linked to degradation of small myofilament proteins (62), we did not find a role for caspase-3 in doxorubicin-induced titin degradation. Consistent with our results, Chen et al (63) reported a reduction in CK release and a decrease in infarct size following calpain but not caspase inhibition in the ischemic reperfused heart, although no clear distinction was made between necrotic and apoptotic cell death.…”

“…At the end of the experimental protocol, myocytes were harvested and sonicated in calcium-free assay buffer containing 63 10 mmol/liter 2-mercaptoethanol, pH 7.3) in a 96-well fluorescent plate. Samples were incubated at 37°C for 10 min, and N-succinyl-Leu-LeuVal-Tyr-7-amido-4-methylcoumarin was added to the samples for a final concentration of 50 mol/liter.…”

Calpain in Atrial Fibrillation: Friend or Foe?

“…This channel has been suggested to preserve calcium homeostasis and would therefore limit calpain activation [16]. Indeed, calpain inhibition was protective in models of ischemia/reperfusion which suggests that pore opening, if it occurs, is downstream of calpain activation [17]. The study of Yue Li et al implies that similar mechanisms apply during AF.…”

Calpastatin Exon 1B-Derived Peptide, a Selective Inhibitor of Calpain: Enhancing Cell Permeability by Conjugation with Penetratin

“…Bordone and Campbell, 2002;Rutledge and Whiteheart, 2002;Chen et al, 2002, among many others). The new selective and cell-permeable inhibitor described here may help to confirm or question results obtained with less specific inhibitors and to discriminate between the effects of calpain and other cellular proteases (Yamashima, 2000).…”