Calpain is activated during hypoxic myocardial cell injury.

Cell death during hypoxia rose to 80% after 6 h. Calpain activity increased to 4 units during hypoxia, much higher than the 0.7 units seen in aerobic condition at 6 h. This activity was markedly inhibited by calpain-specific inhibitor I (n-acetyl-leucyle-leucyle-norleucinal). beta-Adrenergic blocking agents and calcium antagonists suppressed the calpain activity and decreased cell death during hypoxia. On the other hand, alpha-adrenergic blocking agents did not affect calpain activity and cell death under hypoxic conditions. These results prove that beta-adrenergic blocking agents and calcium antagonists prevent protein degradation during hypoxic cell injury.     

Calpain-mediated proteolytic cleavage of troponin I induced by hypoxia or metabolic inhibition in cultured neonatal cardiomyocytes

While ischemic damage to myofibrillar proteins is thought to be responsible in part for depressed cardiac function, the relation between myofilament protein breakdown and chronic hypoxia has not been defined. We previously characterized a chemical hypoxia model of neonatal cardiomyocytes mediated by 1 mM azide that exhibits features of calpain activation (Mol Cell Biochem 178:141-149, 1998). We here show that both hypoxia and azide-mediated metabolic inhibition induced heme oxygenase-1 expression, and caused cell death associated with lipid peroxidation. While blocking calcium influx or inhibiting calpain activity efficiently attenuated hypoxia-induced cell injury, it failed to prevent cell injury caused by adenoviral overexpression of the tumor suppressor protein p53. Inhibitors of caspases, on the other hand, suppressed cell injury caused by p53 overexpression. Hypoxia caused selective cleavage of troponin I (TnI), which could be suppressed by either nifedipine or calpeptin. Other myofilament proteins such as troponin T, myosin heavy chain, and actin appeared to remain largely intact. p53-mediated cell injury exhibited proteolysis of the caspase protein substrate lamin B without appreciable breakdown of TnI. We suggest that calpain-induced TnI breakdown may constitute a unique biochemical marker associated with chronically hypoxic cardiomyocytes.     

Hypoxia-induced cell death of HepG2 cells involves a necrotic cell death mediated by calpain

To elucidate mechanism of cell death in response to hypoxia, we attempted to compare hypoxia-induced cell death of HepG2 cells with cisplatin-induced cell death, which has been well characterized as a typical apoptosis. Cell death induced by hypoxia turned out to be different from cisplatin-mediated apoptosis in cell viability and cleavage patterns of caspases. Hypoxia-induced cell death was not associated with the activation of p53 while cisplatin-induced apoptosis is p53 dependent. In order to explain these differences, we tested involvement of μ-calpain and m-calpain in hypoxia-induced cell death. Calpains, especially μ-calpain, were initially cleaved by hypoxia, but not by cisplatin. Interestingly, the treatment of a calpain inhibitor restored PARP cleavage that was absent during hypoxia, indicating the recovery of activated caspase-3. The inhibition of calpains prevented proteolysis induced by hypoxia. In addition, hypoxia resulted in a necrosis-like morphology while cisplatin induced an apoptotic morphology. The calpain inhibitor prevented necrotic morphology induced by hypoxia and converted partially to apoptotic morphology with nuclear segmentation. Our result suggests that calpains are involved in hypoxia-induced cell death that is likely to be necrotic in nature and the inhibition of calpain switches hypoxia-induced cell death to apoptotic cell death without affecting cell viability.     

Analysis of Events Associated with Serum Deprivation-Induced Apoptosis in C3H/Sol8 Muscle Satellite Cells

Satellite cells are the source of new muscle fibers in postnatal skeletal muscle growth and regeneration. Regulation of satellite cell survival is of fundamental importance in maintaining normal muscle function. Here we describe and characterize a tissue culture model of satellite cell apoptosis. Kinetic studies indicate that serum deprivation triggers a set of sequential events: early cell death, transient cell cycle traverse, and delayed cell death. The satellite cell death occurs by apoptosis based on the internucleosomal DNA laddering,in situDNA end-labeling, and the requirements forde novoprotein synthesis and extracellular calcium influx. The transient period of cell cycle progression (7–11 h after serum withdrawal) is accompanied by temporal induction of members of the immediate early gene family, such as c-myc,c-fos,and SRF, and appears to precede the delayed phase of cell death. Satellite cell apoptosis can be suppressed by several growth factors and by blocking the activity of calpain, a calcium-regulated protease. The late phase of apoptosis is marked by selective activation of ubiquitin-mediated protein conjugation and degradation. This study defines several control points where satellite cell apoptosis may be genetically or pharmacologically intervened.     

Cardiac myocyte apoptosis provokes adverse cardiac remodeling in transgenic mice with targeted TNF overexpression

Although cardiac myocyte apoptosis has been detected in explanted hearts from patients with end-stage dilated and ischemic cardiomyopathy, the relative contribution of apoptotic cell death to left ventricular (LV) remodeling and cardiac decompensation is not known. To determine whether progressive cardiac myocyte apoptosis contributes to the transition from a hypertrophic to a dilated cardiac phenotype that is observed in transgenic myosin heavy chain secreted TNF (MHCsTNF) mice with cardiac restricted overexpression of tumor necrosis factor (TNF), we assessed cardiac myocyte apoptosis (using a DNA ligase technique) in MHCsTNF mice and littermate control mice in relation to serial changes in LV structure, which was assessed using MRI. The prevalence of cardiac myocyte apoptosis increased progressively from 4 to 12 wk as the hearts of the MHCsTNF mice underwent the transition from a concentric hypertrophic to a dilated cardiac phenotype. Treatment of the MHCsTNF mice with the broad-based caspase inhibitor N-[(1,3-dimethylindole-2-carbonyl)-valinyl]-3-amino4-oxo-5-fluoropentanoic acid significantly decreased cardiac myocyte apoptosis and significantly attenuated LV wall thinning and adverse cardiac remodeling. Additional studies suggested that the TNF-induced decrease in Bcl-2 expression and activation of the intrinsic mitochondrial death pathway were responsible for the cardiac myocyte apoptosis observed in the MHCsTNF mice. These studies show that progressive cardiac myocyte apoptosis is sufficient to contribute to adverse cardiac remodeling in the adult mammalian heart through progressive LV wall thinning.     

Calcium-induced calpain mediates apoptosis via caspase-3 in a mouse photoreceptor cell line

The rd mouse, an accepted animal model for photoreceptor degeneration in retinitis pigmentosa, has a recessive mutation for the gene encoding the beta-subunit of the cGMP phosphodiesterase. This mutation results in high levels of cGMP, which leaves an increased number of the cGMP-gated channels in the open state, thus allowing intracellular calcium (Ca(2+)) to rise to toxic levels, and rapid photoreceptor degeneration follows. To delineate the events in rd photoreceptor degeneration, we demonstrated an increase in calpain and caspase-3 activity, hypothesizing that Ca(2+)-mediated apoptosis in photoreceptors is mediated by calpain, involving mitochondrial depolarization and caspase-3 activation. To examine this hypothesis further, a murine photoreceptor-derived cell line (661W) was treated with the Ca(2+) ionophore A23187, cGMP-gated channel agonist 8-bromo-cGMP, or phosphodiesterase inhibitor isobutylmethylxanthine to mimic the increased Ca(2+) influx seen in the rd photoreceptors. Ca(2+)-induced cell death in 661W cells was found to be mediated by calpain and caspase-3 and could be completely inhibited by the calpain inhibitor SJA6017, implicating both calpain and caspases in the apoptotic process. The apoptotic events correlated in an SJA6017-inhibitable manner with bid cleavage, mitochondrial depolarization, cytochrome c release, and caspase-3 and -9 activation. We concluded that Ca(2+) influx in the rd model of photoreceptor degeneration leads to the activation of the cysteine protease calpain, which executes apoptosis via modulation of caspase-3 activity.     

Calpain and calpastatin regulate neutrophil apoptosis

The average polymorphonuclear neutrophil (PMN) lives only a day and then dies by apoptosis. We previously found that the calcium-dependent protease calpain is required for apoptosis in several mouse models of cell death. Here we identify calpain, and its endogenous inhibitor calpastatin, as regulators of human neutrophil apoptosis. Cell death triggered by the translation inhibitor cycloheximide is calpain-dependent, as evidenced using either a calpain active site inhibitor (N-acetyl-leucyl-leucyl-norleucinal) or agents that target calpain's calcium binding sites (PD150606, PD151746). No significant effect on cycloheximide-triggered apoptosis was found by using inhibitors of the proteasome or of other papain-like cysteine proteases, providing further evidence that the active site calpain inhibitor prevents apoptosis via its action on calpain. In addition, we find that potentiation of calpain activity by depleting its endogenous inhibitor, calpastatin, is sufficient to cause apoptosis of neutrophils. Nevertheless, apoptosis signalled via the Fas antigen proceeds regardless of the presence of calpain inhibitor. These experiments support a growing body of work, indicating an upstream regulatory role for calpain in many, but not all, forms of apoptotic cell death. They also identify calpastatin as a participant in apoptotic cell death and suggest that for at least one cell type, a decrease in calpastatin is a sufficient stimulus to initiate calpain-dependent apoptosis. J. Cell. Physiol. 178:311–319, 1999. © 1999 Wiley-Liss, Inc.     

TLR4 mutation and HSP60-induced cell death in adult mouse cardiac myocytes

Extracellular (ex) HSP60 is increasingly recognized as an agent of cell injury. Previously, we reported that low endotoxin exHSP60 causes cardiac myocyte apoptosis. Our findings supported a role for Toll-like receptor (TLR) 4 in HSP60 mediated apoptosis. To further investigate the involvement of TLR4 in cardiac injury, we studied adult cardiac myocytes from C3H/HeJ (HeJ) mice, which have a mutant, nonfunctional TLR4, and compared the results with parallel studies using wild-type (WT) mice. Nuclear factor κB (NFκB) activation is an early step downstream of TLR4. NFκB was activated 1 h after treatment with HSP60 in WT, but not HeJ mouse myocytes. ExHSP60 caused apoptosis in cardiac myocytes from WT mice, but not in myocytes from the HeJ mutants. To further elucidate the importance of exHSP60 in cardiac myocyte injury, both WT and HeJ mutant isolated mouse adult cardiac myocytes were exposed to hypoxia/reoxygenation. Anti-HSP60 antibody treatment reduced apoptosis in the WT group, but had no effect on the HeJ mutant myocytes. Unexpectedly, necrosis was also decreased in the HeJ mutants. Necrosis after hypoxia/reoxygenation in WT cardiac myocytes was mediated in part by TLR2 and TLR4 through rapid activation of PKCα, followed by increased expression of Nox2, and this was ameliorated by blocking antibodies to TLR2/4. These studies provide further evidence that TLR4 mediates exHSP60-associated apoptosis and that exHSP60 has an important role in cardiac myocyte injury, both apoptotic and necrotic.     

Anthracyclines induce calpain-dependent titin proteolysis and necrosis in cardiomyocytes

Titin, the largest myofilament protein, serves as a template for sarcomere assembly and acts as a molecular spring to contribute to diastolic function. Titin is known to be extremely susceptible to calcium-dependent protease degradation in vitro. We hypothesized that titin degradation is an early event in doxorubicin-induced cardiac injury and that titin degradation occurs by activation of the calcium-dependent proteases, the calpains. Treatment of cultured adult rat cardiomyocytes with 1 or 3 micromol/liter doxorubicin for 24 h resulted in degradation of titin in myocyte lysates, which was confirmed by a reduction in immunostaining of an antibody to the spring-like (PEVK) domain of titin at the I-band of the sarcomere. The elastic domain of titin appears to be most susceptible to proteolysis because co-immunostaining with an antibody to titin at the M-line was preserved, suggesting targeted proteolysis of the spring-like domain of titin. Doxorubicin treatment for 1 h resulted in approximately 3-fold increase in calpain activity, which remained elevated at 48 h. Co-treatment with calpain inhibitors resulted in preservation of titin, reduction in myofibrillar disarray, and attenuation of cardiomyocyte necrosis but not apoptosis. Co-treatment with a caspase inhibitor did not prevent the degradation of titin, which precludes caspase-3 as an early mechanism of titin proteolysis. We conclude that calpain activation is an early event after doxorubicin treatment in cardiomyocytes and appears to target the degradation of titin. Proteolysis of the spring-like domain of titin may predispose cardiomyocytes to diastolic dysfunction, myofilament instability, and cell death by necrosis.     

Calpain activation after mitochondrial permeability transition in microcystin-induced cell death in rat hepatocytes

Previous studies have shown that microcystin-LR (MLR), a specific hepatotoxin, induces onset of mitochondrial permeability transition (MPT) and apoptosis in cultured rat hepatocytes. Here we attempted to investigate the downstream events after the onset of MPT in MLR-treated hepatocytes. Various mitochondrial electron transport chain (ETC) inhibitors effectively prevented the onset of MPT, suggesting that the mitochondrial ETC plays an important role in MLR-induced MPT. MLR also induced mitochondrial cytochrome c release, which can be prevented by a specific MPT inhibitor (cyclosporin A, CsA), and by various ETC inhibitors. Interestingly, the release of cytochrome c did not activate caspase-9 and -3, the main caspases involved in apoptosis. Instead, MLR activated calpain in rat hepatocytes, probably through the increase of intracellular Ca(2+) released from mitochondria. Both ALLN and ALLM, two calpain inhibitors, significantly blocked MLR-induced calpain activation and subsequent cell death. CsA also prevented MLR-induced calpain activation and cell death, suggesting that the activation of calpain may be a post-mitochondrial event. These data demonstrate for the first time that calpain rather than caspases plays an important role in MLR-induced apoptosis.     

Activation of ER stress and apoptosis by hydrogen peroxide in HeLa cells: protective role of mild heat preconditioning at 40°C

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) during stress conditions causes activation of the unfolded protein response (UPR). If this adaptive response cannot restore ER homeostasis, cells undergo ER-mediated apoptosis. This study determines whether thermotolerance developed at a mild temperature (40°C) can alter induction of ER-mediated stress and apoptosis by H(2)O(2) in HeLa cells. Protein expression of PERK, p-PERK, eIF2α and p-eIF2α was increased in thermotolerant compared to non-thermotolerant cells. Thus, mild thermotolerance enhanced pro-survival effects of the PERK/eIF2α branch of the UPR. A short exposure (15 min) of cells to H(2)O(2) (15-50 μM) activated the UPR: expression of p-PERK, p-eIF2α and p-IRE1α increased, and ATF6 cleavage occurred. Longer exposure (1-3h) to H(2)O(2) induced ER-mediated apoptosis, whereby CHOP expression increased, and enzymatic activity of calpain, caspase-7, -4, -12 and -9 also increased. These pro-apoptotic events and clonogenic cell killing were all diminished in thermotolerant cells. Activation of caspases-4/-12 was decreased by the calcium chelator BAPTA-AM, and by inhibitors of calpain and caspase-7, confirming the roles of calcium, calpain and caspase-7 in activation of ER-mediated apoptosis by H(2)O(2). In thermotolerant cells with decreased levels of PERK by siRNA, there was partial reversal of resistance to H(2)O(2)-induced apoptosis. Hence, a causal connection exists between the ER stress response and resistance to H(2)O(2)-induced apoptosis. Mild thermotolerance plays a protective, anti-apoptotic role by increasing the threshold for induction of ER-mediated apoptosis by H(2)O(2). Moreover, the adaptive response (UPR) dominates during milder H(2)O(2) stress, whereas ER-mediated apoptosis occurs during more severe stress.     

Hypoxia sensitizes cells to nitric oxide-induced apoptosis

Nitric oxide (NO) can induce apoptosis in a variety of cell types. A non-toxic concentration of nitric oxide under normal oxygen conditions triggered cell death under hypoxic conditions (1.5% O(2)) in fibroblasts. Nitric oxide administered during hypoxia induced the release of cytochrome c, caspase-9 activation, and the loss of mitochondrial membrane potential followed by DNA fragmentation and lactate dehydrogenase release (markers of cell death). Bcl-X(L) protected cells from nitric oxide-induced apoptosis during hypoxia by preventing the release of cytochrome c, caspase-9 activation, and by maintaining a mitochondrial membrane potential. Murine embryonic fibroblasts from bax(-/-) bak(-/-) mice exposed to nitric oxide during hypoxia did not die, indicating that pro-apoptotic Bcl-2 family members are required for NO-induced apoptosis during hypoxia. The nitric oxide-induced cell death during hypoxia was independent of cGMP and peroxynitrite. Cells devoid of mitochondrial DNA (rho secondary-cells) lack a functional electron transport chain and were resistant to nitric oxide-induced cell death during hypoxia, suggesting that a functional electron transport chain is required for nitric oxide-induced apoptosis during hypoxia.     

GLUT-1 reduces hypoxia-induced apoptosis and JNK pathway activation

Many studies have suggested that enhanced glucose uptake protects cells from hypoxic injury. More recently, it has become clear that hypoxia induces apoptosis as well as necrotic cell death. We have previously shown that hypoxia-induced apoptosis can be prevented by glucose uptake and glycolytic metabolism in cardiac myocytes. To test whether increasing the number of glucose transporters on the plasma membrane of cells could elicit a similar protective response, independent of the levels of extracellular glucose, we overexpressed the facilitative glucose transporter GLUT-1 in a vascular smooth muscle cell line. After 4 h of hypoxia, the percentage of cells that showed morphological changes of apoptosis was 30.5 +/- 2.6% in control cells and only 6.0 +/- 1.1 and 3.9 +/- 0.3% in GLUT-1-overexpressing cells. Similar protection against cell death and apoptosis was seen in GLUT-1-overexpressing cells treated for 6 h with the electron transport inhibitor rotenone. In addition, hypoxia and rotenone stimulated c-Jun-NH(2)-terminal kinase (JNK) activity >10-fold in control cell lines, and this activation was markedly reduced in GLUT-1-overexpressing cell lines. A catalytically inactive mutant of MEKK1, an upstream kinase in the JNK pathway, reduced hypoxia-induced apoptosis by 39%. These findings show that GLUT-1 overexpression prevents hypoxia-induced apoptosis possibly via inhibition of stress-activated protein kinase pathway activation.     

Ablation of telomerase and telomere loss leads to cardiac dilatation and heart failure associated with p53 upregulation

Cardiac failure is a frequent cause of death in the aging human population. Telomere attrition occurs with age, and is proposed to be causal for the aging process. To determine whether telomere shortening leads to a cardiac phenotype, we studied heart function in the telomerase knockout mouse, Terc-/-. We studied Terc-/- mice at the second, G2, and fifth, G5, generation. Telomere shortening in G2 and G5 Terc-/- mice was coupled with attenuation in cardiac myocyte proliferation, increased apoptosis and cardiac myocyte hypertrophy. On a single-cell basis, telomere shortening was coincidental with increased expression of p53, indicating the presence of dysfunctional telomeres in cardiac myocytes from G5 Terc-/- mice. The impairment in cell division, the enhanced cardiac myocyte death and cellular hypertrophy, are concomitant with ventricular dilation, thinning of the wall and cardiac dysfunction. Thus, inhibition of cardiac myocyte replication provoked by telomere shortening, results in de-compensated eccentric hypertrophy and heart failure in mice. Telomere shortening with age could also contribute to cardiac failure in humans, opening the possibility for new therapies.     

Reduction of Na/K-ATPase potentiates marinobufagenin-induced cardiac dysfunction and myocyte apoptosis

Decreases in cardiac Na/K-ATPase have been documented in patients with heart failure. Reduction of Na/K-ATPase α1 also contributes to the deficiency in cardiac contractility in animal models. Our previous studies demonstrate that reduction of cellular Na/K-ATPase causes cell growth inhibition and cell death in renal proximal tubule cells. To test whether reduction of Na/K-ATPase in combination with increased cardiotonic steroids causes cardiac myocyte death and cardiac dysfunction, we examined heart function in Na/K-ATPase α1 heterozygote knock-out mice (α1(+/-)) in comparison to wild type (WT) littermates after infusion of marinobufagenin (MBG). Adult cardiac myocytes were also isolated from both WT and α1(+/-) mice for in vitro experiments. The results demonstrated that MBG infusion increased myocyte apoptosis and induced significant left ventricle dilation in α1(+/-) mice but not in their WT littermates. Mechanistically, it was found that in WT myocytes MBG activated the Src/Akt/mTOR signaling pathway, which further increased phosphorylation of ribosome S6 kinase (S6K) and BAD (Bcl-2-associated death promoter) and protected cells from apoptosis. In α1(+/-) myocytes, the basal level of phospho-BAD is higher compared with WT myocytes, but MBG failed to induce further activation of the mTOR pathway. Reduction of Na/K-ATPase also caused the activation of caspase 9 but not caspase 8 in these cells. Using cultures of neonatal cardiac myocytes, we demonstrated that inhibition of the mTOR pathway by rapamycin also enabled MBG to activate caspase 9 and induce myocyte apoptosis.     

High glucose-induced Ca overload and oxidative stress contribute to apoptosis of cardiac cells through mitochondrial dependent and independent pathways

Background

Cardiac cell apoptosis is the initiating factor of cardiac complications especially diabetic cardiomyopathy. Mitochondria are susceptible to the damaging effects of elevated glucose condition. Calcium overload and oxidative insult are the two mutually non-exclusive phenomena suggested to cause cardiac dysfunction. Here, we examined the effect of high-glucose induced calcium overload in calpain-1 mediated cardiac apoptosis in an in vitro setting.

Methods

H9c2, rat ventricular myoblast cell line was treated with elevated glucose condition and the cellular consequences were studied. Intracellular calcium trafficking, ROS generation, calpain-1 activation and caspase-12 and caspase-9 pathway were studied using flow cytometry, confocal microscopy and Western blot analysis.

Results

High-glucose treatment resulted in increased intracellular calcium ([Ca^2 +]i) which was mobilized to the mitochondria. Concomitant intra-mitochondrial calcium ([Ca^2 +]m) increase resulted in enhanced reactive oxygen and nitrogen species generation. These events led to mitochondrial dysfunction and apoptosis. Cardiomyocyte death exhibited several classical markers of apoptosis, including activation of caspases, appearance of annexin V on the outer plasma membrane, increased population of cells with sub-G₀/G₁ DNA content and nuclear condensation. Key findings include elucidation of cell signaling mechanism of high-glucose induced calcium-dependent cysteine protease calpain-1 activation, which triggers non-conventional caspases as alternate mode of cell death.

Conclusion

This information increases the understanding of cardiac cell death under hyperglycemic condition and can possibly be extended for designing new therapeutic strategies for diabetic cardiomyopathy.

General significance

The novel findings of the study reveal that high glucose induces apoptosis by both mitochondria-dependent and independent pathways via concomitant rise in intracellular calcium.     

Calpain activation in apoptosis

Programmed cell death is an active process wherein the cell initiates a sequence of events culminating in the fragmentation of its DNA, nuclear collapse, and disintegration of the cell into small, membrane-bound apoptotic bodies. Examination of the death program in various models has shown common themes, including a rise in cytoplasmic calcium, cytoskeletal changes, and redistribution of membrane lipids. The calcium-dependent neutral protease calpain has putative roles in cytoskeletal and membrane changes in other cellular processes; this fact led us to test the role of calpain in a well-known model of apoptotic cell death, that of thymocytes after treatment with dexamethasone. Assays for calcium-dependent proteolysis in thymocyte extracts reveal a rise in activity with a peak at about 1 hr of incubation with dexamethasone, falling to background at approximately 2 hr. Western blots indicate autolytic cleavage of the proenzyme precursor to the calpain I isozyme, providing additional evidence for calpain activation. We have also found that apoptosis in thymocytes, whether induced by dexamethasone or by low-level irradiation, is blocked by specific inhibitors of calpain. Apoptosis of metamyelocytes incubated with cycloheximide is also blocked by calpain inhibitors. These studies suggest a required role for calpain in both “induction” and “release” models of apoptotic cell death. © 1994 wiley-Liss, Inc.     

Cross-talk between calpain and caspase proteolytic systems during neuronal apoptosis

Cross-talk between calpain and caspase proteolytic systems has complicated efforts to determine their distinct roles in apoptotic cell death. This study examined the effect of overexpressing calpastatin, the specific endogenous calpain inhibitor, on the activity of the two proteolytic systems following an apoptotic stimulus. Human SH-SY5Y neuroblastoma cells were stably transfected with full-length human calpastatin cDNA resulting in 20-fold overexpression based on Western blot and 5-fold greater calpain inhibitory activity in cell extracts. Wild type and calpastatin overexpressing (CST1) cells were neuronally differentiated and apoptosis-induced with staurosporine (0.1-1.0 microm). Calpastatin overexpression decreased calpain activation, increased caspase-3-like activity, and accelerated the appearance of apoptotic nuclear morphology. Following 0.1-0.2 microm staurosporine, plasma membrane integrity based on calcein-acetoxymethyl fluorescence was significantly greater at 24 h in differentiated CST1 compared with differentiated wild type cells. However, this protective effect was lost at higher staurosporine doses (0.5-1.0 microm), which resulted in pronounced caspase-mediated degradation of the overexpressed calpastatin. These results suggest a dual role for calpains during neuronal apoptosis. In the early execution phase, calpain down-regulates caspase-3-like activity and slows progression of apoptotic nuclear morphology. Subsequent calpain activity, facilitated by caspase-mediated degradation of calpastatin, contributes to plasma membrane disruption and secondary necrosis.     

Cardiac release of urocortin precedes the occurrence of irreversible myocardial damage in the rat heart exposed to ischemia/reperfusion injury

This study evaluates whether cardiac ischemia induces release of urocortin, before and independently from myocyte cell death. Urocortin levels rose after 5-min ischemia and peaked after 10-min ischemia, when cell death was not detected. However, myocyte apoptosis and/or necrosis occurred following 20- and 30-min ischemia, which paralleled a fall in urocortin levels, suggesting that urocortin expression and release are mainly sustained by metabolically challenged, though still viable myocytes. Hence, since cardiac release of urocortin, unlike that of conventional biomarkers, occurs before and apart from cell death, urocortin levels may be clinically useful in the diagnosis of sublethal myocardial ischemia.