calpain and Myocardial-Ischemia

Loss of calcium (Ca(2+)) homeostasis contributes through different mechanisms to cell death occurring during the first minutes of reperfusion. One of them is an unregulated activation of a variety of Ca(2+)-dependent enzymes, including the non-lysosomal cysteine proteases known as calpains. This review analyses the involvement of the calpain family in reperfusion-induced cardiomyocyte death. Calpains remain inactive before reperfusion due to the acidic pHi and increased ionic strength in the ischaemic myocardium. However, inappropriate calpain activation occurs during myocardial reperfusion, and subsequent proteolysis of a wide variety of proteins contributes to the development of contractile dysfunction and necrotic cell death by different mechanisms, including increased membrane fragility, further impairment of Na(+) and Ca(2+) handling, and mitochondrial dysfunction. Recent studies demonstrating that calpain inhibition contributes to the cardioprotective effects of preconditioning and postconditioning, and the beneficial effects obtained with new and more selective calpain inhibitors added at the onset of reperfusion, point to the potential cardioprotective value of therapeutic strategies designed to prevent calpain activation.

Topics: Animals; Calcium; Calpain; Enzyme Activation; Humans; Myocardial Ischemia; Myocardial Reperfusion Injury

This article reviews what our colleagues have found as to how ischemic injury or cell death develop in myocardium through Ca(2+)-dependent protease calpain and how compensatory responses evolve through activation of intracellular signaling molecules including PKC isoforms, MAP kinase family enzymes and PI3 kinase. We also addressed how restraint or other psychological stress evokes hypertension and cardiovascular responses in signaling molecules or genes. Unexpectedly, carbon monoxide protects heart and cardiogenic cells against ischemia-resperfusion injury. When I think back, the unresolved cases of autopsies provided ideas for experimental study, which then taught us how the other cases died.

Topics: Animals; Ankyrins; Calcium; Calpain; Carbon Monoxide; Carrier Proteins; Central Nervous System Stimulants; Connexin 43; Death, Sudden, Cardiac; Gap Junctions; Humans; Ischemic Preconditioning, Myocardial; Methamphetamine; Microfilament Proteins; Mitogen-Activated Protein Kinases; Myocardial Contraction; Myocardial Ischemia; Myocardial Reperfusion Injury; Nitric Oxide; Phosphatidylinositol 3-Kinases; Protein Kinase C; Reactive Oxygen Species; Restraint, Physical; Stress, Psychological

Topics: Animals; Apoptosis; Calcium-Calmodulin-Dependent Protein Kinases; Calpain; Death, Sudden, Cardiac; Forensic Medicine; Humans; Myocardial Ischemia; Stress, Psychological

Topics: Animals; Calcium; Calpain; Cataract; Central Nervous System Diseases; Humans; Muscular Dystrophies; Myocardial Ischemia; Proteins

Increasing evidence now suggests that excessive activation of the Ca(2+)-dependent protease calpain could play a key or contributory role in the pathology of a variety of disorders, including cerebral ischaemia, cataract, myocardial ischaemia, muscular dystrophy and platelet aggregation. In this review, Kevin Wang and Po-Wai Yuen discuss the evidence linking these disorders to calpain overactivation. At present, it is difficult to confirm the exact role of calpain in these disorders because of the lack of potent, selective and cell-permeable calpain inhibitors. However, given the multiple therapeutic indications for calpain, it appears that achievement of selective calpain inhibition is an important pharmacological goal.

Topics: Amino Acid Sequence; Brain Ischemia; Calpain; Central Nervous System Diseases; Humans; Molecular Sequence Data; Myocardial Ischemia

Calpain overexpression is implicated in mitochondrial damage leading to tissue oxidative stress and myocardial ischemic injury. The aim of this study was to determine the effects of calpain inhibition (CI) on mitochondrial impairment and oxidative stress in a swine model of chronic myocardial ischemia and metabolic syndrome.. Yorkshire swine were fed a high-fat diet for 4 weeks to induce metabolic syndrome then underwent placement of an ameroid constrictor to the left circumflex artery. Three weeks later, animals received: no drug (control, "CON"; n= 7); a low-dose calpain inhibitor (0.12 mg/kg; "LCI", n= 7); or high-dose calpain inhibitor (0.25 mg/kg; "HCI", n=7). Treatment continued for 5 weeks, followed by tissue harvest. Cardiac tissue was assayed for protein carbonyl content, as well as antioxidant and mitochondrial protein expression. Reactive oxygen species (ROS) and mitochondrial respiration was measured in H9c2 cells following exposure to normoxia or hypoxia (1%) for 24 h with or without CI.. In ischemic myocardial tissue, CI was associated with decreased total oxidative stress compared to control. CI was also associated with increased expression of mitochondrial proteins superoxide dismutase 1, SDHA, and pyruvate dehydrogenase compared to control. 100 nM of calpain inhibitor decreased ROS levels and respiration in both normoxic and hypoxic H9c2 cardiomyoblasts.. In the setting of metabolic syndrome, CI improves oxidative stress in chronically ischemic myocardial tissue. Decreased oxidative stress may be via modulation of mitochondrial proteins involved in free radical scavenging and production.

Topics: Animals; Calpain; Disease Models, Animal; Metabolic Syndrome; Mitochondrial Proteins; Myocardial Ischemia; Myocardium; Oxidative Stress; Protein Carbonylation; Reactive Oxygen Species; Swine

Topics: Calpain; Humans; Myocardial Ischemia; Myocardial Reperfusion Injury

Calpain activation during ischemia is known to play critical roles in myocardial remodeling. We hypothesize that calpain inhibition (CI) may serve to reverse and/or prevent fibrosis in chronically ischemic myocardium.. Yorkshire swine were fed a high-cholesterol diet for 4 weeks followed by placement of an ameroid constrictor on the left circumflex artery to induce myocardial ischemia. 3 weeks later, animals received either: no drug; high-cholesterol control group (CON; n = 8); low-dose CI (0.12 mg/kg; LCI, n = 9); or high-dose CI (0.25 mg/kg; HCI, n = 8). The high-cholesterol diet and CI were continued for 5 weeks, after which myocardial tissue was harvested. Tissue samples were analyzed by western blot for changes in protein content.. In the setting of hypercholesterolemia and chronic myocardial ischemia, CI decreased the expression of collagen in ischemic and nonischemic myocardial tissue. This reduced collagen content was associated with a corresponding decrease in Jak/STAT/MCP-1 signaling pathway, suggesting a role for Jak 2 signaling in calpain activity. CI also decreases the expression of focal adhesion proteins (vinculin) and stabilizes the expression of cytoskeletal and structural proteins (N-cadherin, α-fodrin, desmin, vimentin, filamin, troponin-I). CI had no significant effect on metabolic and hemodynamic parameters.. Calpain inhibition may be a beneficial medical therapy to decrease collagen formation in patients with coronary artery disease and associated comorbidities.

Topics: Animals; Calpain; Chemokine CCL2; Collagen; Coronary Artery Disease; Disease Models, Animal; Fibrosis; Glycoproteins; Hypercholesterolemia; Janus Kinase 2; Myocardial Ischemia; Myocardium; Signal Transduction; STAT Transcription Factors; Swine; Ventricular Remodeling

Topics: Animals; Calpain; Coronary Artery Disease; Drug Discovery; Fibrosis; Glycoproteins; Hypercholesterolemia; Myocardial Ischemia; Myocardium; Swine; Ventricular Remodeling

Myocardial infarction remains the single leading cause of death worldwide. Upon reperfusion of occluded arteries, deleterious cellular mediators particularly located at the mitochondria level can be activated, thus limiting the outcome in patients. This may lead to the so-called ischemia/reperfusion (I/R) injury. Calpains are cysteine proteases and mediators of caspase-independent cell death. Recently, they have emerged as central transmitters of cellular injury in several cardiac pathologies e.g. hypertrophy and acute I/R injury.. Here we investigated the role of cardiac calpains in acute I/R in relation to mitochondrial integrity and whether calpains can be effectively inhibited by posttranslational modification by S-nitrosation. Taking advantage of the a cardiomyocyte cell line (HL1), we determined S-nitrosation by the Biotin-switch approach, cell viability and intracellular calcium concentration after simulated ischemia and reoxygenation - all in dependence of supplementation with nitrite, which is known as an 'hypoxic nitric oxide (NO) donor'. Likewise, using an in vivo I/R model, calpain S-nitrosation, calpain activity and myocardial I/R injury were characterized in vivo.. Nitrite administration resulted in an increased S-nitrosation of calpains, and this was associated with an improved cell-survival. No impact was detected on calcium levels. In line with these in vitro experiments, nitrite initiated calpain S-nitrosation in vivo and caused an infarct sparing effect in an in vivo myocardial I/R model. Using electron microscopy in combination with immuno-gold labeling we determined that calpain 10 increased, while calpain 2 decreased in the course of I/R. Nitrite, in turn, prevented an I/R induced increase of calpains 10 at mitochondria and reduced levels of calpain 1.. Lethal myocardial injury remains a key aspect of myocardial I/R. We show that calpains, as key players in caspase-independent apoptosis, increasingly locate at mitochondria following I/R. Inhibitory post-translational modification by S-nitrosation of calpains reduces deleterious calpain activity in murine cardiomyocytes and in vivo.

Topics: Animals; Calpain; Cell Line; Male; Mice, Inbred C57BL; Mitochondria; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion Injury; Nitrates; Nitrites; Nitrosation

Topics: Animals; Calpain; Collateral Circulation; Coronary Circulation; Coronary Vessels; Hypercholesterolemia; Male; Microvessels; Myocardial Ischemia; Protease Inhibitors

Calpain overexpression is implicated in aberrant angiogenesis. We hypothesized that calpain inhibition (MDL28170) would improve collateral perfusion in a swine model with hypercholesterolemia and chronic myocardial ischemia.. Yorkshire swine fed a high cholesterol diet for 4 weeks underwent surgical placement of an ameroid constrictor to their left circumflex coronary artery. Three weeks later, animals received no drug, high cholesterol control group (n = 8); low-dose calpain inhibition (0.12 mg/kg; n = 9); or high-dose calpain inhibition (0.25 mg/kg; n = 8). The heart was harvested after 5 weeks.. Myocardial perfusion in ischemic myocardium significantly improved with high-dose calpain inhibition at rest and with demand pacing (P = .016 and .011). Endothelium-dependent microvessel relaxation was significantly improved with low-dose calpain inhibition (P = .001). There was a significant increase in capillary density, with low-dose calpain inhibition and high-dose calpain inhibition (P = .01 and .01), and arteriolar density with low-dose calpain inhibition (P = .001). Calpain inhibition significantly increased several proangiogenic proteins, including vascular endothelial growth factor (P = .02), vascular endothelial growth factor receptor 1 (P = .003), vascular endothelial growth factor receptor 2 (P = .003), and talin, a microvascular structural protein (P = .0002). There was a slight increase in proteins implicated in endothelial-dependent (nitric oxide mediated) relaxation, including extracellular signal-regulated kinase, phosphorylated extracellular signal-regulated kinase, and inducible nitric oxide synthase with calpain inhibition.. In the setting of hypercholesterolemia, calpain inhibition improved perfusion, with a trend toward increased collateralization on angiography and increased capillary and arteriolar densities in ischemic myocardium. Calpain inhibition also improved endothelium-dependent microvessel relaxation and increased expression of proteins implicated in angiogenesis and vasodilatation.

Topics: Angiogenic Proteins; Animals; Calpain; Chronic Disease; Collateral Circulation; Coronary Angiography; Coronary Circulation; Coronary Vessels; Disease Models, Animal; Dose-Response Relationship, Drug; Hypercholesterolemia; Male; Microcirculation; Microvessels; Myocardial Ischemia; Myocardial Perfusion Imaging; Neovascularization, Physiologic; Protease Inhibitors; Time Factors; Vasodilation

The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (IKr), which is important for cardiac repolarization. Dysfunction of hERG causes long QT syndrome and sudden death, which occur in patients with cardiac ischemia. Cardiac ischemia is also associated with activation, up-regulation, and secretion of various proteolytic enzymes. Here, using whole-cell patch clamp and Western blotting analysis, we demonstrate that the hERG/IKr channel was selectively cleaved by the serine protease, proteinase K (PK). Using molecular biology techniques including making a chimeric channel between protease-sensitive hERG and insensitive human ether-a-go-go (hEAG), as well as application of the scorpion toxin BeKm-1, we identified that the S5-pore linker of hERG is the target domain for proteinase K cleavage. To investigate the physiological relevance of the unique susceptibility of hERG to proteases, we show that cardiac ischemia in a rabbit model was associated with a reduction in mature ERG expression and an increase in the expression of several proteases, including calpain. Using cell biology approaches, we found that calpain-1 was actively released into the extracellular milieu and cleaved hERG at the S5-pore linker. Using protease cleavage-predicting software and site-directed mutagenesis, we identified that calpain-1 cleaves hERG at position Gly-603 in the S5-pore linker of hERG. Clarification of protease-mediated damage of hERG extends our understanding of hERG regulation. Damage of hERG mediated by proteases such as calpain may contribute to ischemia-associated QT prolongation and sudden cardiac death.

Topics: Animals; Calpain; Endopeptidase K; ERG1 Potassium Channel; HEK293 Cells; Humans; Male; Myocardial Ischemia; Proteolysis; Rabbits; Scorpion Venoms

Calpain is a family of cysteine proteases that has an important role in the initiation, regulation, and execution of cell death. Our recent studies using a hypercholesterolemic swine model demonstrated that in the setting of the metabolic syndrome, calpain inhibition (CI) improved collateral-dependent perfusion and increased expression of proteins implicated in angiogenesis and vasodilation. In this study, we hypothesized that CI (by MLD28170) would decrease myocardial apoptosis in the same model.. Yorkshire swine, all fed a high-cholesterol diet for 4 weeks underwent placement of an ameroid constrictor on the left circumflex coronary artery. Three weeks later, animals received either no drug, termed the high-cholesterol control group (HCC; n = 8); low-dose CI (0.12 mg/kg; LCI, n = 9); or high-dose CI (0.25 mg/kg; HCI, n = 8). The high-cholesterol diet and the CI were continued for 5 weeks, after which the pig was humanely killed and the left ventricular myocardium was harvested and analyzed via terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, oxyblot analysis, and Western blots. Data were analyzed using the Kruskal-Wallis test.. The percentage of apoptotic cells to total cells in ischemic myocardial territory was decreased in the LCI and HCI groups compared with the HCC group as shown by TUNEL staining (P = .018). There was a decrease in proapoptotic proteins, including cleaved caspase 3, caspase 9, cleaved caspase 9, Bax, BAD, p-BAD, and Erk 1/2 (P ≤ .049 each), but no decrease in caspase 3 (P = .737). There was also an increase in antiapoptotic proteins, including BCL-2 and p-BCL2 (P ≤ .025 each). In the ischemic myocardium, several proangiogenic proteins were increased in the LCI and HCI groups compared with the HCC group, including p-AKT, p-eNOS, and eNOS (P ≤ .006 each) but there was no increase in AKT (P = .311). CI decreased tissue oxidative stress in both the LCI and HCI groups compared to the HCC group as shown by oxyblot analysis (P = .021).. In the setting of hypercholesterolemia, CI decreases apoptosis and the expression of proteins in proapoptotic signaling pathways. CI also increased expression of proteins implicated in anti apoptotic pathways and improves oxidative stress in ischemic myocardial tissue.

Topics: Animals; Apoptosis; Biomarkers; Blotting, Western; Calpain; Cardiotonic Agents; Chronic Disease; Dose-Response Relationship, Drug; Glycoproteins; Heart; Hypercholesterolemia; In Situ Nick-End Labeling; Metabolic Syndrome; Myocardial Ischemia; Myocardium; Oxidative Stress; Swine

Loss of cardiac muscle by programmed cell death contributes to the progression of ischemic heart disease. Hypoxia, metabolite waste buildup and energy depletion are components of ischemia which may initiate caspase dependent and independent cell death pathways. Previous work from our laboratory has shown that combined hypoxia with acidosis, a hallmark of ischemia promotes cardiac myocyte injury with increasing severity as the pH declines. Hypoxia-acidosis was demonstrated to activate the pro-apoptotic Bcl-2 protein BNIP3 which initiated opening of the mitochondrial permeability transition pore and cell death in the absence of caspase activation. Because calpains are known to contribute to ischemic myocardial damage in some models, we hypothesized that they are intermediates in the BNIP3-mediated death caused by hypoxia-acidosis.. Neonatal rat cardiac myocytes were subjected to hypoxia with and without acidosis and the contribution of calpains to hypoxia-acidosis cell death determined.. Here we report that the death pathway activated by hypoxia-acidosis is driven by a combination of calcium-activated calpains and pro-death factors (DNases) secreted by the mitochondria. Cytochrome c accumulated in the cytoplasm during hypoxia-acidosis but caspase activity was repressed through a calpain-dependent process that prevents the cleavage of procaspase 3. Calpain inhibitors provide vigorous protection against hypoxia-acidosis-induced programmed death. Knockdown of BNIP3 with siRNA prevented calpain activation confirming a central role of BNIP3 in this pathway.. The results implicate BNIP3 and calpain as dependent components of cardiac myocyte death caused by hypoxia-acidosis.

Topics: Animals; Apoptosis; Calcium; Calpain; Caspase 3; Membrane Proteins; Mitochondrial Proteins; Myocardial Ischemia; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley; Signal Transduction

In the heart, calpastatin (Calp) and its homologue high molecular weight calmodulin-binding protein (HMWCaMBP) regulate calpains (Calpn) by inhibition. A rise in intracellular myocardial Ca2+ during cardiac ischemia activates Calpn thereby causing damage to myocardial proteins, which leads to myocyte death and consequently to loss of myocardial structure and function. The present study aims to elucidate expression of Calp and HMWCaMBP with respect to Calpn during induced ischemia and reperfusion in primary murine cardiomyocyte cultures. Ischemia and subsequently reperfusion was induced in ∼ 80% confluent cultures of neonatal murine cardiomyocytes (NMCC). Flow cytometric analysis (FACS) has been used for analyzing protein expression concurrently with viability. Confocal fluorescent microscopy was used to observe protein localization. We observed that ischemia induces increased expression of Calp, HMWCaMBP and Calpn. Calpn expressing NMCC on co-expressing Calp survived ischemic induction compared to NMCC co-expressing HMWCaMBP. Similarly, living cells expressed Calp in contrast to dead cells which expressed HMWCaMBP following reperfusion. A significant difference in the expression of Calp and its homologue HMWCaMBP was observed in localization studies during ischemia. The current study adds to the existing knowledge that HMWCaMBP could be a putative isoform of Calp. NMCC on co-expressing Calp and Calpn-1 survived ischemic and reperfusion inductions compared to NMCC co-expressing HMWCaMBP and Calpn-1. A significant difference in expression of Calp and HMWCaMBP was observed in localization studies during ischemia.

Topics: Animals; Animals, Newborn; Blotting, Western; Calcium-Binding Proteins; Calmodulin-Binding Proteins; Calpain; Cells, Cultured; Female; Flow Cytometry; Mice; Microscopy, Confocal; Molecular Weight; Myocardial Ischemia; Myocytes, Cardiac; Protein Binding; Protein Isoforms; Reperfusion

We have previously shown that preischemic treatment with glucosamine improved cardiac functional recovery following ischemia-reperfusion, and this was mediated, at least in part, via enhanced flux through the hexosamine biosynthesis pathway and subsequently elevated O-linked N-acetylglucosamine (O-GlcNAc) protein levels. However, preischemic treatment is typically impractical in a clinical setting; therefore, the goal of this study was to investigate whether increasing protein O-GlcNAc levels only during reperfusion also improved recovery. Isolated perfused rat hearts were subjected to 20 min of global, no-flow ischemia followed by 60 min of reperfusion. Administration of glucosamine (10 mM) or an inhibitor of O-GlcNAcase, O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc; 200 microM), during the first 20 min of reperfusion significantly improved cardiac functional recovery and reduced troponin release during reperfusion compared with untreated control. Both interventions also significantly increased the levels of protein O-GlcNAc and ATP levels. We also found that both glucosamine and PUGNAc attenuated calpain-mediated proteolysis of alpha-fodrin as well as Ca(2+)/calmodulin-dependent protein kinase II during reperfusion. Thus two independent strategies for increasing protein O-GlcNAc levels in the heart during reperfusion significantly improved recovery, and this was correlated with attenuation of calcium-mediated proteolysis. These data provide further support for the concept that increasing cardiac O-GlcNAc levels may be a clinically relevant cardioprotective strategy and suggest that this protection could be due, at least in part, to inhibition of calcium-mediated stress responses.

Topics: Acetylglucosamine; Adenosine Triphosphate; Animals; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calpain; Carrier Proteins; Glucosamine; Microfilament Proteins; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Oximes; Phenylcarbamates; Rats

Na+ overload and secondary Ca2+ influx via Na+/Ca2+ exchanger are key mechanisms in cardiomyocyte contracture and necrosis during reperfusion. Impaired Na+/K+-ATPase activity contributes to Na+ overload, but the mechanism has not been established. Because Na+/K+-ATPase is connected to the cytoskeleton protein fodrin through ankyrin, which are substrates of calpains, we tested the hypothesis that calpain mediates Na+/K+-ATPase impairment in reperfused cardiomyocytes. In isolated rat hearts reperfused for 5 minutes after 60 minutes of ischemia, Na+/K+-ATPase activity was reduced by 80%, in parallel with loss of alpha-fodrin and ankyrin-B and detachment of alpha1 and alpha2 subunits of Na+/K+-ATPase from the membrane-cytoskeleton complex. Calpain inhibition with MDL-7943 during reperfusion prevented the loss of these proteins, increased Na+/K+-ATPase activity, attenuated lactate dehydrogenase release, and improved contractile recovery, and these beneficial effects of MDL-7943 were reverted by ouabain. The impairment of Na+/K+-ATPase was not a mere consequence of cell death because it was not altered in hearts in which contracture and cell death had been prevented by contractile blockade with 2,3-butanedione monoxime. In these hearts, concomitant calpain inhibition preserved Na+/K+-ATPase content and function and attenuated cell death occurring on withdrawal of 2,3-butanedione monoxime. In vitro assay showed no detectable degradation of Na+/K+-ATPase subunits after 10 minutes of incubation with activated calpain. Thus, we conclude that calpain activation contributes to the impairment of Na+/K+-ATPase during early reperfusion and that this effect is mainly mediated by degradation of the anchorage of Na+/K+-ATPase to the membrane cytoskeleton.

Topics: Animals; Ankyrins; Calcium; Calpain; Carrier Proteins; Cell Death; L-Lactate Dehydrogenase; Male; Microfilament Proteins; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Ouabain; Rats; Rats, Sprague-Dawley; Sodium-Potassium-Exchanging ATPase; Ventricular Function, Left

Calcineurin (CaN) has been reported as a critical mediator for cardiac hypertrophy and cardiac myocyte apoptosis. In the present study, we investigated the activity and expression of CaN and the effect of calpain in rat heart after ischemia and reperfusion. Rat ischemic heart showed significant increase in CaN activity. Western blot analysis of normal rat heart extract with a polyclonal antibody raised against bovine CaN indicated a prominent immunoreactive band of 60 kDa (CaN A). In ischemic-reperfused hearts, the expression of CaN A was significantly low and immunoreactivity was observed in proteolytic bands of 46 kDa. This may be due to the proteolytic degradation of CaN A in ischemic tissues by m-calpain. We also noticed in vitro proteolysis of bovine cardiac CaN A by m-calpain. Immunohistochemical studies showed strong staining of immunoreactivity in rat hearts that had gone under 30 min ischemia followed by 30 min reperfusion similar to that found in human ischemic heart. Ischemia is associated with multiple alterations in the extracellular and intracellular signaling of cardiomyocytes and may act as an inducer of apoptosis. The increase in CaN activity and strong immunostaining observed in ischemic/perfused rat heart may be due to the calpain-mediated proteolysis of this phosphatase.

Topics: Animals; Blotting, Western; Calcineurin; Calpain; Cattle; Heart; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Rats; Rats, Sprague-Dawley

The purpose of this study was to test the hypothesis that myocardial ischemia-reperfusion (I/R) is accompanied by an early burst in calpain activity, resulting in decreased calpastatin activity and an increased calpain/calpastatin ratio, thereby promoting increased protein release. To determine the possibility of a 'calpain burst' impacting cardiac calpastatin inhibitory activity, rat hearts were subjected (Langendorff) to either 45 or 60 min of ischemia followed by 30 min of reperfusion with and without pre-administration (s.c.) of a cysteine protease inhibitor (E-64c). Myocardial function, calpain activities (casein release assay), calpastatin inhibitory activity and release of CK, LDH, cTnI and cTnT were determined (n = 8 for all groups). No detectable changes in calpain activities were observed following I/R with and without E-64c (p > 0.05). Both I/R conditions reduced calpastatin activity (p < 0.05) while E-64c pre-treatment was without effect, implicating a non-proteolytic event underlying the calpastatin changes. A similar result was noted for calpain-calpastatin ratios and the release of all marker proteins (p < 0.05). In regard to cardiac function, E-64c resulted in transient improvements (15 min) for left ventricular developed pressure (LVDP) and rate of pressure development (p < 0.05). E-64c had no effect on end diastolic pressure (LVEDP) or coronary pressure (CP) during I/R. These findings demonstrate that restricting the putative early burst in calpain activity, suggested for I/R, by pre-treatment of rats with E-64c does not prevent downregulation of calpastatin inhibitory activity and/or protein release despite a transient improvement in cardiac function. It is concluded that increases in calpain isoform activities are not a primary feature of l/R changes, although the role of calpastatin downregulation remains to be elucidated.

Topics: Animals; Calcium-Binding Proteins; Calpain; Male; Myocardial Ischemia; Rats; Rats, Wistar; Reperfusion Injury

Although global ischemia induces troponin I (TnI) degradation, regional ischemia does not. We hypothesized that this disparity is related to preload-induced proteolysis, which varies as a function of the amount of myocardium at risk of ischemia.. Isolated rat hearts were buffer-perfused at controlled levels of preload. Increasing preload to 25 mm Hg in the absence of ischemia produced pronounced TnI degradation (27 kDa versus 31 kDa bands: 16.4 +/- 3.6% versus 4.7 +/- 1.9% in immediately excised controls, P<0.05). TnI degradation could be blocked by preventing the activation of endogenous calpains with 25 micromol/L calpeptin (4.3 +/- 0.6%). This improved function, with left ventricular systolic pressure increasing from 103 +/- 4 mm Hg to 137 +/- 7 mm Hg (P<0.05). Eliminating elevations in preload after global ischemia-induced stunning also prevented TnI degradation.. Calpain-mediated TnI proteolysis can be dissociated from stunning and arises from elevations in preload rather than ischemia. This raises the possibility that ongoing preload-induced TnI degradation could impair myocardial function long-term.

Topics: Animals; Blood Pressure; Calpain; Cardiac Pacing, Artificial; Cysteine Proteinase Inhibitors; Dipeptides; Heart; Hemodynamics; In Vitro Techniques; Myocardial Ischemia; Myocardium; Rats; Rats, Sprague-Dawley; Troponin I; Ventricular Function, Left

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.

Topics: Amino Acid Sequence; Animals; BH3 Interacting Domain Death Agonist Protein; Calpain; Carrier Proteins; Male; Molecular Sequence Data; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Rabbits; Recombinant Proteins

The aim of this study was to investigate the role of secondary free radicals and calpain, a calcium-activated cysteine protease, in the development of reperfusion injury in the heart. The time course of radical generation was assessed directly by Electron Paramagnetic Resonance (EPR) and spin trapping with N-ter butyl-alpha-phenylnitrone (PBN), in isolated perfused rat heart subjected to 30 minutes of global ischemia and 30 minutes of reperfusion. The effect of leupeptin, a calpain inhibitor, was assessed on postischemic dysfunction. The antioxidant properties of leupeptin were also investigated by using allophycocyanin, a fluorescent protein sensitive to oxidative stress generated by the H2O2 + Cu++ system. Moreover, we measured the capacities of leupeptin to scavenge hydroxyl (.OH) and superoxide (O2-.) radicals using EPR technique. Our results show that myocardial reperfusion is associated with an increase of alkyl, alkoxyl free radicals release; the administration of catalase 5.10(5) UI/L significantly reduces this release, but didn't improve the postischemic contractile function of the heart. In our study leupeptin 50 microM possess, in vitro, antioxidant properties and scavenging abilities against .OH and O2-., in return leupeptin does not influence the cardiac functions during reperfusion period. In conclusion, our results confirm that myocardial reperfusion induces an important production of secondary free radicals associated with contractile dysfunction. The role of calpain in myocardial ischemia-reperfusion injury remains to be clarified 1) by assessing the activities of calpain and calpastain, its main endogenous inhibitor, during these periods, 2) by measuring the ability of leupeptin in inhibiting the calpain dependent proteolysis.

Topics: Animals; Antioxidants; Calcium-Binding Proteins; Calpain; Catalase; Cathepsins; Cyclic N-Oxides; Cysteine Proteinase Inhibitors; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Free Radicals; Hydroxyl Radical; Leupeptins; Magnetic Resonance Spectroscopy; Male; Myocardial Contraction; Myocardial Ischemia; Myocardial Reperfusion Injury; Nitrogen Oxides; Oxidative Stress; Phycocyanin; Rats; Rats, Wistar; Spin Labels; Superoxides; Time Factors

This paper tests the hypothesis that calpains are activated in the ischemic (I)/reperfused (R) heart and contribute to myocardial stunning.. Isolated ferret hearts were Langendorff perfused isovolumically, and subjected to 20 min of global I followed by 30 min of R in the presence or absence of 0.2 microM MDL-28170, a membrane-permeant calpain inhibitor. Right trabeculae then were isolated from these hearts, skinned chemically, and pCa(2+)-force curves obtained. Samples of left ventricle were extracted subjected to SDS-PAGE, and Western analyzed for PKC epsilon and PKM epsilon.. Perfused ferret hearts exhibit a 43% decline in left ventricular developed pressure during R. Pre-treatment of hearts with MDL-28170 prior to I significantly improves function during R. Trabecular myofilaments from normal hearts have a KD for Ca2+ of 6.27 +/- 0.06; I/R decreased the KD to 6.09 +/- 0.04; trabeculae from I/R hearts pre-treated with MDL-28170 have a KD of 6.28 +/- 0.04. Western analysis shows ferret hearts to contain a single approximately equal to 96 kDa species of PKC epsilon. I/R hearts contain the native PKC epsilon and a approximately equal to 25 kDa smaller species of PKC epsilon which corresponds to PKM epsilon, the calpain proteolyzed form of PKC epsilon. Pre-treatment of I/R hearts with MDL-28170 markedly diminishes PKM epsilon in reperfused hearts.. Mechanical stunning during R is sensitive to MDL-28170. Depressed mechanical function is reflected in a hyposensitization of trabecular myofilaments to Ca2+. Western analysis shows that PKM epsilon is present in R hearts.

Topics: Animals; Calcium; Calpain; Cell Membrane Permeability; Dipeptides; Ferrets; In Vitro Techniques; Isoenzymes; Myocardial Ischemia; Myocardial Stunning; Myocardium; Perfusion; Protein Kinase C; Protein Kinase C-epsilon

Ankyrin links cytoskeleton and integral membrane proteins and is proteolyzed in vitro by calpain, a Ca2+-dependent protease. In the present study, we examined the localization of two ankyrin isoforms, erythrocyte (red blood cell)-type (ankyrin(R)) and brain-type (ankyrin(B)), and their proteolysis after ischemia-reperfusion in the subcellular fractions of perfused rat heart by immunoblotting and by immunohistochemistry using specific antibodies. Both isoforms were observed to be distributed chiefly in the myofibril-nucleus (1,OOOx g pellet: P1) fraction, while ankyrin(R) was located substantially in the membrane (100,000x g pellet: P2) fraction. Reperfusion after 10 min or more of global ischemia induced preferential proteolysis of ankyrin(R) in the P2 fraction and ankyrin(B) in the P1 fraction. The proteolysis of ankyrin(R), but not ankyrin(B), was effectively inhibited by the synthetic calpain inhibitor acethyl-leucyl-leucyl-norleucinal. The immunohistochemical examination showed that anti-ankyrin(R) delineated striations, sarcolemma and nuclei, and the staining was decreased after ischemia-reperfusion, while anti-ankyrin(B) showed diffuse staining. The proteolysis of ankyrin(R) may interfere with force conduction through disruption of the linkage between integral membrane proteins and the myofibril-cytoskeleton.

Topics: Animals; Ankyrins; Brain; Calpain; Cell Fractionation; Cysteine Proteinase Inhibitors; Erythrocytes; In Vitro Techniques; Leupeptins; Male; Molecular Weight; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Peptide Fragments; Rats; Rats, Wistar

The activities of calpain and its endogenous inhibitor, calpastatin, were measured in the soluble fraction of perfused rat heart after ischemia for 5-20 min and reperfusion for up to 30 min. The method for m-calpain measurement was modified: washing of the DEAE-cellulose column with 0.18 M NaCl instead of 0.15 M NaCl increased the m-calpain activity 12.5-fold. Ischemia for 20 min followed by reperfusion for 30 min did not affect the m-calpain activity but decreased the calpastatin activity. m-Calpain was enriched in the nucleus-myofibril fraction but was not further translocated on ischemia-reperfusion. Mu-calpain was below the limit of detection on immunoblotting or casein zymography, but its mRNA was substantially expressed, as detected on Northern blotting. Casein zymography also revealed a novel Ca2+-dependent protease without the typical characteristics of mu- or m-calpain. The immunoblotting of myocardial fractions showed that calpastatin was proteolyzed on ischemia-reperfusion. The calpastatin proteolysis was suppressed by a calpain inhibitor, Ac-Leu-Leu-norleucinal. Calpastatin may sequester calpain from its substrates in the normal myocardium, but may be proteolyzed by calpain in the presence of an unidentified activator in the early phase of calpain activation during ischemia-reperfusion, resulting in the proteolysis of calpastatin and then other calpain substrates.

Topics: Animals; Blotting, Northern; Blotting, Western; Calcium-Binding Proteins; Calpain; Chromatography, DEAE-Cellulose; Cysteine Proteinase Inhibitors; Down-Regulation; Hydrolysis; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Rats; Rats, Wistar; RNA, Messenger

We investigated the mechanism of the decreased myofilament Ca2+ responsiveness in stunned myocardium. The steady state force-[Ca2+] relationship was measured before and after skinning in thin ventricular trabeculae from control or stunned (20 minutes of ischemia, 20 minutes of reperfusion) rat hearts.[Ca2+]i was determined using microinjected fura 2 salt in intact muscles, whereas the myofilaments of chemically skinned trabeculae were activated directly with solutions of varied [Ca2+]. Maximal Ca2+- activated force (F max) before and after skinning was identical within either the control or stunned groups but was markedly depressed in both groups of stunned trabeculae (P < .001)). After ischemia and reperfusion, the [Ca2+] required for 50% of maximal activation (Ca50) was increased in both intact (control, 0.60 +/- 0.09 micromol/L; stunned, 0.85 +/- 0.09 micromol/L;P < .001) and skinned (control, 1.13 +/- 0.24 micromol/L; stunned 1.39 +/- 0.21 micromol/L; P = .0025) trabeculae. These data indicate that the decreased Ca2+ responsiveness of stunned myocardium is due to intrinsic alterations of the myofilaments. Therefore, we tested the hypothesis that activation of proteases by reperfusion-induced Ca2+ overload decreases the Ca2+ responsiveness of the cardiac myofilaments. Force-[Ca2+] relations were compared before and 5 to 30 minutes after direct exposure of skinned trabeculae to calpain I (18 microgram/mL, 20 minutes at [Ca2+]=10.8 micromol/L), a Ca2+-activated protease that is present in myocardium. Calpain I reduced F max from 94.3 +/- 8.3 to 56 +/- 8.5 mN/mm2 while increasing Ca50 from 0.94 +/- 0.11 to 1.36 +/- 0.21 micromol/L (P < .01). Calpastatin, a specific calpain inhibitor prevented the effects of calpain I on skinned trabeculae. The results show that the reduced Ca2+ responsiveness of stunned myocardium reflects alteration of the myofilaments themselves, not of soluble cytosolic factors, which can be faithfully reproduced by exposure to Ca2+-dependent protease.

Topics: Actin Cytoskeleton; Animals; Calcium; Calpain; Endopeptidases; Female; Hydrolysis; In Vitro Techniques; Kinetics; Male; Myocardial Contraction; Myocardial Ischemia; Myocardial Reperfusion Injury; Rats; Rats, Sprague-Dawley

During myocardial ischemia, the increase in cytosolic Ca2+ promotes the activation of neutral proteases such as calpains. Since the troponin T subunit is a substrate for calpains, we investigated the effects of irreversible myocyte damage on troponin T immunoreactivity.. Hearts from adult guinea pigs (n=32) were perfused under conditions of normoxia, ischemia, postischemic reperfusion, or Ca2+ paradox. Hearts were frozen and processed for immunohistochemistry and Western blot with three anti-troponin T monoclonal antibodies. Two of these antibodies are unreactive on cryosections of freshly isolated and normoxic hearts and of hearts exposed to 30 minutes of no-flow ischemia. In contrast, reactivity is detected in rare myocytes after 60 minutes of ischemia, in a large population of myocytes after 60 minutes of ischemia followed by 30 minutes of reperfusion, and in every myocyte exposed to Ca2+ paradox. In Western blots, samples from ischemia-reperfusion and Ca2+ overloaded hearts show reactive polypeptides of about 240 to 260 kD and 65 to 66 kD in addition to troponin T. A similar pattern of immunoreactivity is observed with an anti-troponin I antibody. Histochemical troponin T immunoreactivity and reactivity on high-molecular-weight polypeptides are detectable in normal heart samples after preincubation with 10 mmol/L Ca2+ or with transglutaminase, whereas they are not if either transglutaminase or calpain is inhibited.. The evolution of the ischemic injury is accompanied by changes in troponin T immunoreactivity as a consequence of the calcium-dependent activation of both calpain proteolysis and transglutaminase cross-linking.

Topics: Animals; Calcium; Calpain; Guinea Pigs; Immunohistochemistry; Male; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; Osmolar Concentration; Transglutaminases; Troponin; Troponin T

To explore the spatial and temporal localization of PKC isoforms during ischemia, we quantified PKC isoforms in the subcellular fractions in perfused rat heart by immunoblotting using specific antibodies against PKC isoforms. PKCs-alpha and epsilon translocated from the 100000 x g supernatant (S, cytosolic) fraction to the 1000 x g pellet (PI, nucleus-myofibril) and the 1000-100000 x g pellet (P2, membrane) fractions during 5-40 min of ischemia. PKC-delta redistributed from the P2 to the S fraction. A 50-kDa fragment of PKC-alpha appeared during ischemia possibly through calpain action. Immunohistochemical observations showed the different localizations of PKC-alpha, delta, and epsilon in the myocytes. The PKC assay displayed high basal levels of Ca(2+)-independent PKC, the activation of Ca(2+)-dependent PKC in the P1 and P2 fractions, and the activation of Ca(2+)-independent PKC in the P1 fraction after 20 min of ischemia. These observations show that ischemia induces different patterns of translocation of the three PKC isoforms, suggesting differences in their roles.

Topics: Animals; Calcium; Calpain; Cell Fractionation; Cell Membrane; Cell Nucleus; Cytosol; Glycoproteins; Immunoblotting; Immunoenzyme Techniques; Isoenzymes; Male; Molecular Weight; Myocardial Ischemia; Myocardium; Myofibrils; Protein Kinase C; Rats; Rats, Wistar

Rat myocardium expresses the 240- and 235-kD polypeptides antigenically related to alpha- and beta-subunits of brain calspectin (nonerythroid spectrin or fodrin), respectively. In the subcellular fractions of the myocardium, alpha-calspectin was found in the 600g, 10,000g, and 100,000g pellets, whereas beta-calspectin was localized to the 10,000g pellet. On the basis of the Na+,K(+)-ATPase activity and the contents of a gap junction protein, the sarcolemma was distributed to the 10,000g and 100,000g pellets, and the intercalated disks were enriched in the 10,000g pellet. Both alpha- and beta-calspectin were proteolyzed by calpain in vitro. The two subunits were also proteolyzed in vivo, when the rat hearts underwent 10 to 60 minutes of global ischemia followed by 30 minutes of reperfusion. The reperfusion following the ischemia induced the proteolysis of alpha-calspectin in the 10,000g and 100,000g pellets, producing the 150-kD fragment. A synthetic calpain inhibitor, calpain inhibitor-1, suppressed the degradation of calspectin in vivo, which indicates that calpain is responsible for the reperfusion-induced proteolysis of calspectin. The inhibitor also improved myocardial stunning. Immunohistochemical study revealed that the proteolysis of alpha-calspectin occurs at the intercalated disks and the sarcolemma after postischemic reperfusion, in accord with the biochemical data. These results suggest that degradation of calspectin partly accounts for the contractile failure of the myocardium after postischemic reperfusion by disrupting the membrane skeleton and the intercalated disks.

Topics: Animals; Calmodulin-Binding Proteins; Calpain; Myocardial Ischemia; Myocardial Reperfusion; Rats; Rats, Wistar

Calpain activity was measured in the various subfractions of rat myocardia after global ischemia for 60 min or after ischemia followed by 30 min of reperfusion after the chromatographic separation of mu- and m-calpains. The activity of m-calpain after ischemia and that of mu-calpain after reperfusion were both higher than that in the control. The activity of the endogenous calpain inhibitor calpastatin in 10,000 x g supernatant was decreased after both ischemia and ischemia-reperfusion. The increase in m- and mu-calpain activities was suppressed by pre-ischemic perfusion with a synthetic calpain inhibitor, transepoxysuccinyl-L-leucylamido (4-guanidino) butane (E64d, 100 micrograms/ml). After reperfusion, the calpain activity in the 10,000 x g pellet was also increased, which was inhibited by pre-ischemic perfusion with E64d or dimethylsulfoxide (a solvent for E64d) or by reperfusion with 1 mmol/L ethyleneglycol bis (beta-aminoethylether)-N, N, N', N'-tetraacetic acid. SDS-polyacrylamide gel electrophoresis revealed the proteolysis of several proteins, including fodrin, in the 10,000 x g and 100,000 x g pellet fractions after ischemia and reperfusion, some of which were confirmed to be in vitro substrates of calpain. The creatine kinase release during the reperfusion was also partially inhibited by E64d or dimethylsulfoxide. Thus, calpain activity in the soluble or particulate fractions was altered during ischemia or reperfusion, and appeared to be implicated in the proteolysis of the membrane proteins, which may contribute to myocardial injury.

Topics: Animals; Calcium-Binding Proteins; Calpain; Creatine Kinase; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; Leucine; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Rats; Rats, Wistar