calpain and Myocardial-Infarction

Reperfusion may induce additional cell death in patients with acute myocardial infarction receiving primary angioplasty or thrombolysis. Altered intracellular Ca(2+) handling was initially considered an essential mechanism of reperfusion-induced cardiomyocyte death. However, more recent studies have demonstrated the importance of Ca(2+)-independent mechanisms that converge on mitochondrial permeability transition (MPT) and are shared by cardiomyocytes and other cell types. This article analyses the importance of Ca(2+)-dependent cell death in light of these new observations. Altered Ca(2+) handling includes increased cytosolic Ca(2+) levels, leading to activation of calpain-mediated proteolysis and sarcoplasmic reticulum-driven oscillations; this can induce hypercontracture, but also MPT due to the privileged Ca(2+) transfer between sarcoplasmic reticulum and mitochondria through cytosolic Ca(2+) microdomains. In the opposite direction, permeability transition can worsen altered Ca(2+) handling and favour hypercontracture. Ca(2+) appears to play an important role in cell death during the initial minutes of reperfusion, particularly after brief periods of ischaemia. Developing effective and safe treatments to prevent Ca(2+)-mediated cardiomyocyte death in patients with transient ischaemia, by targeting Ca(2+) influx, intracellular Ca(2+) handling, or Ca(2+)-induced cell death effectors, is an unmet challenge with important therapeutic implications and large potential clinical impact.

Topics: Calcium; Calpain; Cell Death; Humans; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Myocardial Infarction; Myocardial Reperfusion; Myocardial Reperfusion Injury; Myocardium; Myocytes, Cardiac; Sarcoplasmic Reticulum

The precise mechanism(s) of the progression of advanced heart failure (HF) should be determined to establish strategies for its treatment or prevention. Based on pathological, molecular, and physiological findings in 3 animal models and human cases, we propose a novel scheme that a vicious cycle formed by increased sarcolemma (SL) permeability, preferential activation of calpain over calpastatin, and translocation and cleavage of dystrophin (Dys) commonly lead to advanced HF. The aim of this article was to assess our recent paradigm that disruption of myocardial Dys is a final common pathway to advanced HF, irrespective of its hereditary or acquired origin, but not intended to provide a comprehensive overview of the various factors that may be involved in the course of HF in different clinical settings. In addition, each component of Dys-associated proteins (DAP) was heterogeneously degraded in vivo and in vitro, i.e. Dys and alpha-sarcoglycan (SG) were markedly destroyed using isolated calpain 2, while delta-SG was not degraded at all. The up-regulation of calpain 2 was confirmed through previously published data that remain insufficient for precise evaluation, supporting our new scheme that the activation of calpain(s) is involved in the steady process of Dys cleavage. In addition, somatic gene therapy is discussed as a potential option to ameliorate the physiological/metabolic indices and to improve the prognosis.

Topics: Animals; Calpain; Cardiomyopathy, Dilated; Disease Models, Animal; Dystrophin; Genetic Therapy; Heart Failure; Humans; Myocardial Infarction; Sarcoglycans; Transduction, Genetic

Topics: Alcohol Drinking; Alcoholism; Animals; Apoptosis; Calpain; Cardiolipins; Caspase 3; Cytochromes c; Dimethyl Sulfoxide; Ethanol; Isoproterenol; Male; Myocardial Infarction; Myocardium; Rats; Rats, Wistar

Aging chronically increases endoplasmic reticulum (ER) stress that contributes to mitochondrial dysfunction. Activation of calpain 1 (CPN1) impairs mitochondrial function during acute ER stress. We proposed that aging-induced ER stress led to mitochondrial dysfunction by activating CPN1. We posit that attenuation of the ER stress or direct inhibition of CPN1 in aged hearts can decrease cardiac injury during ischemia-reperfusion by improving mitochondrial function. Male young (3 mo) and aged mice (24 mo) were used in the present study, and 4-phenylbutyrate (4-PBA) was used to decrease the ER stress in aged mice. Subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) were isolated. Chronic 4-PBA treatment for 2 wk decreased CPN1 activation as shown by the decreased cleavage of spectrin in cytosol and apoptosis inducing factor (AIF) and the α1 subunit of pyruvate dehydrogenase (PDH) in mitochondria. Treatment improved oxidative phosphorylation in 24-mo-old SSM and IFM at baseline compared with vehicle. When 4-PBA-treated 24-mo-old hearts were subjected to ischemia-reperfusion, infarct size was decreased. These results support that attenuation of the ER stress decreased cardiac injury in aged hearts by improving mitochondrial function before ischemia. To challenge the role of CPN1 as an effector of the ER stress, aged mice were treated with MDL-28170 (MDL, an inhibitor of calpain 1). MDL treatment improved mitochondrial function in aged SSM and IFM. MDL-treated 24-mo-old hearts sustained less cardiac injury following ischemia-reperfusion. These results support that age-induced ER stress augments cardiac injury during ischemia-reperfusion by impairing mitochondrial function through activation of CPN1.

Topics: Age Factors; Animals; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Disease Models, Animal; Endoplasmic Reticulum Stress; Enzyme Activation; Isolated Heart Preparation; Male; Mice, Inbred C57BL; Mitochondria, Heart; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Oxidative Phosphorylation; Phenylbutyrates

Ischemia and anoxia-induced mitochondrial impairment may be a key factor leading to heart injury during myocardial infarction (MI). Calpain 1 and 2 are involved in the MI-induced mitochondria injury. G protein-coupled receptor 35 (GPR35) could be triggered by hypoxia. Whether or not GPR35 regulates calpain 1/2 in the pathogenesis of MI is still unclear. In this study, we determined that MI increases GPR35 expression in myocardial tissue. Suppression of GPR35 protects heart from MI injury in mice through reduction of reactive oxygen species activity and mitochondria-dependent apoptosis. Further studies show that GPR35 regulates calpain 1/2. Suppression of GPR35 reduces the expression and activity of calpain 1/2, and alleviates calpain 1/2-associated mitochondrial injury to preserve cardiac function. Based on these data, we conclude that a functional inhibition of GPR35 downregulates calpain 1/2 and contributes to maintenance of cardiac function under pathologic conditions with mitochondrial disorder. In conclusion, our study showed that the identified regulation by GPR35 of calpain 1/2 has important implications for the pathogenesis of MI. Targeting the action of GPR35 and calpain 1/2 in mitochondria presents a potential therapeutic intervention for MI.

Topics: Animals; Apoptosis; Calpain; Cells, Cultured; Disease Models, Animal; Male; Mice, Inbred C57BL; Mitochondria, Heart; Myocardial Infarction; Myocytes, Cardiac; Oxidative Stress; Reactive Oxygen Species; Receptors, G-Protein-Coupled; RNA, Small Interfering; RNAi Therapeutics; Signal Transduction

Mitochondrial dynamic disorder is involved in myocardial ischemia/reperfusion (I/R) injury. To explore the effect of mitochondrial calcium uniporter (MCU) on mitochondrial dynamic imbalance under I/R and its related signal pathways, a mouse myocardial I/R model and hypoxia/reoxygenation model of mouse cardiomyocytes were established. The expression of MCU during I/R increased and related to myocardial injury, enhancement of mitochondrial fission, inhibition of mitochondrial fusion and mitophagy. Suppressing MCU functions by Ru360 during I/R could reduce myocardial infarction area and cardiomyocyte apoptosis, alleviate mitochondrial fission and restore mitochondrial fusion and mitophagy. However, spermine administration, which could enhance MCU function, deteriorated the above-mentioned myocardial cell injury and mitochondrial dynamic imbalanced. In addition, up-regulation of MCU promoted the expression and activation of calpain-1/2 and down-regulated the expression of Optic atrophy type 1 (OPA1). Meantime, in transgenic mice (overexpression calpastatin, the endogenous inhibitor of calpain) I/R model and OPA1 knock-down cultured cell. In I/R models of transgenic mice over-expressing calpastatin, which is the endogenous inhibitor of calpain, and in H/R models with siOPA1 transfection, inhibition of calpains could enhance mitochondrial fusion and mitophagy, and inhibit excessive mitochondrion fission and apoptosis through OPA1. Therefore, we conclude that during I/R, MCU up-regulation induces calpain activation, which down-regulates OPA1, consequently leading to mitochondrial dynamic imbalance.

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Apoptosis; Calcium Channels; Calcium-Binding Proteins; Calpain; Enzyme Inhibitors; GTP Phosphohydrolases; Homeostasis; Male; Mice, Inbred C57BL; Mice, Transgenic; Mitochondria, Heart; Mitochondrial Dynamics; Mitophagy; Myocardial Infarction; Myocardial Reperfusion Injury; Protective Agents; Rats; Up-Regulation

Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is essential for normal heart function and protects the heart from ischemia-reperfusion (I/R) injury. It is known that protein kinase-A (PKA)-mediated phosphorylation of cMyBP-C prevents I/R-dependent proteolysis, whereas dephosphorylation of cMyBP-C at PKA sites correlates with its degradation. While sites on cMyBP-C associated with phosphorylation and proteolysis co-localize, the mechanisms that link cMyBP-C phosphorylation and proteolysis during cardioprotection are not well understood. Therefore, we aimed to determine if abrogation of cMyBP-C proteolysis in association with calpain, a calcium-activated protease, confers cardioprotection during I/R injury. Calpain is activated in both human ischemic heart samples and ischemic mouse myocardium where cMyBP-C is dephosphorylated and undergoes proteolysis. Moreover, cMyBP-C is a substrate for calpain proteolysis and cleaved by calpain at residues 272-TSLAGAGRR-280, a domain termed as the calpain-target site (CTS). Cardiac-specific transgenic (Tg) mice in which the CTS motif was ablated were bred into a cMyBP-C null background. These Tg mice were conclusively shown to possess a normal basal structure and function by analysis of histology, electron microscopy, immunofluorescence microscopy, Q-space MRI of tissue architecture, echocardiography, and hemodynamics. However, the genetic ablation of the CTS motif conferred resistance to calpain-mediated proteolysis of cMyBP-C. Following I/R injury, the loss of the CTS reduced infarct size compared to non-transgenic controls. Collectively, these findings demonstrate the physiological significance of calpain-targeted cMyBP-C proteolysis and provide a rationale for studying inhibition of calpain-mediated proteolysis of cMyBP-C as a therapeutic target for cardioprotection.

Topics: Animals; Calpain; Cardiotonic Agents; Carrier Proteins; Female; Heart Function Tests; Humans; Male; Mice, Transgenic; Middle Aged; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Phosphorylation; Proteolysis

Topics: Animals; Apoptosis; Bone Marrow; Calcium; Calpain; Cardiotonic Agents; Caspase 12; Cell Hypoxia; Cellular Reprogramming; Doxorubicin; Endoplasmic Reticulum Stress; Enzyme Activation; Fibroblasts; Gene Deletion; GTP-Binding Protein alpha Subunits, Gq-G11; Humans; Male; Mice; Myocardial Infarction; Myocytes, Cardiac; Prostaglandin D2; Receptors, Immunologic; Receptors, Prostaglandin; Regeneration; Tetrazoles

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

Calpains activate during myocardial ischemia-reperfusion and contribute to reperfusion injury. Studies in transgenic animals with altered calpain/calpastatin system subjected to permanent ischemia suggest that calpains are also involved in post-infarction remodelling and heart failure.. To determine whether delayed oral administration of the calpain inhibitor SNJ-1945 reduces adverse myocardial remodelling and dysfunction following transient coronary occlusion.. Male Sprague-Dawley rats were subjected to 30 min of ischemia followed by 21 days of reperfusion and received the calpain inhibitor SNJ-1945 intraperitoneally at the onset of reperfusion (Acute group), orally starting after 24 h of reperfusion and for 14 days (Chronic group), or the combination of both treatments. Calpain-1 and calpain-2 protein content increased and correlated with higher calpain activity in control hearts. Administration of SNJ-1945 attenuated calpain activation, and reduced scar expansion, ventricular dilation and dysfunction in both acute and chronic groups. Acute treatment reduced infarct size in hearts reperfused for 24 h and inflammation measured after 3 days. Delayed, chronic oral administration of SNJ-1945 attenuated inflammation, cardiomyocyte hypertrophy and collagen infiltration in the non-infarcted myocardium at 21 days in correlation with increased levels of IĸB and reduced NF-ĸB activation. In cultured fibroblasts, SNJ-1945 attenuated TGF-β1-induced fibroblast activation.. Our data demonstrate for the first time that long-term calpain inhibition is possible with delayed oral treatment, attenuates adverse post-infarction remodelling, likely through prevention of NF-ĸB activation, and may be a promising therapeutic intervention to prevent adverse remodelling and heart failure in patients with acute myocardial infarction.

Topics: Animals; Calcium-Binding Proteins; Calpain; Carbamates; Glycoproteins; Heart; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Rats, Sprague-Dawley

Cardioprotection represents one of the most important and realistic aspects of preventive therapy today. Quercetin, a naturally occurring dietary flavone, has been studied extensively for its antioxidant properties. The objective of present study is to find out the cardioprotective activity and to explore the underlying mechanisms of quercetin pretreatment (50 mg/kg body weight, orally) for 14 days against isoproterenol (ISO; 100 mg/kg body weight, subcutaneously) induced myocardial infarction in Wistar rats. Cardiac diagnostic markers, oxidative stress, inflammatory cytokines, histopathology along with gene expression analysis of calpain 1 and 2 were carried out in experimental rats. Quercetin pretreatment showed protective effects on heart by significantly attenuating the ISO-induced oxidative stress, inflammation, protecting heart architecture, and by downregulation of the expression of calpain. Overall, these findings revealed the cardio-protective potential of quercetin and its mechanism of action against ISO-induced MI in rats.

Topics: Animals; Calpain; Cardiotonic Agents; Gene Expression Regulation, Enzymologic; Isoproterenol; Male; Myocardial Infarction; Myocardium; Oxidative Stress; Quercetin; Rats; Rats, Wistar

Calpain is activated following myocardial infarction and ablation of calpastatin (CAST), an endogenous inhibitor of calpains, promotes left ventricular remodeling after myocardial infarction (MI). The present study aimed to investigate the effect of transgenic over-expression of CAST on the post-infarction myocardial remodeling process.. We established transgenic mice (TG) ubiquitously over-expressing human CAST protein and produced MI in TG mice and C57BL/6J wild-type (WT) littermates.. The CAST protein expression was profoundly upregulated in the myocardial tissue of TG mice compared with WT littermates (P < 0.01). Overexpression of CAST significantly reduced the infarct size (P < 0.01) and blunted MI-induced interventricular hypertrophy, global myocardial fibrosis and collagen I and collagen III deposition, hypotension and hemodynamic disturbances at 21 days after MI. Moreover, the MI-induced up-regulation and activation of calpains were obviously attenuated in CAST TG mice. MI-induced down-regulation of CAST was partially reversed in TG mice. Additionally, the MI-caused imbalance of matrix metalloproteinases and their inhibitors was improved in TG mice.. Transgenic over-expression of CAST inhibits calpain activation and attenuates post-infarction myocardial remodeling.

Topics: Animals; Calpain; Cardiomegaly; Collagen Type I; Collagen Type III; Cytoskeletal Proteins; Disease Models, Animal; Enzyme Activation; Female; Fibrosis; Gene Expression Regulation; Humans; Isoenzymes; Male; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases, Secreted; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myocardial Infarction; Survival Analysis; Ventricular Function, Left; Ventricular Remodeling

Pro-free radical oxidative stresses, as well as regulatory factors, are believed to be the key players in the development of diabetes and heart-related disorders such as myocardial infarction. The aim of the present study was to highlight the role of oxidative stress-responsive factors (reactive oxygen species [ROS], super oxide dismutase [SOD], and calpain-1) in type 2 diabetes and myocardial infarction.. A total of 100 type 2 diabetes patients with myocardial infarction and 50 normal individuals were selected for this analysis. The levels of ROS and activities of SOD in the serum were determined. Serum calpain-1 expression was checked using western blotting.. The serum level of ROS and the expression of calpain-1 were significantly higher while the activity of SOD was significantly lower in diabetic patients with myocardial infraction compared to normal individuals.. These findings suggest a possible link between decreased antioxidant (SOD) and increased ROS levels as well as calpain-1 expression, supporting the role of oxidative stress-regulatory factors in diabetes and myocardial infraction.

Topics: Calpain; Case-Control Studies; Diabetes Mellitus, Type 2; Female; Humans; Male; Middle Aged; Myocardial Infarction; Oxidative Stress; Reactive Oxygen Species; Superoxide Dismutase

The activation of the calpain system is involved in the repair process following myocardial infarction (MI). However, the impact of the inhibition of calpain by calpastatin, its natural inhibitor, on scar healing and left ventricular (LV) remodeling is elusive. Male mice ubiquitously overexpressing calpastatin (TG) and wild-type (WT) controls were subjected to an anterior coronary artery ligation. Mortality at 6 wk was higher in TG mice (24% in WT vs. 44% in TG, P < 0.05) driven by a significantly higher incidence of cardiac rupture during the first week post-MI, despite comparable infarct size and LV dysfunction and dilatation. Calpain activation post-MI was blunted in TG myocardium. In TG mice, inflammatory cell infiltration and activation were reduced in the infarct zone (IZ), particularly affecting M2 macrophages and CD4(+) T cells, which are crucial for scar healing. To elucidate the role of calpastatin overexpression in macrophages, we stimulated peritoneal macrophages obtained from TG and WT mice in vitro with IL-4, yielding an abrogated M2 polarization in TG but not in WT cells. Lymphopenic Rag1(-/-) mice receiving TG splenocytes before MI demonstrated decreased T-cell recruitment and M2 macrophage activation in the IZ day 5 after MI compared with those receiving WT splenocytes. Calpastatin overexpression prevented the activation of the calpain system after MI. It also impaired scar healing, promoted LV rupture, and increased mortality. Defective scar formation was associated with blunted CD4(+) T-cell and M2-macrophage recruitment.

Topics: Animals; Calcium-Binding Proteins; Calpain; CD4-Positive T-Lymphocytes; Chemotaxis, Leukocyte; Disease Models, Animal; Enzyme Activation; Genotype; Heart Rupture, Post-Infarction; Homeodomain Proteins; Lymphocyte Activation; Macrophage Activation; Macrophages; Male; Mice, Inbred C57BL; Mice, Knockout; Myocardial Infarction; Myocardium; Phenotype; Time Factors; Up-Regulation; Ventricular Dysfunction, Left; Ventricular Function, Left; Ventricular Remodeling; Wound Healing

Enzymatic proteolysis by calpains, Ca(2+)-dependent intracellular cysteine proteases, has been implicated in pathological processes such as cellular degeneration or death. Here, we investigated the role of calpain activation in the hearts subjected to myocardial infarction. We produced myocardial infarction in Cast(-/-) mice deficient for calpastatin, the specific endogenous inhibitory protein for calpains, and Cast(+/+) mice. The activity of cardiac calpains in Cast(+/+) mice was not elevated within 1 day but showed a gradual elevation after 7 days following myocardial infarction, which was further pronounced in Cast(-/-) mice. Although the prevalence of cardiomyocyte death was indistinguishable between Cast(-/-) and Cast(+/+) mice, Cast(-/-) mice exhibited profound contractile dysfunction and chamber dilatation and showed a significant reduction in survival rate after myocardial infarction as compared with Cast(+/+) mice. Notably, immunofluorescence revealed that at 28 days after myocardial infarction, calpains were activated in cardiomyocytes exclusively at the border zone and that Cast(-/-) mice showed higher intensity and a broader extent of calpain activation at the border zone than Cast(+/+) mice. In the border zone of Cast(-/-) mice, pronounced activation of calpains was associated with a decrease in N-cadherin expression and up-regulation of molecular markers for cardiac hypertrophy and fibrosis. In cultured rat neonatal cardiomyocytes, calpain activation by treatment with ionomycin induced cleavage of N-cadherin and decreased expression levels of β-catenin and connexin 43, which was attenuated by calpain inhibitor. These results thus demonstrate that activation of calpains disassembles cell-cell adhesion at intercalated discs by degrading N-cadherin and thereby promotes left ventricular remodeling after myocardial infarction.

Topics: Animals; beta Catenin; Cadherins; Calcium-Binding Proteins; Calpain; Connexin 43; Enzyme Activation; Mice; Mice, Knockout; Muscle Proteins; Myocardial Infarction; Myocytes, Cardiac; Nerve Tissue Proteins; Rats; Rats, Wistar; Ventricular Remodeling

Junctin and triadin are calsequestrin-binding proteins that regulate sarcoplasmic reticulum (SR) Ca(2+) release by interacting with the ryanodine receptor. The levels of these proteins are significantly down-regulated in failing human hearts. However, the significance of such decreases is currently unknown. Here, we addressed the functional role of these accessory proteins in the heart's responses to ischaemia/reperfusion (I/R) injury.. Isolated mouse hearts were subjected to global I/R, and contractile parameters were assessed in wild-type (WT), junctin-knockout (JKO), and triadin-knockout (TKO) hearts. Both JKO and TKO were associated with significantly depressed post-I/R contractile recovery. However, ablation of triadin resulted in the most severe post-I/R phenotype. The additional contractile impairment of TKO hearts was not related to a mitochondrial death pathway, but attributed to endoplasmic reticulum (ER) stress-mediated apoptosis. Activation of the X-box-binding protein-1 and transcriptional up-regulation of C/EBP-homologous protein (CHOP) provided a molecular mechanism of caspase-12-dependent apoptosis in myocytes. In addition, elevation of cytosolic Ca(2+) during reperfusion was associated with the activation of calpain proteases and troponin I breakdown. Accordingly, treatment with the calpain inhibitor MDL-28170 significantly ameliorated post-I/R impairment of contractile recovery in intact hearts.. These findings indicate that deficiency of either junctin or triadin impairs the contractile recovery in post-ischaemic hearts, which appears to be primarily attributed to increased ER stress and activation of calpain.

Topics: Animals; Apoptosis; Blotting, Western; Calcium; Calcium Signaling; Calcium-Binding Proteins; Calpain; Calsequestrin; Carrier Proteins; Cysteine Proteinase Inhibitors; Dipeptides; Humans; Membrane Proteins; Mice; Mixed Function Oxygenases; Muscle Proteins; Myocardial Contraction; Myocardial Infarction; Reperfusion Injury

A prolonged myocardial ischemia, which results from a total deprivation of blood supply to an area of cardiac muscle for an appreciable period of time, is the leading mechanism responsible for acute myocardial infarction (AMI). The irreversible injury of myocardiocytes and the subsequent release of a variety of intracellular components into blood is the cornerstone of the diagnosis of AMI. Cardiac troponins are advocated as the biochemical gold standards among the various biomarkers of plaque instability, plaque rupture, ischemia, reversible cellular injury, and early and late necrosis (i.e., irreversible injury). The assessment of cardiac troponins in the diagnostic approach of patients with chest pain presents, however, some specific challenges due to the complex mechanisms of release from the injured myocardium, as well as to the enzymatic degradation by cardiac and extracardiac proteases (i.e., calpains, caspases, cathepsin L, and gelatinase A) that might alter the immunoreactivity (and thus laboratory detection) of the molecules. These two aspects will be discussed in this article, with specific focus on cardiac troponin I, as a variety of immunoassays based on antibodies which recognize different epitopes on the molecule is available for the measurement of this important cardiac biomarker.

Topics: Biomarkers; Calpain; Cathepsin L; Chest Pain; Epitope Mapping; Epitopes; Humans; Immunoassay; Leukocytes; Metalloproteases; Myocardial Infarction; Myocardium; Protein Processing, Post-Translational; Troponin I

Calpain has been implicated in acute myocardial injury after myocardial infarction (MI). However, the causal relationship between calpain and post-MI myocardial remodeling has not been fully understood. This study examined whether deletion of Capn4, essential for calpain-1 and calpain-2 activities, reduces myocardial remodeling and dysfunction following MI, and if yes, whether these effects of Capn4 deletion are associated with NF-κB signaling and inflammatory responses in the MI heart. A novel mouse model with cardiomyocyte-specific deletion of Capn4 (Capn4-ko) was employed. MI was induced by left coronary artery ligation. Deficiency of Capn4 dramatically reduced the protein levels and activities of calpain-1 and calpain-2 in the Capn4-ko heart. In vivo cardiac function was relatively improved in Capn4-ko mice at 7 and 30 days after MI when compared with their wild-type littermates. Deletion of Capn4 reduced apoptosis, limited infarct expansion, prevented left ventricle dilation, and reduced mortality in Capn4-ko mice. Furthermore, cardiomyocyte cross-sectional areas and myocardial collagen deposition were significantly attenuated in Capn4-ko mice, which were accompanied by down-regulation of hypertrophic genes and profibrotic genes. These effects of Capn4 knock-out correlated with restoration of IκB protein and inhibition of NF-κB activation, leading to suppression of proinflammatory cytokine expression and inflammatory cell infiltration in the Capn4-ko heart after MI. In conclusion, deficiency of Capn4 reduces adverse myocardial remodeling and myocardial dysfunction after MI. These effects of Capn4 deletion may be mediated through prevention of IκB degradation and NF-κB activation, resulting in inhibition of inflammatory responses.

Topics: Animals; Apoptosis; Calpain; Cells, Cultured; Cytokines; Disease Models, Animal; Endomyocardial Fibrosis; Gene Deletion; Gene Expression Regulation; I-kappa B Proteins; Inflammation; Mice; Mice, Knockout; Myocardial Infarction; Myocardium; Myocytes, Cardiac; NF-kappa B; Signal Transduction; Time Factors

The Ca(2+) paradox represents a good model to study Ca(2+) overload injury in ischemic heart diseases. We and others have demonstrated that contracture and calpain are involved in the Ca(2+) paradox-induced injury. This study aimed to elucidate their roles in this model. The Ca(2+) paradox was elicited by perfusing isolated rat hearts with Ca(2+)-free KH media for 3 min or 5 min followed by 30 min of Ca(2+) repletion. The LVDP was measured to reflect contractile function, and the LVEDP was measured to indicate contracture. TTC staining and the quantification of LDH release were used to define cell death. Calpain activity and troponin I release were measured after Ca(2+) repletion. Ca(2+) repletion of the once 3-min Ca(2+) depleted hearts resulted in almost no viable tissues and the disappearance of contractile function. Compared to the effects of the calpain inhibitor MDL28170, KB-R7943, an inhibitor of the Na(+)/Ca(2+) exchanger, reduced the LVEDP level to a greater extent, which was well correlated with improved contractile function recovery and tissue survival. The depletion of Ca(2+) for 5 min had the same effects on injury as the 3-min Ca(2+) depletion, except that the LVEDP in the 5-min Ca(2+) depletion group was lower than the level in the 3-min Ca(2+) depletion group. KB-R7943 failed to reduce the level of LVEDP, with no improvement in the LVDP recovery in the hearts subjected to the 5-min Ca(2+) depletion treatment; however, KB-R7943 preserved its protective effects in surviving tissue. Both KB-R7943 and MDL28170 attenuated the Ca(2+) repletion-induced increase in calpain activity in 3 min or 5 min Ca(2+) depleted hearts. However, only KB-R7943 reduced the release of troponin I from the Ca(2+) paradoxic heart. These results provide evidence suggesting that contracture is the main cause for contractile dysfunction, while activation of calpain mediates cell death in the Ca(2+) paradox.

Topics: Animals; Blotting, Western; Calcium; Calpain; Contracture; Dipeptides; Heart Injuries; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Rats; Rats, Sprague-Dawley; Sodium-Calcium Exchanger; Thiourea

Calpains contribute to reperfusion-induced myocardial cell death. However, it remains controversial whether its activation occurs during ischemia or reperfusion. We investigated the regulation and time-course of calpain activation secondary to transient ischemia and the efficacy of its inhibition at reperfusion as a therapeutic strategy to limit infarct size. In isolated rat hearts (Sprague-Dawley), ischemia induced a time-dependent translocation of m-calpain to the membrane that was not associated with calpain activation as assessed by proteolysis of its substrate alpha-fodrin. Translocation of calpain was dependent on Ca(2+) entry through reverse mode Na(+)/Ca(2+)-exchange and was independent of acidosis. Calpain activation occurred during reperfusion, but only after intracellular pH (pHi) normalization, and was not prevented by inhibiting its translocation during ischemia with methyl-beta-cyclodextrin. The intravenous infusion of MDL-28170 in an in vivo rat model with transient coronary occlusion during the first minutes of reperfusion resulted in a reduction of infarct size (43.9+/-3.9% vs. 60.2+/-4.7, P=0.046, n=18) and alpha-fodrin degradation. These results suggest that (1) Ca(2+)-induced calpain translocation to the membrane during ischemia is independent of its activation, (2) intracellular acidosis inhibits calpain activation during ischemia and pHi normalization allows activation upon reperfusion, and (3) calpain inhibition at the time of reperfusion appears as a potentially useful strategy to limit infarct size.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Death; Enzyme Activation; Hemodynamics; Hydrogen-Ion Concentration; In Vitro Techniques; Intracellular Space; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Protease Inhibitors; Protein Transport; Rats; Rats, Sprague-Dawley

Protein kinase (PK)Cs and calpain cysteine proteases are highly expressed in myocardium. Ischemia produces calcium overload that activates calpains and conventional PKCs. However, calpains can proteolytically process PKCs, and the potential in vivo consequences of this interaction are unknown.. To determine the biochemical and pathophysiological consequences of calpain-mediated cardiac PKCα proteolysis.. Isolated mouse hearts subjected to global ischemia/reperfusion demonstrated cleavage of PKCα. Calpain 1 overexpression was not sufficient to produce PKCα cleavage in normal hearts, but ischemia-induced myocardial PKCα cleavage and myocardial injury were greatly increased by cardiac-specific expression of calpain 1. In contrast, calpain 1 gene ablation or inhibition with calpastatin prevented ischemia/reperfusion induced PKCα cleavage; infarct size was decreased and ventricular function enhanced in infarcted calpain 1 knockout hearts. To determine consequences of PKCα fragmentation on myocardial protein phosphorylation, transgenic mice were created conditionally expressing full-length PKCα or its N-terminal and C-terminal calpain 1 cleavage fragments. Two-dimensional mapping of ventricular protein extracts showed a distinct PKCα phosphorylation profile that was exaggerated and distorted in hearts expressing the PKCα C-terminal fragment. MALDI mass spectroscopy revealed hyperphosphorylation of myosin-binding protein C and phosphorylation of atypical substrates by the PKCα C-terminal fragment. Expression of parent PKCα produced a mild cardiomyopathy, whereas myocardial expression of the C-terminal PKCα fragment induced a disproportionately severe, rapidly lethal cardiomyopathy.. Proteolytic processing of PKCα by calcium-activated calpain activates pathological cardiac signaling through generation of an unregulated and/or mistargeted kinase. Production of the PKCα C-terminal fragment in ischemic hearts occurs via a receptor-independent mechanism.

Topics: Animals; Calcium; Calpain; Cardiomyopathies; Carrier Proteins; Humans; Mice; Mice, Transgenic; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Phosphorylation; Protein Kinase C-alpha; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Signal Transduction

Cardiac pathology, such as myocardial infarction (MI), activates intracellular proteases that often trigger programmed cell death and contribute to maladaptive changes in myocardial structure and function. To test whether inhibition of calpain, a Ca(2+)-dependent cysteine protease, would prevent these changes, we used a mouse MI model. Calpeptin, an aldehydic inhibitor of calpain, was intravenously administered at 0.5 mg/kg body wt before MI induction and then at the same dose subcutaneously once per day. Both calpeptin-treated (n = 6) and untreated (n = 6) MI mice were used to study changes in myocardial structure and function after 4 days of MI, where end-diastolic volume (EDV) and left ventricular ejection fraction (EF) were measured by echocardiography. Calpain activation and programmed cell death were measured by immunohistochemistry, Western blotting, and TdT-mediated dUTP nick-end labeling (TUNEL). In MI mice, calpeptin treatment resulted in a significant improvement in EF [EF decreased from 67 + or - 2% pre-MI to 30 + or - 4% with MI only vs. 41 + or - 2% with MI + calpeptin] and attenuated the increase in EDV [EDV increased from 42 + or - 2 microl pre-MI to 73 + or - 4 microl with MI only vs. 55 + or - 4 microl with MI + calpeptin]. Furthermore, calpeptin treatment resulted in marked reduction in calpain- and caspase-3-associated changes and TUNEL staining. These studies indicate that calpain contributes to MI-induced alterations in myocardial structure and function and that it could be a potential therapeutic target in treating MI patients.

Topics: Animals; Apoptosis; Calpain; Cardiotonic Agents; Caspase 3; Cysteine Proteinase Inhibitors; Dipeptides; Disease Models, Animal; Enzyme Activation; Injections, Intravenous; Injections, Subcutaneous; Mice; Mice, Inbred C57BL; Myocardial Contraction; Myocardial Infarction; Myocardium; Stroke Volume; Time Factors; Ultrasonography; Ventricular Function, Left; Ventricular Remodeling

The calpain inhibitor A-705253 and the Na(+)/H(+)-exchange inhibitor Cariporide were studied in isolated perfused rabbit hearts subjected to 60 min occlusion of the ramus interventricularis of the left coronary artery (below the origin of the first diagonal branch), followed by 120 min of reperfusion. The inhibitors were added to the perfusion fluid solely or in combination at the beginning of reperfusion. Hemodynamic monitoring and biochemical analysis of perfusion fluid from the coronary outflow were performed. Myocardial infarct size and area at risk (transiently not perfused myocardium) were determined from left ventricular slices after a special staining procedure with Evans blue and 2,3,5-triphenyltetrazolium chloride. The infarcted area (dead myocardium) was 72.7+/-4.0% of the area at risk in untreated controls, but was significantly smaller in the presence of the inhibitors. The largest effect was seen with 10(-6) m A-705253, which reduced the infarcted area to 49.2+/-4.1% of the area at risk, corresponding to a reduction of 33.6%. Cariporide at 10(-6) m reduced the infarct size to the same extent. The combination of both inhibitors, however, did not further improve cardioprotection. No statistical difference was observed between the experimental groups in coronary perfusion, left ventricular pressure, heart rate, and in the release of lactate dehydrogenase and creatin kinase from heart muscle.

Topics: Animals; Anti-Arrhythmia Agents; Benzamides; Blood Pressure; Calpain; Coronary Circulation; Female; Guanidines; Heart; Heart Rate; In Vitro Techniques; Male; Myocardial Infarction; Myocardium; Potassium; Rabbits; Sodium-Hydrogen Exchangers; Sulfones; Ventricular Function, Left

Changes in proteolytic activity of the myocardium during the development of heart failure after left coronary artery ligation (CAL) of rats were examined. Hemodynamics of the rats at the eighth week (8w-CAL rat), but not at the second week (2w-CAL rat), after CAL showed the symptoms of chronic heart failure. Contents of mu-calpin and m-calpain, but not an intrinsic calpain inhibitor calpastatin, in the viable left ventricular muscle (viable LV) and the right ventricular muscle (RV) of the 2w-CAL and 8w-CAL rats were increased, which was associated with an elevation of intrinsic activities of leupeptin-sensitive, Ca(2+)-activated proteolysis in the cytosolic fractions of the viable LV and RV. Oral administration of 3 mg/kg/d trandolapril or 1 mg/kg/d candesartan from the second to eighth week after CAL improved the hemodynamics of 8w-CAL rats. The drug treatment attenuated the increases in mu-calpain and m-calpain contents and the elevation of the proteolytic activity of the viable LV and RV in the 8w-CAL rat. The drug treatment increased calpastatin content of the RV in the 8w-CAL rat. These results suggest that sustained activation of calpain is involved in the development of chronic heart failure and that trandolapril and candesartan prevent the activation of calpains after CAL.

Topics: Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Pressure; Calcium-Binding Proteins; Calpain; Cytosol; Enzyme Activation; Heart Failure; Male; Myocardial Infarction; Myocardium; Organ Size; Rats; Rats, Wistar

We examined the effects of trandolapril and candesartan on changes in the levels of sarcoglycans and dystrophin in the right ventricle of rats with the left coronary artery ligation. Hemodynamic and morphological alterations suggested the development of hypertrophy of the right ventricle and chronic heart failure by the 8th week. By the end of the 8th week, alpha- and beta-sarcoglycans and dystrophin were decreased. Increases in mu- and m-calpains in the hypertrophied right ventricle were associated with an elevation of casein-proteolytic activity in the cytosolic fraction. Oral administration of 3 mg/kg/day trandolapril or 1 mg/kg/day candesartan from the 2nd to 8th week after the left coronary artery ligation attenuated decreases in alpha-sarcoglycan and dystrophin and reduced the increased proteolytic activity. The results suggest that attenuation of decreases in sarcoglycans and dystrophin is a possible mechanism underlying trandolapril- and candesartan-mediated improvement of structural and functional alterations of the right ventricle in the coronary artery-ligated rat.

Topics: Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Animals; Benzimidazoles; Biphenyl Compounds; Blood Pressure; Blotting, Western; Body Weight; Calcium-Binding Proteins; Calpain; Coronary Vessels; Cytosol; Dystrophin; Gene Expression; Heart Rate; Heart Ventricles; Indoles; Ligation; Male; Myocardial Infarction; Protein Isoforms; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sarcoglycans; Tetrazoles; Time Factors; Transcription, Genetic

Calpains, a family of Ca2+-dependent cysteine proteases, are activated during myocardial ischemia and reperfusion. This study investigates the cardioprotective effects of calpain inhibition on infarct size and global hemodynamics in an ischemia/reperfusion model in pigs, using the calpain inhibitor A-705253. The left anterior descending coronary artery was occluded for 45 min and reperfused for 6 h. A bolus of 1.0 mg/kg A-705253 or distilled water was given intravenously 15 min prior to induction of ischemia and a constant plasma level of A-705253 was maintained by continuous infusion of 1.0 mg/kg A-705253 during reperfusion. Infarct size was assessed histochemically using triphenyltetrazolium chloride staining. Macromorphometric findings were verified by light microscopy on hematoxylin-eosin- and Tunel-stained serial sections. Global hemodynamics, including the first derivate of the left ventricular pressure (dP / dtmax), were measured continuously throughout the experiment. A-705253 reduced the infarct size by 35% compared to controls (P < 0.05). Hemodynamic alterations, including heart rate, aortic blood pressure, central venous pressure and left atrial pressure, were attenuated mainly during ischemia and the first 2 h during reperfusion by A-705253. Cardiac function improved, as determined by dP / dtmax, after 6 h of reperfusion (P < 0.003). Our results demonstrate that myocardial protection can be achieved by calpain inhibition, which decreases infarct size and improves left ventricular contractility and global hemodynamic function. Hence, the calpain-calpastatin system might play an important pathophysiological role in porcine myocardial ischemia and reperfusion damage and A-705253 could be a promising cardioprotective agent.

Topics: Animals; Benzamides; Blood Pressure; Calpain; Cardiotonic Agents; Disease Models, Animal; Heart Rate; Hemodynamics; Infusions, Intravenous; Injections, Intravenous; Myocardial Contraction; Myocardial Infarction; Myocardial Reperfusion Injury; Sus scrofa; Ventricular Dysfunction, Left

Two novel calpain inhibitors (A-705239 and A-705253) were studied in isolated perfused rabbit hearts subjected to 60-min occlusion of the ramus interventricularis of the left coronary artery (below the origin of the first diagonal branch), followed by 120 min of reperfusion. The inhibitors were added to the perfusion fluid in various final concentrations from the beginning of the experiments before the coronary artery was blocked. Hemodynamic monitoring and biochemical analysis of perfusion fluid from the coronary outflow were carried out. Myocardial infarct size and the area at risk (transiently non-perfused myocardium) were determined from left ventricular slices after a special staining procedure with Evans blue and 2,3,5-triphenyltetrazolium chloride. The infarcted area (dead myocardium) was 77.9+/-2.3% of the area at risk in untreated controls ( n =12). The infarct size was significantly reduced in the presence of both calpain inhibitors. The best effect was achieved with 10 -8 M A-705253 ( n =8), which reduced ( p <0.001) the infarcted area to 49.3+/-3.9% of the area at risk, corresponding to an infarct reduction of 61.8%. No statistical difference was observed between the experimental groups in coronary perfusion, left ventricular pressure, and in the release of lactate dehydrogenase and creatine kinase from heart muscle.

Topics: Animals; Benzamides; Calpain; Enzyme Inhibitors; Female; Heart; In Vitro Techniques; Male; Myocardial Infarction; Potassium; Rabbits

Genetic defects in several sarcoglycans (SGs) and dystrophin (Dys) play a critical role in cardiomyopathy. The present study was designed to determine whether changes in SGs and Dys might occur in animals with chronic heart failure (CHF) induced by acute myocardial infarction (AMI), which have no genetic defects.. AMI was induced by the left coronary artery ligation (CAL) in rats. The hemodynamic parameters of the 2- and 8-week CAL (2w- and 8w-CAL) rats were measured and the myocardial SGs, Dys, calpain, and calpastatin levels were determined by the Western blot method. Myocardial calpain-like protease activity was evaluated as caseinolysis activity.. Increases in left ventricular end-diastolic pressure (LVEDP) and right ventricular systolic pressure, and a decrease in +/-dP/dt were observed at the 2nd week, whereas cardiac output index (COI) was preserved. In contrast, the 8w-CAL rats showed a further increment in LVEDP with low COI. alpha-SG of the viable left ventricle (LV), and septum (Sep) of the 8w-CAL rat decreased (60-70% of the control). The alpha- and beta-SGs of the right ventricle (RV) of the 2w- and 8w-CAL rats were reduced, while gamma- and delta-SGs in the three regions did not change significantly. Dys in the viable LV and RV of the 8w-CAL rat decreased (75% of the control). The amount of m-calpain in the three regions of the 2w- and 8w-CAL rats increased (140-200% of the control), whereas the endogenous calpain inhibitor, calpastatin, did not change significantly. The in vitro degradation studies using purified m-calpain or cytosolic fractions of the 8w-CAL rat heart suggested a reduction in SGs and Dys by calpain.. The results suggest that a decrease in SGs and Dys may play an important role in the pathophysiology of CHF following AMI.

Topics: Animals; Blood Pressure; Blotting, Western; Calcium-Binding Proteins; Calpain; Cardiac Output, Low; Culture Techniques; Cytoskeletal Proteins; Cytosol; Dystroglycans; Dystrophin; Heart Rate; Male; Membrane Glycoproteins; Models, Animal; Myocardial Infarction; Myocardium; Rats; Rats, Wistar; Sarcoglycans; Sarcolemma

Abnormal Ca(2+) inward current through cardiac Ca(2+) channels during ischemia has been shown to be an initial signal for activation of myocardial Ca(2+)-dependent enzymes. This study investigated the contribution of cardiac L- and T-type Ca(2+) channels in the calpain-mediated myocardial damage following myocardial infarction. Myocardial infarction was induced by permanent ligation of the left coronary artery. Infarcted rats were orally treated with placebo, amlodipine (L-channel blockade; 4 mg/kg/day) or mibefradil (L-/T-channel blockade; 10 mg/kg/day) beginning 7 days before induction of myocardial infarction. Gene expression, protein levels and enzyme activity of calpains I and II were measured 1, 3, 7 and 14 days postcoronary occlusion in the noninfarcted and infarcted myocardium. Infarct size, left ventricular dilation and interstitial collagen volume fraction were determined in picrosirius red-stained hearts. Myocardial infarction induced an up-regulation of calpain I mRNA, protein and activity in the noninfarcted myocardium (maximum 14 days postinfarction), whereas mRNA, protein and activity of calpain II were maximally increased in the infarcted myocardium 3 days postinfarction. Fourteen days postinfarction, infarct size was 49%, the left ventricle was dilated and interstitial collagen volume fraction was increased. Amlodipine-inhibited mRNA, protein and activity up-regulation of calpain I decreased interstitial collagen volume fraction and infarct size. Mibefradil-attenuated mRNA, protein and activity up-regulation of calpain II at all four time points measured and of calpain I at 7 and 14 days postinfarction reduced infarct size and prevented left ventricular dilation. Infarction-induced cardiac hypertrophy was accompanied by an up-regulation of calpain I, whereas calpain II was up-regulated in the infarcted myocardium. Cardiac L- and T-type Ca(2+) channel blockade differentially reduced postinfarction remodeling associated with selective inhibition of cardiac calpains I and II, respectively.

Topics: Animals; Calcium Channel Blockers; Calpain; Gene Expression Regulation; Male; Myocardial Infarction; Myocardium; Protein Biosynthesis; Rats; Rats, Wistar; RNA, Messenger; Transcription, Genetic; Up-Regulation

1. The calpains have been proposed to be activated following cardiac ischaemia and to contribute to myocyte damage after myocardial infarction (MI). In this study, the activity of calpains I and II in the infarcted and non-infarcted rat myocardium and the action of the selective calpain inhibitor, CAL 9961, has been investigated. 2. MI was induced by permanent ligation of the left coronary artery. One, 3, 7 and 14 days post MI, the enzymes calpain I and II were separated from homogenates of the interventricular septum (IS) and left ventricular free wall (LVFW) by chromatography on DEAE-Sepharose. The activity of the calpains was measured in sham-operated and MI animals chronically treated with placebo or CAL 9961 (15 mg kg(-1) d(-1) s.c.) in a synthetic substrate assay. Treatment was started 3 days before MI induction. 3. Calpain I activity reached highest values in IS 14 days post MI, whereas maximum activity of calpain II was measured in LVFW 3 days post MI. In experiments in vitro, CAL 9961 completely inhibited both calpains. In vivo, chronic treatment of MI animals with CAL 9961 partially prevented the increase in calpain I activity in IS and reduced calpain II activity in LVFW to sham levels. 4. Our findings demonstrate that calpains I and II are activated after MI, however, both enzymes differ in their regional and temporal activation within the infarcted myocardium. Chronic inhibition of these enzymes with CAL 9961 might limit the calpain-induced myocardial damage and preserve cardiac structural integrity post MI.

Topics: Animals; Calpain; Chronic Disease; Cysteine Proteinase Inhibitors; Enzyme Activation; Glycoproteins; Male; Myocardial Infarction; Myocardium; Rats; Rats, Wistar; Ventricular Function, Left

1. Recent studies demonstrated that the cardiac calpain system is activated during ischaemic events and is involved in cardiomyocyte injury. The aim of this study was to investigate the contribution of AT(1) and AT(2) receptors in the regulation of calpain-mediated myocardial damage following myocardial infarction (MI). 2. Infarcted animals were treated either with placebo, the ACE inhibitor ramipril (1 mg kg(-1) d(-1)), the AT(1) receptor antagonist valsartan (10 mg kg(-1) d(-1)) or the AT(2) receptor antagonist PD 123319 (30 mg kg(-1) d(-1)). Treatment was started 7 days prior to surgery. On day 1, 3, 7 and 14 after MI, gene expression and protein levels of calpain I, II and calpastatin were determined in left ventricular free wall (LVFW) and interventricular septum (IS). At day 3 and 14 post MI, morphological investigations were performed. 3. Calpain I mRNA expression and protein levels were increased in IS 14 days post MI, whereas mRNA expression and protein levels of calpain II were maximally increased in LVFW 3 days post MI. Ramipril and valsartan decreased mRNA and protein up-regulation of calpain I and II, and reduced infarct size and interstitial fibrosis. PD 123319 did not affect calpain I or II up-regulation in the infarcted myocardium, but decreased interstitial fibrosis. Calpastatin expression and translation were not affected by AT receptor antagonists or ACE inhibitor. 4. Our data demonstrate a distinct, temporary-spatial up-regulation of calpain I and II following MI confer with the hypothesis of calpain I being involved in cardiac remodelling in the late and calpain II contributing to cardiac tissue damage in the early phase of MI. The up-regulation of calpain I and II is partly mediated via the AT(1) receptor and can be reduced by ACE inhibitors and AT(1) receptor antagonists.

Topics: Angiotensin II; Angiotensin Receptor Antagonists; Angiotensin-Converting Enzyme Inhibitors; Animals; Antihypertensive Agents; Blood Pressure; Calcium-Binding Proteins; Calpain; Disease Models, Animal; Gene Expression Regulation; Heart; Heart Rate; Imidazoles; Male; Myocardial Infarction; Protein Biosynthesis; Pyridines; Rats; Rats, Wistar; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; RNA, Messenger; Tetrazoles; Transcription, Genetic; Valine; Valsartan

Drs. John Elce and Peter Davies, biochemists at Queen's University, Kingston, Ont., are investigating the molecular structure of calpain, an enzyme that has been implicated in the cellular damage that occurs after such events as myocardial infarction and stroke. This damage is precipitated by an imbalance in the regulation of calpain that arises as an indirect result of ischemia. Elce and Davies hope that their research, which involves techniques such as recombinant DNA technology and x-ray crystallography, will lead to the development of a calpain inhibitor that will prevent such damage from occurring and enhance recovery.

Topics: Calpain; Cerebrovascular Disorders; Humans; Myocardial Infarction

We examined the efficacy of the combination of coronary reperfusion and calcium-activated neutral protease (CANP) inhibitor (E-64c) for the treatment of acute myocardial infarction in dogs. In 34 dogs, the left anterior descending artery (LAD) was occluded and reperfused after 1 hour (Groups A and B). In the remaining 49 dogs, the LAD was ligated (Groups C and D). E-64c (100 mg/kg, Groups A and C) or vehicle (Groups B and D) was injected intravenously before and after the coronary occlusion or ligation. After 24 hours the hearts were removed. The proportion of the infarct size in the LAD perfusing area (risk zone) in Group A was 47.3 +/- 9.7%, significantly lower than in Group C (54.8 +/- 8.2%, p less than 0.05) or Group D (58.7 +/- 10.0%, p less than 0.01). There was a significant difference between Group B (52.9 +/- 8.6%) and Group D as well (p less than 0.05). The effects of reperfusion (p = 0.0016) and E-64c (p = 0.0226) per se on infarct size were significant, but the combination of reperfusion and E-64c was not additive. The decrease in CPK activity in the risk zone was significantly lower in the reperfused group (p = 0.0001). The mCANP activity was higher in the border zone and lower in the infarct zone. The trend in the mu CANP activity was similar to that of mCANP. Thus, treatment with a CANP inhibitor in the early phase of acute myocardial infarction may be marginally beneficial in combination with reperfusion.

Topics: Animals; Calpain; Creatine Kinase; Dogs; Glycoproteins; Myocardial Infarction; Myocardial Reperfusion; Myocardium; Protease Inhibitors

The purpose of this study is to clarify whether cysteine proteinases play an important role in the degradation of myocardial proteins in the infarcted tissue. We studied the effects of a cysteine proteinase inhibitor, Ep459, on degradation of cardiac structural proteins caused by ischemia due to coronary artery ligation for 24 h. Proteolytic effects of purified cysteine proteinases on isolated cardiac tissue were also examined. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, degradation of cardiac structural proteins, particularly of myosin heavy chain, alpha-actinin and troponin-I was observed in the infarcted tissue. Treatment with Ep459 significantly reduced protein degradation and total activity of cathepsins B and L in the infarcted tissue, compared with the findings in the untreated group. The electrophoretic pattern of the infarcted myocardium was similar to that of myofibrillar proteins degraded by cathepsins B and L. These results suggest that cysteine proteinases, particularly cathepsins B and L, are involved in degradation of myofibrillar proteins in myocardial infarction.

Topics: Animals; Calpain; Cathepsin B; Cathepsin L; Cathepsins; Coronary Disease; Cysteine Endopeptidases; Dogs; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Female; Kinetics; Leucine; Male; Muscle Proteins; Myocardial Infarction; Myocardium; Protease Inhibitors

Calcium-activated neutral protease (CANP) might be involved in the irreversible degradation of myocardial proteins in the ischemic region, leading to the loss of contractility. The new compound, NCO-700, and its analogues were synthetized against CANP. Among these analogues, NCO-700 was the most potent to reduce the size of acute myocardial infarction, which was produced by coronary artery ligation in rabbits, in vivo, although it showed less powerful action to inhibit CANP activity in vitro. The new reagent, NCO-700 might be promising to reduce acute myocardial infarction size and beneficial for the clinical studies, because it had no action to reduce cardiac muscle contractility, compared with beta antagonist or calcium-channel blockades.

Topics: Animals; Calpain; Male; Myocardial Infarction; Myocardium; Piperazines; Protease Inhibitors; Rabbits