Calpain Substrate Research Articles

Calpain inhibition protects against atrial fibrillation by mitigating diabetes-associated atrial fibrosis and calcium handling dysfunction in type 2 diabetes mice

BackgroundDiabetes mellitus (DM) is a major risk factor for atrial structural remodeling and atrial fibrillation (AF). Calpain activity is hypothesized to promote atrial remodeling and AF. ObjectiveThe purpose of this study was to investigate the role of calpain in diabetes-associated AF, fibrosis, and calcium handling dysfunction. MethodsDM-associated AF was induced in wild-type (WT) mice and in mice overexpressing the calpain inhibitor calpastatin (CAST-OE) using high-fat diet feeding followed by low-dose streptozotocin injection (75 mg/kg). DM and AF outcomes were assessed by measuring blood glucose levels, fibrosis, and AF susceptibility during transesophageal atrial pacing. Intracellular Ca2+ transients, spontaneous Ca2+ release events, and intracellular T-tubule membranes were measured by in situ confocal microscopy. ResultsWT mice with DM had significant hyperglycemia, atrial fibrosis, and AF susceptibility with increased atrial myocyte calpain activity and Ca2+ handling dysfunction relative to control treated animals. CAST-OE mice with DM had a similar level of hyperglycemia as diabetic WT littermates but lacked significant atrial fibrosis and AF susceptibility. DM-induced atrial calpain activity and downregulation of the calpain substrate junctophilin-2 were prevented by CAST-OE. Atrial myocytes of diabetic CAST-OE mice exhibited improved T-tubule membrane organization, Ca2+ handling, and reduced spontaneous Ca2+ release events compared to littermate controls. ConclusionThis study confirmed that DM promotes calpain activation, atrial fibrosis, and AF in mice. CAST-OE effectively inhibits DM-induced calpain activation and reduces atrial remodeling and AF incidence through improved intracellular Ca2+ homeostasis. Our results support calpain inhibition as a potential therapy for preventing and treating AF in DM patients.

N-Terminomic Changes in Neurons During Excitotoxicity Reveal Proteolytic Events Associated With Synaptic Dysfunctions and Potential Targets for Neuroprotection

Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIβ (CaMKIIβ). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIβ, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.

Comparative effectiveness of the new calpain inhibitor NPO-2270 versus enalapril in pressure overload-induced heart failure

Abstract Background Studies performed in multiple preclinical models support the contribution of the Ca2+-dependent cysteine proteases calpains to ventricular remodelling and heart failure (HF). However, pharmacological calpain inhibition has not yet been tested in patients with HF mainly due to the limitations of available inhibitors. Purpose To determine the effect of NPO-2270 (NPO), a new ketoamide derivative calpain inhibitor, in a mouse model of pressure-overload and compare its effectiveness with that of enalapril. Methods C57BL6 mice were subjected to transverse aortic constriction (TAC) for 4 weeks. Mice were randomised to receive orally administered NPO or enalapril at the dose of 10 mg/kg/day once a day, or vehicle, starting at day 7 after TAC surgery. The combination of both drugs and the effect of NPO starting 1 day after surgery were tested in additional TAC mice. Echocardiographic data, markers of hypertrophy, fibrosis, calpain activity and cleavage of calpain substrates were measured at different time points. Results TAC increased calpain-1 and -2 expression and activity. Administration of NPO and enalapril prevented the progression of hypertrophy and interstitial fibrosis induced by TAC with no statistically significant differences between the two treatments. However, ongoing ventricular dysfunction was less severe in the NPO group than in the enalapril group (27% of LVEF reduction in control group, 6% in NPO group and 16% in the enalapril group after TAC, P=0.024 between NPO and enalapril groups). The combined treatment or the administration of NPO from the first day after TAC surgery was not superior to NPO alone starting 7 days after TAC. These differences in LVEF correlated with better preservation of cadherin-based cell adhesion complex in mice treated with NPO-2270. No adverse effects associated with long-term NPO administration were observed in a sham group. Conclusions The new calpain inhibitor NPO-2270 prevents the development of hypertrophy and fibrosis with similar efficacy than enalapril but prevents cardiac dysfunction more effectively in a preclinical model of pressure overload when given orally at equivalent doses. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Instituto de Salud Carlos III of the Spanish Ministry of Health

Visualizing Cell Death in Live Retina: Using Calpain Activity Detection as a Biomarker for Retinal Degeneration

Calpains are a family of calcium-activated proteases involved in numerous disorders. Notably, previous studies have shown that calpain activity was substantially increased in various models for inherited retinal degeneration (RD). In the present study, we tested the capacity of the calpain-specific substrate t-BOC-Leu-Met-CMAC to detect calpain activity in living retina, in organotypic retinal explant cultures derived from wild-type mice, as well as from rd1 and RhoP23H/+ RD-mutant mice. Test conditions were refined until the calpain substrate readily detected large numbers of cells in the photoreceptor layer of RD retina but not in wild-type retina. At the same time, the calpain substrate was not obviously toxic to photoreceptor cells. Comparison of calpain activity with immunostaining for activated calpain-2 furthermore suggested that individual calpain isoforms may be active in distinct temporal stages of photoreceptor cell death. Notably, calpain-2 activity may be a relatively short-lived event, occurring only towards the end of the cell-death process. Finally, our results support the development of calpain activity detection as a novel in vivo biomarker for RD suitable for combination with non-invasive imaging techniques.

Calcium-Mediated Calpain Activation and Microtubule Dissociation in Cell Model of Hereditary Sensory Neuropathy Type-1 Expressing V144D SPTLC1 Mutation.

Hereditary sensory neuropathy type 1A (HSN1A) is an autosomal, dominantly inherited peripheral neuropathy caused by mutations in serine palmitoyl transferase long chain 1 (SPTLC1), involved in the de novo synthesis of sphingolipids. We have previously reported calcium imbalance, as well as mitochondrial and ER stress in both HSN1 patient lymphoblasts and a transiently transfected cell model. In this study, we investigated the role of the Ca2+-activated protease calpain in destabilizing the cell cytoskeleton, by examining calpain activity in SH-SY5Y cells overexpressing the V144D mutant and changes in microtubule-associated proteins (MAP). Intramitochondrial Ca2+ was found to be significantly depleted and cytoplasmic Ca2+ increased in the V144D mutant. Subsequently, calpain and proteasome activity were increased and calpain substrates, microtubule associated proteins MAP2, and tau were significantly reduced in the microtubule fraction of the mutant. Significant changes were also found in motor proteins dynein and KIF2A detected in the microtubule fraction of cells overexpressing the V144D mutation. There was also a reduction in anterograde and retrograde mitochondrial transport velocities in the V144D mutant. These findings strongly implicate cytoskeletal aberration caused by Ca2+ dysregulation and subsequent loss of microtubule transport functions as the cause of axonal dying back that is characteristic of HSN1.

Abstract 10154: Efficacy of the New Calpain Inhibitor Npo2270 Compared with Enalapril in Mouse Models of Ventricular Remodeling and Heart Failure

Introduction: Ca 2+ -dependent proteases calpains contribute to adverse ventricular remodeling and heart failure (HF) in multiple experimental models. However, the efficacy of pharmacological calpain inhibition for treating HF in patients has not yet been tested mainly due to the limitations of available inhibitors. Hypothesis:: To assess the effect of NPO-2270, a new ketoamide derivative with calpain inhibitory properties, against myocardial remodeling and HF, and compare its effectiveness with enalapril. Methods: C57BL6/J mice were subjected to transverse aortic constriction (TAC) for 28 days or to coronary occlusion for 45 min followed by 21 of reperfusion (IR). NPO-2270 and enalapril were orally administered once a day at 10 mg/Kg/day, starting 7 days after TAC surgery or at the onset of reperfusion. Echocardiographic data, markers of hypertrophy, fibrosis, calpain activity and calpain substrates were measured at different time points. Results: TAC and IR increased calpain expression and activity. Chronic oral administration of NPO-2270 and enalapril prevented progression of hypertrophy and interstitial fibrosis induced by TAC, and reduced scar area by 40% in mice subjected to IR with no significant differences between treatments. However, NPO-2270 attenuated myocardial dysfunction to a greater extent than enalapril in both experimental models (27% of EF reduction in the control group, 6% in NPO-2270 group and 16% in the enalapril group subjected to TAC, P=0.024 between NPO-2270 and enalapril groups; and 11.9% in the control group, 1.8% in NPO-2270 group and 8.8% in the enalapril group subjected to IR, P=0.160 between NPO-2270 and enalapril groups). These results correlated with better preservation of cell adhesion complex in the group treated with NPO-2270. Conclusions: Our data demonstrate that the calpain inhibitor NPO-2270 prevents the progression of hypertrophy and fibrosis with similar efficacy to enalapril but ameliorates cardiac dysfunction more effectively in two clinically relevant experimental models of HF. This study provides compelling evidence that the new calpain inhibitor NPO-2270 is an attractive candidate to determine the potential of calpain inhibition as a strategy for treating HF in patients.

Inhibition of the Shear-Induced Platelet Microparticle Formation by a mAb Against GPIb alpha Is Associated with Protein Tyrosine Dephosphorylation and the Blockade of Filamin Cleavage.

Platelet microparticle (MP) formation during shear-induced platelet aggregation (SIPA) contributes to a relevant part of thrombin generation. A monoclonal antibody (mAb) Ib-23 against platelet GPIb alpha (provided by B.S.) inhibits von Willebrand factor binding, ristocetin-induced platelet aggregation (RIPA), shear induced MP formation and thrombin generation. Platelet calpain affects several functions including MP-formation. One of several target substrates of calpain, filamin, binds to the cytoplasmic tail of GPIbalpha and mediates the assembly of the contractile elements during platelet activation. We therefore hypothesized that the inhibitory mechanism of our mAb Ib-23 on MP formation is a “calpeptin-like effect”, i.e. an inhibition of calpain affecting the protein tyrosine phosphorylation.PPACK-anticoagulated platelet-rich plasma or washed platelets (250′000/μl) were exposed to a shear rate of 5000 sec−1 for 5 min in a cone-plate viscometer. The mAb Ib-23 was added at the RIPA-inhibiting IC80–90. The samples were fluorescence-labelled with an anti-GPIIb-PE mAb to monitor SIPA and MP formation and analyzed by flow cytometry. Resting and sheared platelets were lysed in SDS-PAGE loading buffer for the analysis of filamin degradation and protein tyrosine phosphorylation.Results: Epitope mapping showed that mAb Ib-23 binds between residues 234 and 456 on GP Ib alpha. Exposure of platelets to high shear rates induced the activation of calpain as demonstrated by the degradation of filamin, a phenomenon blocked by the mAb Ib-23. Ib-23 did not inhibit the SIPA induced tyrosine phosphorylation of high molecular weight proteins like FAK, Syk and alpha-actinin, it even weakly enhanced the tyrosine phosphorylation of these proteins. Moreover, Ib-23 prevented the dephosphorylation of low molecular weight proteins (appr. 40 and 33kDa). Annexin V-binding was unaffected by Ib-23. In contrast, abciximab, a GP IIb/IIIa antagonist, partially inhibited SIPA and annexin binding, but not MP generation. Abciximab weakly inhibited the degradation of filamin, inhibited SIPA-induced tyrosine phosphorylation of FAK and Syk, but did not prevent the tyrosine dephosphorylation of the low molecular weight proteins. Control-experiments with calpeptin demonstrated a similar inhibition of filamin degradation as with mAb Ib-23. An isotype control antibody was without effects. The intact antibody Ib-23 was more effective than the Fab2 fragment. A mAb against the FcgammaRII receptor (IV.3, to exclude a bridging of the FcgammaRII and GpIbalpha via the antibody's Fc part) did not affect MP-formation.We conclude that binding of mAb Ib-23 on the outside of GP Ib alpha inhibits SIPA-induced MP formation and thrombin generation possibly via a “calpeptin-like effect” which inhibits the activation of calpain and of protein tyrosine phosphatases.

DNA polymerase η is a substrate for calpain: a possible mechanism for pol η retention in UV-induced replication foci.

DNA polymerase η (pol η) is specifically required for translesion DNA synthesis across UV-induced DNA lesions. Recruitment of this error-prone DNA polymerase is tightly regulated during replication to avoid mutagenesis and perturbation of fork progression. Here, we report that pol η interacts with the calpain small subunit-1 (CAPNS1) in a yeast two-hybrid screening. This interaction is functional, as demonstrated by the ability of endogenous calpain to mediate calcium-dependent cleavage of pol η in cell-free extracts and in living cells treated with a calcium ionophore. The proteolysis of pol η was found to occur at position 465, leading to a catalytically active truncated protein containing the PCNA-interacting motif PIP1. Unexpectedly, cell treatment with the specific calpain inhibitor calpeptin resulted in a decreased extent of pol η foci after UV irradiation, indicating that calpain positively regulates pol η accumulation in replication foci.

M-Calpain is released from striatal synaptosomes

Purpose of the study: We aimed to investigate whether m-calpain (a Ca2+-dependent neutral cysteine protease) is released from synaptosomes. Materials and methods: This research was carry on Wistar male rats and isolated nerve endings – synaptosomes. The synaptosomal integrity was checked by the method of measuring LDH activity. Activity of calpains was measured by the casein zymography in gel and in solution. Extracellular calpain was detected by immunoprecipitation and immunoblotting procedures Prediction of secreted proteins peptide on a protein sequence through a local version of the PrediSi tool (http://www.predisi.de). The probability of calpain isoform nonclassical secretion was analyzed by using SecretomeP (http://www.cbs.dtu.dk/services/SecretomeP2.0) software. Results: It has been shown that calcium- and time-dependent m-calpain is released from synaptosomes in an activated form or in a form capable of activation, and this process is not a result of a violation of the integrity of synaptosomes. Analysis of the probability of secretion of the small catalytic subunit of rat m-calpain along a nonclassical pathway showed a high probability of its secretion. Additionally, the release of calpain from synaptosomes revealed by us is suppressed by the addition of glyburide, an ABC transporter inhibitor, to the incubation medium. Among extracellular proteins, potential substrates of calpains are of calpains are found, for example, matrix metalloprotease-2 and -9, alpha-synuclein, etc. Conclusions: Active m-calpain is present in the media generated from striatal synaptosomes. Glyburide prevents m-calpain release from striatal synaptosomes.

The lipid phosphatase Synaptojanin 1 undergoes a significant alteration in expression and solubility and is associated with brain lesions in Alzheimer\u2019s disease

Synaptojanin 1 (SYNJ1) is a brain-enriched lipid phosphatase critically involved in autophagosomal/endosomal trafficking, synaptic vesicle recycling and metabolism of phosphoinositides. Previous studies suggest that SYNJ1 polymorphisms have significant impact on the age of onset of Alzheimer’s disease (AD) and that SYNJ1 is involved in amyloid-induced toxicity. Yet SYNJ1 protein level and cellular localization in post-mortem human AD brain tissues have remained elusive. This study aimed to examine whether SYNJ1 localization and expression are altered in post-mortem AD brains. We found that SYNJ1 is accumulated in Hirano bodies, plaque-associated dystrophic neurites and some neurofibrillary tangles (NFTs). SYNJ1 immunoreactivity was higher in neurons and in the senile plaques in AD patients carrying one or two ApolipoproteinE (APOE) ε4 allele(s). In two large cohorts of APOE-genotyped controls and AD patients, SYNJ1 transcripts were significantly increased in AD temporal isocortex compared to control. There was a significant increase in SYNJ1 transcript in APOEε4 carriers compared to non-carriers in AD cohort. SYNJ1 was systematically co-enriched with PHF-tau in the sarkosyl-insoluble fraction of AD brain. In the RIPA-insoluble fraction containing protein aggregates, SYNJ1 proteins were significantly increased and observed as a smear containing full-length and cleaved fragments in AD brains. In vitro cleavage assay showed that SYNJ1 is a substrate of calpain, which is highly activated in AD brains. Our study provides evidence of alterations in SYNJ1 mRNA level and SYNJ1 protein degradation, solubility and localization in AD brains.

Investigating the role of the innate immune response in relapse or blast crisis in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is characterized by expression of the tyrosine kinase oncogene, Bcr–abl. Tyrosine kinase inhibitors (TKI) induce prolonged remission in CML, and therapy discontinuation is an accepted approach to patients with reduction in Bcr–abl transcripts of four logs or greater. Half such individuals sustain a therapy free remission, but molecular mechanisms predicting relapse are undefined. We found relative calpain inhibition in CML cells with stabilization of calpain substrates, including βcatenin and Xiap1. Since the Survivin gene is activated by βcatenin, this identified two apoptosis-resistance mechanisms. We found that Survivin impaired apoptosis in leukemia stem cells (LSCs) and Xiap1 in CML granulocytes. Consistent with this, we determined treatment with an inhibitor of Survivin, but not Xiap1, prevented relapse during TKI treatment and after therapy discontinuation in a murine CML model. By transcriptome profiling, we identified activation of innate immune response pathways in murine CML bone marrow progenitors. This was increased by TKI treatment alone, but normalized with addition of a Survivin inhibitor. We found that activation of the innate immune response induced rapid blast crisis in untreated CML mice, and chronic phase relapse during a TKI discontinuation attempt. These results suggest that extrinsic stress exerts adverse effects on CML-LSCs.

Prevention of calpain-dependent degradation of STK38 by MEKK2-mediated phosphorylation

Serine-threonine kinase 38 (STK38) is a member of the protein kinase A (PKA)/PKG/PKC-family implicated in the regulation of cell division and morphogenesis. However, the molecular mechanisms underlying STK38 stability remain largely unknown. Here, we show that treatment of cells with either heat or the calcium ionophore A23187 induced STK38 degradation. The calpain inhibitor calpeptin suppressed hyperthermia-induced degradation or the appearance of A23187-induced cleaved form of STK38. An in vitro cleavage assay was then used to demonstrate that calpain I directly cleaves STK38 at the proximal N-terminal region. Deletion of the N-terminal region of STK38 increased its stability against hyperthermia. We further demonstrated that the MAPKK kinase (MAP3K) MEKK2 prevented both heat- and calpain-induced cleavage of STK38. MEKK2 knockdown enhanced hyperthermia-induced degradation of STK38. We performed an in vitro MEKK2 assay and identified the key regulatory site in STK38 phosphorylated by MEKK2. Experiments with a phosphorylation-defective mutant demonstrated that phosphorylation of Ser 91 is important for STK38 stability, as the enzyme is susceptible to degradation by the calpain pathway unless this residue is phosphorylated. In summary, we demonstrated that STK38 is a calpain substrate and revealed a novel role of MEKK2 in the process of STK38 degradation by calpain.

Molecular characterization and functional analysis of the Schistosoma mekongi Ca2+-dependent cysteine protease (calpain)

BackgroundSchistosoma mekongi, which causes schistosomiasis in humans, is an important public health issue in Southeast Asia. Treatment with praziquantel is the primary method of control but emergence of praziquantel resistance requires the development of alternative drugs and vaccines. Calcium-dependent cysteine protease (calpain) is a novel vaccine candidate that has been studied in S. mansoni, S. japonicum, and protozoans including malaria, leishmania and trypanosomes. However, limited information is available on the properties and functions of calpain in other Schistosoma spp., including S. mekongi. In this study, we functionally characterized calpain 1 of S. mekongi (SmeCalp1).ResultsCalpain 1 of S. mekongi was obtained from transcriptomic analysis of S. mekongi; it had the highest expression level of all isoforms tested and was predominantly expressed in the adult male. SmeCalp1 cDNA is 2274 bp long and encodes 758 amino acids, with 85% to 90% homology with calpains in other Schistosoma species. Recombinant SmeCalp1 (rSmeCalp1), with a molecular weight of approximately 86.7 kDa, was expressed in bacteria and stimulated a marked antibody response in mice. Native SmeCalp1 was detected in crude worm extract and excretory-secretory product, and it was mainly localized in the tegument of the adult male; less signal was detected in the adult female worm. Thus, SmeCalp1 may play a role in surface membrane synthesis or host–parasite interaction. We assessed the protease activity of rSmeCalp1 and demonstrated that rSmeCalp1 could cleave the calpain substrate N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, that was inhibited by calpain inhibitors (MDL28170 and E64c). Additionally, rSmeCalp1 could degrade the biological substrates fibronectin (blood clotting protein) and human complement C3, indicating important roles in the intravascular system and in host immune evasion.ConclusionsSmeCalp1 is expressed on the tegumental surface of the parasite and can cleave host defense molecules; thus, it might participate in growth, development and survival during the entire life-cycle of S. mekongi. Information on the properties and functions of SmeCalp1 reported herein will be advantageous in the development of effective drugs and vaccines against S. mekongi and other schistosomes.

Single cell measurement of calpain activity in neutrophils reveals link to cytosolic Ca2+ elevation and individual phagocytotic events

It has been proposed that Ca2+ activation of calpain-1 is important for the rapid cell shape changes which accompany phagocytosis. In this paper, we use a fluorogenic calpain substrate, (CBZ-Ala Ala)2 R110, and find that there was a low calpain activity measureable in resting (ie without intentional activation) neutrophils, but that it was accelerated by an elevation of cytosolic free Ca2+ (ionomycin -induced) and inhibited by calpeptin (an established calpain-1 inhibitor). The fluorescence signal was sufficiently bright for detection in individual neutrophils that enabled the quantification of dynamic changes in calpain activity to be related to elevations in cytosolic Ca2+ within individual neutrophils. It was found that during phagocytosis of C3bi-opsonised zymosan particles, calpain activity was elevated incrementally, each step increase corresponding to the phagocytosis of an individual particle. The sub-cellular source of the fluorescent product of calpain activity was the phagocytic site itself and originated at the phagocytic cup. It was thus concluded that calpain was activated locally during the formation of the phagocytic cup. These data were consistent with central role of Ca2+ activated calpain activation in controlling phagocytosis.

Dynamin-like protein 1 cleavage by calpain in Alzheimer's disease.

Abnormal mitochondrial dynamics contributes to mitochondrial dysfunction in Alzheimer's disease (AD), yet the underlying mechanism remains elusive. In the current study, we reported that DLP1, the key mitochondrial fission GTPase, is a substrate of calpain which produced specific N‐terminal DLP1 cleavage fragments. In addition, various AD‐related insults such as exposure to glutamate, soluble amyloid‐β oligomers, or reagents inducing tau hyperphosphorylation (i.e., okadaic acid) led to calpain‐dependent cleavage of DLP1 in primary cortical neurons. DLP1 cleavage fragments were found in cortical neurons of CRND8 APP transgenic mice which can be inhibited by calpeptin, a potent small molecule inhibitor of calpain. Importantly, these N‐terminal DLP1 fragments were also present in the human brains, and the levels of both full‐length and N‐terminal fragments of DLP1 and the full‐length and calpain‐specific cleavage product of spectrin were significantly reduced in AD brains along with significantly increased calpain. These results suggest that calpain‐dependent cleavage is at least one of the posttranscriptional mechanisms that contribute to the dysregulation of mitochondrial dynamics in AD.

Novel Aspects of Cardiac Ischemia and Reperfusion Injury Mechanisms

Introduction.The present article, in which a contemporary analysis of the literature on the pathophysiology of ischemic and reperfusion injury (IRI) of the myocardium is presented, focuses on the possible role played by of the calpain system and oxidative stress. Several process development options were proposed, including cytosolic and mitochondrial Ca2+ overload, reactive oxygen stress release, acute inflammatory response and metabolic degradation. The combined effect of all of the above factors produces irreversible ischemic and reperfused damage of cardiomyocytes.Materials and methods.The role of the calpain system in the creation of myocardial IRI was experimentally investigated. It was found that active calpain substrates play a significant role in the processes of cell cycle, apoptosis and differentiation, adversely affecting cardiomyocyte functionality. The calpain system is part of an integrated proteolytic system that is critical to the relationship between the structure and function of the cardiac sarcomere. Uncontrolled activation of calpain is indicated in the pathophysiology of many cardiovascular disorders. As shown by research, inhibitor calpain reduces the size of the zone of infarction following ischemia reperfusion and thus lessens the risk of “stunning” the myocardium. As is known, a consequence of IRI is acute myocardial infarction (AMI), which is a central factor in cardiovascular disease (CVD) and is one of the primary causes of mortality. Understanding the exact pathophysiological mechanisms remains an urgent problem for clinical physicians. To date, the mechanisms of IRI are not fully known, which creates certain difficulties in further treatment and prevention tactics. In addition, myocardial IRI is also an important issue for pathoanatomical service, since sudden coronary death can occur despite timely reperfusion therapy following AMI.Conclusion.The development of strategies for creating conditions that limit the degree of damage to myocardial tissues significantly increases the ability of the heart to withstand ischemic damage.

Casein Zymography for Analysis of Calpain-1 and Calpain-2 Activity.

Casein zymography is used to detect calpain activity in cell or tissue lysates. In this technique, lysates are loaded into a polyacrylamide gel containing casein, and the enzymes within the lysates are electrophoretically separated. The casein embedded in the gel acts as a substrate for calpains in the lysates, and its degradation reflects the activity of these enzymes. Calpain degradation of the casein is visualized as distinct bands that are devoid of dye when stained with Coomassie Brilliant Blue G-250. We describe here how calpains can be extracted from muscle tissue and assayed for activity using this technique. This technique is also generally applicable to lysates from other types of cells or tissues.

Identification of Calpain-Activated Protein Functions.

As opposed to proteasome-mediated proteolysis that leads to protein degradation, calpain proteases carry out limited proteolytic cleavages of their substrates. The cleavage of some substrates can produce active fragments that perform functions that are different from those performed by the full-length proteins. Therefore, cleavage by calpains can operate as a posttranslational modification and increase the functional diversity of target proteins. Nevertheless, activation of protein function by calpain cleavage is still an understudied area in molecular biology. Identifying and functionally characterizing by products generated by calpain cleavage could lead to the discovery of biomarkers and the identification of novel drug targets for the treatment of human diseases. This chapter contains a workflow designed to experimentally characterize novel calpain substrates, including identification of potential calpain targets via Western blotting, characterization of calpain cleavage sites, and the study of cellular functions played by such cleaved products. We will employ MYC as an example for these experiments.

LabCaS for Ranking Potential Calpain Substrate Cleavage Sites from Amino Acid Sequence.

Calpains are a family of Ca2+-dependent cysteine proteases involved in many important biological processes, where they selectively cleave relevant substrates at specific cleavage sites to regulate the function of the substrate proteins. Presently, our knowledge about the function of calpains and the mechanism of substrate cleavage is still limited due to the fact that the experimental determination and validation on calpain bindings are usually laborious and expensive. This chapter describes LabCaS, an algorithm that is designed for predicting the calpain substrate cleavage sites from amino acid sequences. LabCaS is built on a conditional random field (CRF) statistic model, which trains the cleavage site prediction on multiple features of amino acid residue preference, solvent accessibility information, pair-wise alignment similarity score, secondary structure propensity, and physical-chemistry properties. Large-scale benchmark tests have shown that LabCaS can achieve a reliable recognition of the cleavage sites for most calpain proteins with an average AUC score of 0.862. Due to the fast speed and convenience of use, the protocol should find its usefulness in large-scale calpain-based function annotations of the newly sequenced proteins. The online web server of LabCaS is freely available at http://www.csbio.sjtu.edu.cn/bioinf/LabCaS .

Calpain inhibition ameliorates scald burn-induced acute lung injury in rats

BackgroundThe molecular pattern of severe burn-induced acute lung injury, characterized by cell structure damage and leukocyte infiltration, remains unknown. This study aimed to determine whether calpain, a protease involved in both processes, mediates severe burn-induced acute lung injury.MethodsRats received full-thickness scald burns covering 30% of the total body surface area, followed by instant fluid resuscitation. MDL28170 (Tocris Bioscience), an inhibitor of calpain, was given intravenously 1 h before or after the scald burn. The histological score, wet/dry weight ratio, and caspase-3 activity were examined to evaluate the degree of lung damage. Calpain activity and its source were detected by an assay kit and immunofluorescence staining. The proteolysis of membrane skeleton proteins α-fodrin and ankyrin-B, which are substrates of calpain, was measured by Western blot.ResultsTime-course studies showed that tissue damage reached a peak between 1 and 6 h post-scald burn and gradually diminished at 24 h. More importantly, calpain activity reached peak levels at 1 h and was maintained until 24 h, paralleled by lung damage to some extent. Western blot showed that the levels of the proteolyzed forms of α-fodrin and ankyrin-B correlated well with the degree of damage. MDL28170 at a dose of 3 mg/kg b. w. given 1 h before burn injury not only antagonized the increase in calpain activity but also ameliorated scald burn-induced lung injury, including the degradation of α-fodrin and ankyrin-B. Immunofluorescence images revealed calpain 1 and CD45 double-positive cells in the lung tissue of rats exposed to scald burn injury, suggesting that leukocytes were a dominant source of calpain. Furthermore, this change was blocked by MDL28170. Finally, MDL28170 given at 1 h post-scald burn injury significantly ameliorated the wet/dry weight ratio compared with burn injury alone.ConclusionsCalpain, a product of infiltrating leukocytes, is a mediator of scald burn-induced acute lung injury that involves enhancement of inflammation and proteolysis of membrane skeleton proteins. Its late effects warrant further study.