MCU Up‐regulation contributes to myocardial ischemia‐reperfusion Injury through calpain/OPA‐1–mediated mitochondrial fusion/mitophagy Inhibition

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.     

Distinct mechanistic roles of calpain and caspase activation in neurodegeneration as revealed in mice overexpressing their specific inhibitors

Enzymatic proteolysis has been implicated in diverse neuropathological conditions, including acute/subacute ischemic brain injuries and chronic neurodegeneration such as Alzheimer disease and Parkinson disease. Calcium-dependent proteases, calpains, have been intensively analyzed in relation to these pathological conditions, but in vivo experiments have been hampered by the lack of appropriate experimental systems for a selective regulation of the calpain activity in animals. Here we have generated transgenic (Tg) mice that overexpress human calpastatin, a specific and the only natural inhibitor of calpains. In order to clarify the distinct roles of these cell death-associated cysteine proteases, we dissected neurodegenerative changes in these mice together with Tg mice overexpressing a viral inhibitor of caspases after intrahippocampal injection of kainic acid (KA), an inducer of neuronal excitotoxicity. Immunohistochemical analyses using endo-specific antibodies against calpain- and caspase-cleaved cytoskeletal components revealed that preclusion of KA-induced calpain activation can rescue the hippocampal neurons from disruption of the neuritic cytoskeletons, whereas caspase suppression has no overt effect on the neuritic pathologies. In addition, progressive neuronal loss between the acute and subacute phases of KA-induced injury was largely halted only in human calpastatin Tg mice. The animal models and experimental paradigm employed here unequivocally demonstrate their usefulness for clarifying the distinct contribution of calpain and caspase systems to molecular mechanisms governing neurodegeneration in adult brains, and our results indicate the potentials of specific calpain inhibitors in ameliorating excitotoxic neuronal damages.     

Synergistic activation of caspase-3 by m-calpain after neonatal hypoxia-ischemia: a mechanism of "pathological apoptosis"?

The relative contributions of apoptosis and necrosis in brain injury have been a matter of much debate. Caspase-3 has been identified as a key protease in the execution of apoptosis, whereas calpains have mainly been implicated in excitotoxic neuronal injury. In a model of unilateral hypoxia-ischemia in 7-day-old rats, caspase-3-like activity increased 16-fold 24 h postinsult, coinciding with cleavage of the caspase-3 proenzyme and endogenous caspase-3 substrates. This activation was significantly decreased by pharmacological calpain inhibition, using CX295, a calpain inhibitor that did not inhibit purified caspase-3 in vitro. Activation of caspase-3 by m-calpain, but not mu-calpain, was facilitated in a dose-dependent manner in vitro by incubating cytosolic fractions, containing caspase-3 proform, with calpains. This facilitation required the presence of some active caspase-3 and could be abolished by including the specific calpain inhibitor calpastatin. This indicates that initial cleavage of caspase-3 by m-calpain, producing a 29-kDa fragment, facilitates the subsequent cleavage into active forms. This is the first report to our knowledge suggesting a direct link between the early, excitotoxic, calcium-mediated activation of calpain after cerebral hypoxia-ischemia and the subsequent activation of caspase-3, thus representing a tentative pathway of "pathological apoptosis."     

Sidekicks: synaptic adhesion molecules that promote lamina-specific connectivity in the retina

Ca(2+) signaling by calpains leads to controlled proteolysis during processes ranging from cytoskeleton remodeling in mammals to sex determination in nematodes. Deregulated Ca(2+) levels result in aberrant proteolysis by calpains, which contributes to tissue damage in heart and brain ischemias as well as neurodegeneration in Alzheimer's disease. Here we show that activation of the protease core of mu calpain requires cooperative binding of two Ca(2+) atoms at two non-EF-hand sites revealed in the 2.1 A crystal structure. Conservation of the Ca(2+) binding residues defines an ancestral general mechanism of activation for most calpain isoforms, including some that lack EF-hand domains. The protease region is not affected by the endogenous inhibitor, calpastatin, and may contribute to calpain-mediated pathologies when the core is released by autoproteolysis.     

Calpastatin levels affect calpain activation and calpain proteolytic activity in APP transgenic mouse model of Alzheimer's disease

The intracellular Ca(2+)-dependent protease calpain and the specific calpain endogenous inhibitor calpastatin are widely distributed, with the calpastatin/calpain ratio varying among tissues and species. Increased Ca(2+) and calpain activation have been implicated in Alzheimer's disease (AD), with scant data available on calpastatin/calpain ratio in AD. Information is lacking on calpain activation and calpastatin levels in transgenic mice that exhibit AD-like pathology. We studied calpain and calpastatin in Tg2576 mice and in their wild type littermates (control mice). We found that in control mice calpastatin level varies among brain regions; it is significantly higher in the cerebellum than in the hippocampus, frontal and temporal cortex, whereas calpain levels are similar in all these regions. In the Tg2576 mice, calpain is activated, calpastatin is diminished, and calpain-dependent proteolysis is observed in brain regions affected in AD and in transgenic mice (especially hippocampus). In contrast, no differences are observed between the Tg2576 and the control mice in the cerebellum, which does not exhibit AD-like pathology. The results are consistent with the notion that a high level of calpastatin in the cerebellum renders the calpain in this brain region less liable to be activated; in the other brain parts, in which calpastatin is low, calpain is more easily activated in the presence of increased Ca(2+), and in turn the activated calpain leads to further diminution in calpastatin (a known calpain substrate). The results indicate that calpastatin is an important factor in the regulation of calpain-induced protein degradation in the brains of the affected mice, and imply a role for calpastatin in attenuating AD pathology. Promoting calpastatin expression may be used to ameliorate some manifestations of AD.     

Identification of a novel calpain inhibitor using phage display

Calpains are calcium- and thiol-dependent proteases that cleave a variety of intracellular substrates. Overactivation of the calpains has been implicated in a number of diseases and conditions such as ischemic stroke indicating a need for the development of calpain inhibitors. A major problem with current calpain inhibitors has been specific targeting to calpain. To identify highly specific calpain interacting peptides, we developed a peptide-phage library screening method based on the calcium-dependent conformation change associated with calpain activation. A phage-peptide library representing greater than 2 billion expressed 12-mers was incubated with calpain I in the presence of calcium. The calcium-dependent bound phage was then eluted by addition of EGTA. After four rounds of selection we found a conserved 5-mer sequence represented by LSEAL. Synthetic LSEAL inhibited tau-calpain interaction and in vitro proteolysis of tau- and alpha-synuclein by calpains. Deletion of the portion of the tau protein containing a homologous sequence to LSEAL resulted in decreased calpain-mediated tau degradation. These data suggest that these peptides may represent novel calpastatin mimetics.     

Calpastatin is up-regulated in response to hypoxia and is a suicide substrate to calpain after neonatal cerebral hypoxia-ischemia.

In a model of cerebral hypoxia-ischemia in the immature rat, widespread brain injury is produced in the ipsilateral hemisphere, whereas the contralateral hemisphere is left undamaged. Previously, we found that calpains were equally translocated to cellular membranes (a prerequisite for protease activation) in the ipsilateral and contralateral hemispheres. However, activation, as judged by degradation of fodrin, occurred only in the ipsilateral hemisphere. In this study we demonstrate that calpastatin, the specific, endogenous inhibitor protein to calpain, is up-regulated in response to hypoxia and may be responsible for the halted calpain activation in the contralateral hemisphere. Concomitantly, extensive degradation of calpastatin occurred in the ipsilateral hemisphere, as demonstrated by the appearance of a membrane-bound 50-kDa calpastatin breakdown product. The calpastatin breakdown product accumulated in the synaptosomal fraction, displaying a peak 24 h post-insult, but was not detectable in the cytosolic fraction. The degradation of calpastatin was blocked by administration of CX295, a calpain inhibitor, indicating that calpastatin acts as a suicide substrate to calpain during hypoxia-ischemia. In summary, calpastatin was up-regulated in areas that remain undamaged and degraded in areas where excessive activation of calpains and infarction occurs.     

The critical role of calpain versus caspase activation in excitotoxic injury induced by nitric oxide

The pathogenesis of various acute and chronic neurodegenerative disorders has been linked to excitotoxic processes and excess generation of nitric oxide. We investigated the deleterious effects of calpain activation in nitric oxide-elicited neuronal apoptosis. In this model, nitric oxide triggers apoptosis of murine cerebellar granule cells by an excitotoxic mechanism requiring glutamate exocytosis and receptor-mediated intracellular calcium overload. Here, we found that calcium-dependent cysteine proteases, calpains, were activated early in apoptosis of cerebellar granule cells exposed to nitric oxide. Release of the proapoptogenic factors cytochrome c and apoptosis-inducing factor from mitochondria preceded neuronal death. However, caspases-3 was not activated. We observed that procaspase-9 was cleaved by calpains to proteolytically inactive fragments. Inhibition of calpains by different synthetic calpain inhibitors or by adenovirally mediated expression of the calpastatin inhibitory domain prevented mitochondrial release of cytochrome c and apoptosis-inducing factor, calpain-specific proteolysis and neuronal apoptosis. We conclude that (i) signal transduction pathways exist that prevent the entry of neurons into a caspase-dependent death after mitochondrial release of cytochrome c and (ii) that calpain activation links nitric oxide-triggered excitotoxic events with the execution of caspase-independent apoptosis in neurons.     

Intracellular regulatory system involving calpain and calpastatin

Seven years have elapsed since the terms calpain and calpastatin were introduced. During these years, significant progress in research has been recorded. Thus, cloning and sequencing of cDNAs for calpains I and II and calpastatin have established amino acid sequences of these molecules. Structure-function relationship of calpastatin has been studied using mutated cDNAs expressed in E. coli. Interleukin 2 receptor-linked expression of calpastatin in HTLV-I-infected T-cells has been reported. Evidence for Ca2+-induced translocation of calpain to the cell membrane, followed by its autolytic activation, has been discussed. A great varieties of proteins such as several kinases, membrane and cytoskeletal proteins, and hormone receptors have been reported to be susceptible to calpains. This paper is to summarize our current knowledge on chemistry and biology of calpain and calpastatin and thereby to speculate the true function of calpains and their regulatory mechanisms.     

Calpains (intracellular calcium-activated cysteine proteinases): structure-activity relationships and involvement in normal and abnormal cellular metabolism

1. Calpains (calcium-activated cysteine proteinases) have evolved by gene fusion events involving calmodulin-like genes, cysteine proteinase genes and other sequences of unknown origin. 2. The enzymes are composed of two non-identical subunits, each of which contains functional calcium-binding sequences. 3. Calpains are inhibited by the endogenous protein inhibitor, calpastatin and some calmodulin antagonists are also inhibitors of calpain. A number of synthetic proteinase inhibitors also inhibit calpains. 4. Calpains can be activated by phospholipids, an endogenous protein activator and some amino acid derivatives. 5. Various protein substrates for calpains have been recognized in vitro, but the identity of in situ substrates remains unclear. 6. Proposals have been made for calpain function, including involvement in signal transduction, platelet activation, cell fusion, mitosis and cytoskeleton and contractile protein turnover. 7. Calpain and calpastatin expression is altered in a number of abnormal states including muscular dystrophy, muscle denervation and tenotomy, hypertension and platelet abnormalities.     

Mechanisms of calpain mediated proteolysis of voltage gated sodium channel α-subunits following in vitro dynamic stretch injury

Although enhanced calpain activity is well documented after traumatic brain injury (TBI), the pathways targeting specific substrate proteolysis are less defined. Our past work demonstrated that calpain cleaves voltage gated sodium channel (NaCh) α-subunits in an in vitro TBI model. In this study, we investigated the pathways leading to NaCh cleavage utilizing our previously characterized in vitro TBI model, and determined the location of calpain activation within neuronal regions following stretch injury to micropatterned cultures. Calpain specific breakdown products of α-spectrin appeared within axonal, dendritic, and somatic regions 6 h after injury, concurrent with the appearance of NaCh α-subunit proteolysis in both whole cell or enriched axonal preparations. Direct pharmacological activation of either NMDA receptors (NMDArs) or NaChs resulted in NaCh proteolysis. Likewise, a chronic (6 h) dual inhibition of NMDArs/NaChs but not L-type voltage gated calcium channels significantly reduced NaCh proteolysis 6 h after mechanical injury. Interestingly, an early, transient (30 min) inhibition of NMDArs alone significantly reduced NaCh proteolysis. Although a chronic inhibition of calpain significantly reduced proteolysis, a transient inhibition of calpain immediately after injury failed to significantly attenuate NaCh proteolysis. These data suggest that both NMDArs and NaChs are key contributors to calpain activation after mechanical injury, and that a larger temporal window of sustained calpain activation needs consideration in developing effective treatments for TBI.     

Sepsis stimulates calpain activity in skeletal muscle by decreasing calpastatin activity but does not activate caspase-3

We examined the influence of sepsis on the expression and activity of the calpain and caspase systems in skeletal muscle. Sepsis was induced in rats by cecal ligation and puncture (CLP). Control rats were sham operated. Calpain activity was determined by measuring the calcium-dependent hydrolysis of casein and by casein zymography. The activity of the endogenous calpain inhibitor calpastatin was measured by determining the inhibitory effect on calpain activity in muscle extracts. Protein levels of mu- and m-calpain and calpastatin were determined by Western blotting, and calpastatin mRNA was measured by real-time PCR. Caspase-3 activity was determined by measuring the hydrolysis of the fluorogenic caspase-3 substrate Ac-DEVD-AMC and by determining protein and mRNA expression for caspase-3 by Western blotting and real-time PCR, respectively. In addition, the role of calpains and caspase-3 in sepsis-induced muscle protein breakdown was determined by measuring protein breakdown rates in the presence of specific inhibitors. Sepsis resulted in increased muscle calpain activity caused by reduced calpastatin activity. In contrast, caspase-3 activity, mRNA levels, and activated caspase-3 29-kDa fragment were not altered in muscle from septic rats. Sepsis-induced muscle proteolysis was blocked by the calpain inhibitor calpeptin but was not influenced by the caspase-3 inhibitor Ac-DEVD-CHO. The results suggest that sepsis-induced muscle wasting is associated with increased calpain activity, secondary to reduced calpastatin activity, and that caspase-3 activity is not involved in the catabolic response to sepsis.     

Decreased expression of high-molecular-weight calmodulin-binding protein and its correlation with apoptosis in ischemia-reperfused rat heart

A cardiac high-molecular-weight calmodulin-binding protein (HMWCaMBP) was previously identified as a homologue of the calpain inhibitor, calpastatin. In the present study, we investigated the expression of HMWCaMBP and calpains in rat heart after ischemia and reperfusion. Western blot analysis of normal rat heart extract with a polyclonal antibody raised against bovine HMWCaMBP indicated a prominent immunoreactive band of 140kDa. Both the expression and the activity of HMWCaMBP were decreased by ischemia reperfusion. Immunohistochemical studies showed strong-to-moderate HMWCaMBP immunoreactivity in normal heart and poor immunoreactivity in ischemia-reperfused heart muscle. However, the expression of micro-calpain and m-calpain in ischemia-reperfused heart was increased as compared to normal heart. The calpain inhibitory activity of ischemia-reperfused heart tissues was significantly lower as compared to normal heart tissues. The pre-ischemic and post-ischemic perfusion of hearts with a cell-permeable calpain inhibitor suppressed the increase in calpain expression but increased the HMWCaMBP expression. In-vitro HMWCaMBP was proteolyzed by micro-calpain and m-calpain. We also measured apoptosis in normal and ischemia-reperfused tissues. An increase in the number of apoptotic bodies was observed with increased duration of ischemia and reperfusion. Bcl-2 expression did not change in any of the groups, whereas Bax expression increased with ischemia-reperfusion and correlated well with the degree of apoptosis. Our findings suggest that HMWCaMBP may sequester calpains from its substrates in the normal myocardium, but it is susceptible to proteolysis by calpains during ischemia-reperfusion. Thus, decreased expression of HMWCaMBP may play an important role in myocardial injury.     

Neuroprotective effects of Mycoplasma hyorhinis against amyloid-β-peptide toxicity in SH-SY5Y human neuroblastoma cells are mediated by calpastatin upregulation in the mycoplasma-infected cells

Mycoplasmas are frequent contaminants of cell cultures. Contamination leads to altered synthetic and metabolic pathways. We have found that contamination of neuroblastoma SH-SY5Y cells by a strain of Mycoplasma hyorhinis derived from SH-SY5Y cell culture (NDMh) leads to increased levels of calpastatin (the endogenous inhibitor of the Ca(2+)-dependent protease, calpain) in NDMh-infected cells. We have now examined effects of amyloid-β-peptide (Aβ) (central to the pathogenesis of Alzheimer's disease) on uncontaminated (clean) and NDMh-infected SH-SY5Y cells. Aβ was toxic to clean cells, resulting in necrotic cell damage. Aβ treatment led to activation of calpain and enhanced proteolysis, cell swelling, cell membrane permeability to propidium iodide (PI) (without nuclear apoptotic changes), and diminished mitochondrial enzyme activity (XTT reduction). Aβ-toxicity was attenuated in the high calpastatin-containing NDMh-infected cells, as shown by inhibition of calpain activation and activity, no membrane permeability, normal cell morphology, and maintenance of mitochondrial enzyme activity (similar to attenuation of Aβ-toxicity in non-infected cells overexpressing calpastatin following calpastatin-plasmid introduction into the cells). By contrast, staurosporine affected both clean and infected cells, causing apoptotic damage (cell shrinkage, nuclear apoptotic alterations, caspase-3 activation and caspase-promoted proteolysis, without PI permeability, and without effect on XTT reduction). The results indicate that mycoplasma protects the cells against certain types of insults involving calpain. The ratio of calpastatin to calpain is an important factor in the control of calpain activity. Exogenous pharmacological means, including calpastatin-based inhibitors, have been considered for therapy of various diseases in which calpain is implicated. Mycoplasmas provide the first naturally occurring biological system that upregulates the endogenous calpain inhibitor, and thus may be of interest in devising treatments for some disorders, such as neurodegenerative diseases.     

Calpains and Delayed Calcium Deregulation in Excitotoxicity

Overactivation of glutamate receptors results in neurodegeneration in a variety of brain pathologies, including ischemia, epilepsy, traumatic brain injury and slow-progressing neurodegenerative disorders. In all these pathologies, it is well accepted that the calcium-dependent cysteine proteases calpains are key players in the mechanisms of neuronal cell death. Many research groups have been actively pursuing to establish a link between the deregulation of intracellular Ca²⁺ homeostasis associated with excitotoxicity and calpain activity. It is well established that these two events are connected and interact synergistically to promote neurodegeneration, but whether calpain activity depends on or contributes to Ca²⁺ deregulation is still under debate.     

Age‐related loss of nitric oxide synthase in skeletal muscle causes reductions in calpain S‐nitrosylation that increase myofibril degradation and sarcopenia

Sarcopenia, the age‐related loss of muscle mass, is a highly‐debilitating consequence of aging. In this investigation, we show sarcopenia is greatly reduced by muscle‐specific overexpression of calpastatin, the endogenous inhibitor of calcium‐dependent proteases (calpains). Further, we show that calpain cleavage of specific structural and regulatory proteins in myofibrils is prevented by covalent modification of calpain by nitric oxide (NO) through S‐nitrosylation. We find that calpain in adult, non‐sarcopenic muscles is S‐nitrosylated but that aging leads to loss of S‐nitrosylation, suggesting that reduced S‐nitrosylation during aging leads to increased calpain‐mediated proteolysis of myofibrils. Further, our data show that muscle aging is accompanied by loss of neuronal nitric oxide synthase (nNOS), the primary source of muscle NO, and that expression of a muscle‐specific nNOS transgene restores calpain S‐nitrosylation in aging muscle and prevents sarcopenia. Together, the findings show that in vivo reduction of calpain S‐nitrosylation in muscle may be an important component of sarcopenia, indicating that modulation of NO can provide a therapeutic strategy to slow muscle loss during old age.     

Melatonin,a calpain inhibitor in the central nervous system: Current status and future perspectives

Dysregulation of neuronal Ca²⁺ and oxidative stress plays an important role in the activation of cysteine proteases including calpains and caspases that contribute to neuronal death. In neurodegenerative diseases, traumatic brain injury, stroke, and neuropathic pain calpain activities are markedly increased. Melatonin is a beneficial supplement in the treatment of central nervous system (CNS) disorders. Melatonin is a potent antioxidant and works as a free-radical scavenger to regulate a large number of molecular pathways, including oxidative stress, inflammation, apoptosis, and cell death under different pathological conditions. However, limited studies have evaluated the inhibitory effect of melatonin on calpains. This review summarizes the current knowledge related to the effects of melatonin on calpains in some of the common CNS disorders.     

Calpain is required for the rapid, calcium-dependent repair of wounded plasma membrane

Mammalian cells require extracellular calcium ion to undergo rapid plasma membrane repair seconds after mechanical damage. Utilizing transformed fibroblasts from calpain small subunit knock-out (Capns1-/-) mouse embryos, we now show that the heterodimeric, typical subclass of calpains is required for calcium-mediated survival after plasma membrane damage caused by scraping a cell monolayer. Survival of scrape-damaged Capns1-/- cells was unaffected by calcium in the scraping medium, whereas more Capns1+/+ cells survived when calcium was present. Calcium-mediated survival was increased when Capns1-/- cells were scraped in the presence of purified m- or mu-calpain. Survival rates of scraped Capns1+/+, HFL-1, or Chinese hamster ovary cells were decreased by the calpain inhibitor, calpeptin, or the highly specific calpain inhibitor protein, calpastatin. Capns1-/- cells failed to reseal following laser-induced membrane disruption, demonstrating that their decreased survival after scraping resulted, at least in part, from failed membrane repair. Proteomic and immunologic analyses demonstrated that the known calpain substrates talin and vimentin were exposed at the cell surface and processed by calpain following cell scraping. Autoproteolytic activation of calpain at the scrape site was evident at the earliest time point analyzed and appeared to precede proteolysis of talin and vimentin. The results indicate that conventional calpains are required for calcium-facilitated survival after plasma membrane damage and may act by localized remodeling of the cortical cytoskeleton at the injury site.