Heart mitochondria and calpain 1: Location, Function, and Targets (original) (raw)
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Activation of mitochondrial calpain and increased cardiac injury: Beyond AIF release
American journal of physiology. Heart and circulatory physiology, 2015
Calpain 1 (CPN1) is a ubiquitous cysteine protease that exists in both cytosol and cardiac mitochondria. Mitochondrial calpain 1 (mit-CPN1) is located in the intermembrane space and matrix. Activation of mit-CPN1 within the intermembrane space increases cardiac injury by releasing apoptosis inducing factor (AIF) from mitochondria during ischemia-reperfusion (IR). We asked if activation of mit-CPN1 is involved in mitochondrial injury during IR. MDL-28170 was used to inhibit calpain 1 in buffer perfused hearts following 25 min ischemia and 30 min reperfusion. MDL treatment decreased the release of LDH into coronary effluent compared to untreated hearts, indicating that inhibition of calpain 1 decreases cardiac injury. MDL also prevented the cleavage of spectrin (a substrate of calpain 1) in cytosol during IR, supporting that MDL treatment decreased cytosolic calpain activation. In addition, MDL markedly improved calcium retention capacity (CRC) compared to untreated heart, suggesting ...
Biochemical and Biophysical Research Communications, 2011
Ubiquitous calpains (calpain I & II) are generally recognized as cytosolic proteins. Recently, mitochondrial localized calpain I (μ-calpain) has been identified. Activation of mito-u-calpain cleaves apoptosis inducing factor (AIF), a flavoprotein located within the mitochondrial intermembrane space, in liver mitochondria, but not in brain mitochondria. We first tested if activation of mito-u-calpain cleaves AIF in isolated heart mitochondria. A decrease in AIF content within mitochondria increases cardiac injury during ischemia-reperfusion by augmenting oxidative stress. We hypothesize that the activation of mito-u-calpain by calcium overload during ischemiareperfusion results in decreased AIF content within mitochondria by cleaving AIF. The u-calpain was present within mouse heart mitochondria, mostly in the intermembrane space. Exogenous calcium treatment induced a calpain-dependent decrease of mitochondrial AIF content in isolated mouse heart mitochondria. This process was blocked by a calpain inhibitor (MDL-28170). The Mitochondrial u-calpain activity was increased by 160% ± 15% during ischemia-reperfusion compared to time control. In contrast, the mitochondrial AIF content was decreased by 52% ± 7% during reperfusion vs. time control in the buffer perfused mouse heart. Inhibition of mito-u-calpain using MDL-28170 decreased cardiac injury by preserving AIF content within mitochondria during ischemia-reperfusion. Thus, activation of mito-u-calpain is required to release AIF from cardiac mitochondria. Inhibition of calpains using MDL-28170 decreases cardiac injury by inhibiting both cytosolic calpains and mito-u-calpain during ischemia-reperfusion. Keywords mitochondria; calpastatin; calpain; ischemia-reperfusion; calcium Calpains are a family of Ca 2+-dependent cysteine proteases including 15 ubiquitous isoforms and additional tissue-specific isoforms [1]. There are two ubiquitous calpains: calpain I (μ-calpain) and calpain II (m-calpain). μ-Calpain is activated in the presence of
Targeted inhibition of calpain in mitochondria alleviates oxidative stress-induced myocardial injury
Acta Pharmacologica Sinica, 2020
The protein levels and activities of calpain-1 and calpain-2 are increased in cardiac mitochondria under pathological conditions including ischemia, diabetes, and sepsis, and transgenic overexpression of mitochondrial-targeted calpain-1 induces dilated heart failure, which underscores an important role of increased calpain in mitochondria in mediating myocardial injury. However, it remains to be determined whether selective inhibition of calpain in mitochondria protects the heart under pathological conditions. In this study, we generated transgenic mice overexpressing mitochondrial-targeted calpastatin in cardiomyocytes. Their hearts were isolated and subjected to global ischemia/reperfusion. Hyperglycemia was induced in the transgenic mice by injections of STZ. We showed that transgenic calpastatin was expressed exclusively in mitochondria isolated from their hearts but not from other organs including skeletal muscle and lung tissues. Transgenic overexpression of mitochondrial-targeted calpastatin significantly attenuated mitochondrial oxidative stress and cell death induced by global ischemia/reperfusion in isolated hearts, and ameliorated mitochondrial oxidative stress, cell death, myocardial remodeling and dysfunction in STZ-treated transgenic mice. The protective effects of mitochondrial-targeted calpastatin were correlated with increased ATP5A1 protein expression and ATP synthase activity in isolated hearts subjected to global ischemia/reperfusion and hearts of STZ-treated transgenic mice. In cultured rat myoblast H9c2 cells, overexpression of mitochondrial-targeted calpastatin maintained the protein levels of ATP5A1 and ATP synthase activity, prevented mitochondrial ROS production and decreased cell death following hypoxia/reoxygenation, whereas upregulation of ATP5A1 or scavenging of mitochondrial ROS by mito-TEMPO abrogated mitochondrial ROS production and decreased cell death. These results confirm the role of calpain in myocardial injury, suggesting that selective inhibition of calpain in myocardial mitochondria by mitochondrial-targeted calpastatin is an effective strategy for alleviating myocardial injury and dysfunction in cardiac pathologies.
Mitochondrial calpain system: An overview
Calpain system is generally known to be comprised of three molecules: two Ca 2+ -dependent proteases: land m-calpains, and their endogenous inhibitor, calpastatin. While calpains have previously been considered as the cytoplasmic enzymes, research in the recent past demonstrated that l-calpain, m-calpain and calpain 10 are present in mitochondria, which play important roles in a variety of pathophysiological conditions including necrotic and apoptotic cell death phenomena. Although a number of original research articles on mitochondrial calpain system are available, yet to the best of our knowledge, a precise review article on mitochondrial calpain system has, however, not been available. This review outlines the key features of the mitochondrial calpain system, and its roles in several cellular and biochemical events under normal and some pathophysiological conditions.
Calpains, mitochondria, and apoptosis
Cardiovascular Research, 2012
Mitochondrial activity is critical for efficient function of the cardiovascular system. In response to cardiovascular injury, mitochondrial dysfunction occurs and can lead to apoptosis and necrosis. Calpains are a 15-member family of Ca 2+-activated cysteine proteases localized to the cytosol and mitochondria, and several have been shown to regulate apoptosis and necrosis. For example, in endothelial cells, Ca 2+ overload causes mitochondrial calpain 1 cleavage of the Na + /Ca 2+ exchanger leading to mitochondrial Ca 2+ accumulation. Also, activated calpain 1 cleaves Bid, inducing cytochrome c release and apoptosis. In renal cells, calpains 1 and 2 promote apoptosis and necrosis by cleaving cytoskeletal proteins, which increases plasma membrane permeability and cleavage of caspases. Calpain 10 cleaves electron transport chain proteins, causing decreased mitochondrial respiration and excessive activation, or inhibition of calpain 10 activity induces mitochondrial dysfunction and apoptosis. In cardiomyocytes, calpain 1 activates caspase 3 and poly-ADP ribose polymerase during tumour necrosis factor-a-induced apoptosis, and calpain 1 cleaves apoptosis-inducing factor after Ca 2+ overload. Many of these observations have been elucidated with calpain inhibitors, but most calpain inhibitors are not specific for calpains or a specific calpain family member, creating more questions. The following review will discuss how calpains affect mitochondrial function and apoptosis within the cardiovascular system.
Mitochondrial localization of [mu]-calpain
Biochemical and Biophysical Research Communications, 2005
Calcium-dependent cysteine proteases, calpains, have physiological roles in cell motility and differentiation but also play a pathological role following insult or disease. The ubiquitous calpains are widely considered to be cytosolic enzymes, although there has been speculation of a mitochondrial calpain. Within a highly enriched fraction of mitochondria obtained from rat cortex and SH-SY5Y human neuroblastoma cells, immunoblotting demonstrated enrichment of the 80 kDa μ-calpain large subunit and 28 kDa small subunit. In rat cortex, antibodies against domains II and III of the large μ-calpain subunit also detected a 40 kDa fragment, similar to the autolytic fragment generated following incubation of human erythrocyte μ-calpain with Ca2+. Mitochondrial proteins including apoptosis inducing factor and mitochondrial Bax are calpain substrates, but the mechanism by which calpains gain access to these proteins is uncertain. Mitochondrial localization of μ-calpain places the enzyme in proximity to its mitochondrial substrates and to Ca2+ released from mitochondrial stores.
N terminus of calpain 1 is a mitochondrial targeting sequence
Journal of Biological …, 2008
The ubiquitous m-and -calpains are thought to be localized in the cytosolic compartment, as is their endogenous inhibitor calpastatin. Previously, -calpain was found to be enriched in mitochondrial fractions isolated from rat cerebral cortex and SH-SY5Y neuroblastoma cells, but the submitochondrial localization of -calpain was not determined. In the present study, submitochondrial fractionation and digitonin permeabilization studies indicated that both calpain 1 and calpain small subunit 1, which together form -calpain, are present in the mitochondrial intermembrane space. The N terminus of calpain 1 contains an amphipathic ␣-helical domain, and is distinct from the N terminus of calpain 2. Calpain 1, but not calpain 2, was imported into mitochondria. Removal of the N-terminal 22 amino acids of calpain 1 blocked the mitochondrial calpain import, while addition of this N-terminal region to calpain 2 or green fluorescent protein enabled mitochondrial import. The N terminus of calpain 1 was not processed following mitochondrial import, but was removed by autolysis following calpain activation. Calpain small subunit 1 was not directly imported into mitochondria, but was imported in the presence of calpain 1. The presence of a mitochondrial targeting sequence in the N-terminal region of calpain 1 is consistent with the localization of -calpain to the mitochondrial intermembrane space and provides new insight into the possible functions of this cysteine protease.
Submitochondrial localization of associated μ-calpain and calpastatin
Archives of Biochemistry and Biophysics, 2008
Recently, we have reported the presence of calpain-calpastatin system in mitochondria of bovine pulmonary smooth muscle [P. Kar, T. Chakraborti, S. Roy, R. Choudhury, S. Chakraborti, Arch. Biochem. Biophys. 466 (2007) 290-299]. Herein, we report its localization in the mitochondria. Immunoblot, immunoelectron microscopy and casein zymographic studies suggest that l-calpain and calpastatin are present in the inner mitochondrial membrane; but not in the outer mitochondrial membrane or in the inter membrane space or in the matrix of the mitochondria. Co-immunoprecipitation studies suggest that l-calpain-calpastatin is associated in the inner mitochondrial membrane. Additionally, the proteinase K and sodium carbonate treatments of the mitoplasts revealed that l-calpain is integrally and calpastatin is peripherally embedded to the outer surface of inner mitochondrial membrane. These studies indicate that an association between l-calpain and calpastatin occurs in the inner membrane towards the inter membrane space of the mitochondria, which provides better insight about the protease regulation towards initiation of apoptotic processes mediated by mitochondria.
Submitochondrial localization of associated �-calpain and calpastatin
Arch Biochem Biophys, 2008
Recently, we have reported the presence of calpain-calpastatin system in mitochondria of bovine pulmonary smooth muscle [P. Kar, T. Chakraborti, S. Roy, R. Choudhury, S. Chakraborti, Arch. Biochem. Biophys. 466 (2007) 290-299]. Herein, we report its localization in the mitochondria. Immunoblot, immunoelectron microscopy and casein zymographic studies suggest that l-calpain and calpastatin are present in the inner mitochondrial membrane; but not in the outer mitochondrial membrane or in the inter membrane space or in the matrix of the mitochondria. Co-immunoprecipitation studies suggest that l-calpain-calpastatin is associated in the inner mitochondrial membrane. Additionally, the proteinase K and sodium carbonate treatments of the mitoplasts revealed that l-calpain is integrally and calpastatin is peripherally embedded to the outer surface of inner mitochondrial membrane. These studies indicate that an association between l-calpain and calpastatin occurs in the inner membrane towards the inter membrane space of the mitochondria, which provides better insight about the protease regulation towards initiation of apoptotic processes mediated by mitochondria.
Calpain and mitochondria in ischemia/reperfusion injury
2002
Studies of ischemia/reperfusion (I/R) injury and preconditioning have shown that ion homeostasis, particularly calcium homeostasis, is critical to limiting tissue damage. However, the relationship between ion homeostasis and specific cell death pathways has not been investigated in the context of I/R. Previously we reported that calpain cleaved Bid in the absence of detectable caspase activation (1). In this study, we have shown that an inhibitor of the sodium/hydrogen exchanger prevented calpain activation after I/R. Calpain inhibitors prevented cleavage of Bid as well as the downstream indices of cell death, including DNA strand breaks, creatine kinase (CK) release, and infarction measured by triphenyl tetrazolium chloride (TTC) staining. In contrast, the broad spectrum caspase inhibitor IDN6734 was not protective in this model. To ascertain whether mitochondrial dysfunction downstream of these events was a required step, we utilized a peptide corresponding to residues 4 -23 of Bcl-x L conjugated to the protein transduction domain of HIV TAT (TAT-BH4), which has been shown to protect mitochondria against Ca 2؉ -induced ⌬⌿ m loss (2). TAT-BH4 attenuated CK release and loss of TTC staining, demonstrating the role of mitochondria and a pro-apoptotic Bcl-2 family member in the process leading to cell death. We propose the following pathway. (i) Reperfusion results in sodium influx followed by calcium accumulation. (ii) This leads to calpain activation, which in turn leads to Bid cleavage. (iii) Bid targets the mitochondria, causing dysfunction and release of pro-apoptotic factors, resulting in DNA fragmentation and death of the cell. Ischemia/reperfusion initiates a cell death pathway that is independent of caspases but requires calpain and mitochondrial dysfunction.