Calpain-1 induces endoplasmic reticulum stress in promoting cardiomyocyte apoptosis following hypoxia/reoxygenation (original) (raw)
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Biochimica et biophysica acta, 2016
Diabetes and obesity are prevalent in westernized countries. In both conditions, excessive fatty acid uptake by cardiomyocytes induces cardiac lipotoxicity, an important mechanism contributing to diabetic cardiomyopathy. This study investigated the effect of calpain disruption on cardiac lipotoxicity. Cardiac-specific capns1 knockout mice and their wild-type littermates (male, age of 4weeks) were fed a high fat diet (HFD) or normal diet for 20weeks. HFD increased body weight, altered blood lipid profiles and impaired glucose tolerance comparably in both capns1 knockout mice and their wild-type littermates. Calpain activity, cardiomyocyte cross-sectional areas, collagen deposition and triglyceride were significantly increased in HFD-fed mouse hearts, and these were accompanied by myocardial dysfunction and up-regulation of hypertrophic and fibrotic collagen genes as well as pro-inflammatory cytokines. These effects of HFD were attenuated by disruption of calpain in capns1 knockout mi...
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.
Calpain activation contributes to hyperglycaemia-induced apoptosis in cardiomyocytes
Cardiovascular Research, 2009
Time for primary review: 23 days Aims Cardiomyocyte apoptosis contributes to cardiac complications of diabetes. The aim of this study was to investigate the role of calpain in cardiomyocyte apoptosis induced by hyperglycaemia. Methods and results In cultured adult rat ventricular cardiomyocytes, high glucose (33 mM) increased calpain activity and induced apoptosis, concomitant with the impairment of Na þ /K þ ATPase activity. These effects of high glucose on cardiomyocytes were abolished by various pharmacological calpain inhibitors, knockdown of calpain-1 but not calpain-2 using siRNA, or over-expression of calpastatin, a specific endogenous calpain inhibitor. The effect of calpain inhibition on cardiomyocyte apoptosis was abrogated by ouabain, a selective inhibitor of Na þ /K þ ATPase. Furthermore, blocking gp91 phox-NADPH oxidase activation, L-type calcium channels, or ryanodine receptors prevented calpain activation and apoptosis in high glucose-stimulated cardiomyocytes. In a mouse model of streptozotocin-induced diabetes, administration of different calpain inhibitors blocked calpain activation, increased the Na þ /K þ ATPase activity, and decreased apoptosis in the heart. Conclusion Calpain-1 activation induces apoptosis through down-regulation of the Na þ /K þ ATPase activity in high glucose-stimulated cardiomyocytes and in vivo hyperglycaemic hearts. High glucoseinduced calpain-1 activation is mediated through the NADPH oxidase-dependent pathway and associated with activation of L-type calcium channels and ryanodine receptors. Our data suggest that calpain activation may be important in the development of diabetic cardiomyopathy and thus may represent a potential therapeutic target for diabetic heart diseases.
Contribution of calpains to myocardial ischaemia/reperfusion injury
Cardiovascular Research, 2012
Loss of calcium (Ca 2+) homeostasis contributes through different mechanisms to cell death occurring during the first minutes of reperfusion. One of them is an unregulated activation of a variety of Ca 2+-dependent enzymes, including the non-lysosomal cysteine proteases known as calpains. This review analyses the involvement of the calpain family in reperfusion-induced cardiomyocyte death. Calpains remain inactive before reperfusion due to the acidic pHi and increased ionic strength in the ischaemic myocardium. However, inappropriate calpain activation occurs during myocardial reperfusion, and subsequent proteolysis of a wide variety of proteins contributes to the development of contractile dysfunction and necrotic cell death by different mechanisms, including increased membrane fragility, further impairment of Na + and Ca 2+ handling, and mitochondrial dysfunction. Recent studies demonstrating that calpain inhibition contributes to the cardioprotective effects of preconditioning and postconditioning, and the beneficial effects obtained with new and more selective calpain inhibitors added at the onset of reperfusion, point to the potential cardioprotective value of therapeutic strategies designed to prevent calpain activation.
Cardiovascular Research, 2009
Time for primary review: 46 days Aims Lipopolysaccharide (LPS) induces cardiomyocyte caspase-3 activation and proinflammatory factors, in particular tumour necrosis factor-alpha (TNF-a) production, both of which contribute to myocardial dysfunction during sepsis. The present study was to investigate the roles of calpain/calpastatin system in cardiomyocyte caspase-3 activation, TNF-a expression, and myocardial dysfunction during LPS stimulation. Methods and results In cultured adult rat cardiomyocytes, LPS (1 mg/mL) induced calpain and caspase-3 activity, and up-regulated TNF-a expression. These effects of LPS were abrogated by over-expression of calpastatin, an endogenous calpain inhibitor, transfection of calpain-1 siRNA, or various pharmacological calpain inhibitors. Furthermore, blocking gp91 phox-NADPH oxidase prevented calpain and caspase-3 activation and decreased TNF-a expression in LPS-stimulated cardiomyocytes. To investigate the role of calpastatin in endotoxaemia, transgenic mice with calpastatin over-expression (CAST-Tg) and wild-type mice were treated with LPS (4 mg/kg, i.p.) or saline in the presence of calpain inhibitor-III (10 mg/kg, i.p.) for 4 h, and their heart function was measured with a Langendorff system. Overexpression of calpastatin significantly attenuated myocardial dysfunction (P , 0.05). Consistently, calpain activity, caspase-3 activity, and TNF-a expression were also reduced in CAST-Tg and calpain inhibitor-III compared with wild-type and vehicle-treated hearts, respectively. Conclusion gp91 phox-NADPH oxidase-mediated calpain-1 activation induces caspase-3 activation and TNF-a expression in cardiomyocytes during LPS stimulation. Over-expression of calpastatin inhibits calpain activation and improves myocardial function in endotoxaemia. The present study suggests that targeting calpain/calpastatin system may be a potential therapeutic intervention for septic hearts.
Activation of c-Jun N-terminal kinase promotes survival of cardiac myocytes after oxidative stress
Biochemical Journal, 2002
Reperfusion injury occurs when ischaemic tissue is reperfused. It involves the generation and release of reactive oxygen that activates numerous signalling pathways and initiates cell death. Exposure of isolated cardiac myocytes to chronic hypoxia followed by reoxygenation results in the early activation of c-Jun N-terminal kinase (JNK) and death by apoptosis of approx. 30 % of the myocytes. Although JNK activation has been described in a number of models of ischaemia\reperfusion, the contribution of JNK activation to cell fate has not been established. Here we report that the activation of JNK by reoxygenation correlates with myocyte survival. Transfection of myocytes with JNK pathway interfering plasmid vectors or infection with adenoviral vectors support the hypothesis that JNK is protective. Transfection or infection with JNK inhibitory mutants increased the rates of apoptosis by almost 2-fold compared with control cultures grown aerobically or subjected to hypoxia
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 ...
150-kDa oxygen-regulated protein attenuates myocardial ischemia–reperfusion injury in rat heart
Journal of Molecular and Cellular Cardiology, 2005
Early contractile dysfunction and the later death of cardiomyocytes are two major problems that can follow myocardial infarction or major cardiovascular surgery that demands ischemic arrest of the heart. Here, we found that 24 h of hypoxia and 1 h of reoxygenation induced the expression of the chaperone ORP150 in cultured rat cardiomyocytes. Inhibition of its induction using an adenovirus to express anti-sense ORP150 significantly enhanced the hypoxia-reoxygenation-induced cardiomyocyte death; cell death was reduced by overexpressing ORP150. Decreased levels of ORP150 expression also enhanced caspase-3 and -8 activation, cytochrome-c release, and DNA fragmentation, suggesting that this chaperone regulates apoptotic cell death. In contrast, increasing the expression of ORP150 in the cardiomyocytes had the opposite effect on the expression of these molecules. Moreover, apoptotic cell death initiated by myocardial ischemia-reperfusion (I/R) was significantly inhibited in vivo by transfecting an ORP150 expression plasmid into whole rat heart using the hemagglutinating virus of Japan (HVJ)liposome method. Interestingly, ORP150 seemed to preserve calcium homeostasis in cardiomyocytes that underwent ischemia-reoxygenation in vitro. Calpain activity in the cardiomyocytes was enhanced by anti-sense ORP150 and suppressed by sense ORP150. Finally, we examined the functional recovery of rat hearts that overexpressed ORP150 or GFP protein and were subjected to I/R; we found that ORP150 preserved early contractile function after transient ischemia. Our results indicated cytoprotective roles for ORP150 in rat heart and suggested a therapeutic role for the protein both in preventing cardiomyocyte death and in preserving contractile function after ischemic damage.
Endocrinology, 2012
Increased apoptosis of cardiac progenitor cells (CPCs) has been proposed as a mechanism of myocardial damage and dysfunction. Glucagon-like peptide-1 (GLP-1) has been shown to improve heart recovery and function after ischemia and to promote cell survival. The protective effects of GLP-1 on oxidative stress-induced apoptosis were investigated in human CPCs isolated from human heart biopsies. Mesenchymal-type cells were isolated from human heart biopsies, exhibited the marker profile of CPCs, differentiated toward the myocardiocyte, adipocyte, chondrocyte, and osteocyte lineages under appropriate culture conditions, and expressed functional GLP-1 receptors. CPCs were incubated with GLP-1 with or without hydrogen peroxide (H 2 O 2 ). Phospho-and total proteins were detected by immunoblotting and immunofluorescence analysis. Gene expression was evaluated by quantitative RT-PCR. The role of the canonical GLP-1 receptor was assessed by using the receptor antagonist exendin(9 -39) and receptor-specific silencer small interfering RNAs. Cell apoptosis was quantified by an ELISA assay and by flow cytometry-detected Annexin V. Exposure of CPCs to H 2 O 2 induced a 2-fold increase in cell apoptosis, mediated by activation of the c-Jun N-terminal protein kinase (JNK) pathway. Preincubation of CPCs with GLP-1 avoided H 2 O 2 -triggered JNK phosphorylation and nuclear localization, and protected CPCs from apoptosis. The GLP-1 effects were markedly reduced by coincubation with the receptor antagonist exendin(9 -39), small interfering RNA-mediated silencing of the GLP-1 receptor, and pretreatment with the protein kinase A inhibitor H89. In conclusion, activation of GLP-1 receptors prevents oxidative stress-mediated apoptosis in human CPCs by interfering with JNK activation and may represent an important mechanism for the cardioprotective effects of GLP-1. (Endocrinology 153: 0000 -0000, 2012)