Activation of proteolytic enzymes and depression of the sarcolemmal Na+/K+-ATPase in ischemia-reperfused heart may be mediated through oxidative stress (original) (raw)
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Canadian Journal of Physiology and Pharmacology, 2009
Ischemia–reperfusion (IR) in the heart has been shown to produce myofibrillar remodeling and depress Ca2+ sensitivity of myofilaments; however, the mechanisms for these alterations are not clearly understood. In view of the role of oxidative stress in cardiac dysfunction due to IR, isolated rat hearts were subjected to global ischemia for 30 min followed by a 30-minute period of reperfusion. IR was found to induce cardiac dysfunction, as reflected by depressed LVDP, +dP/dt, and –dP/dt, and elevated LVEDP, and to reduce myofibrillar Ca2+-stimulated ATPase activity. These changes were simulated by perfusing the hearts with a mixture of xanthine plus xanthine oxidase, which is known to generate oxyradicals. The alterations in cardiac function and myofibrillar Ca2+-stimulated ATPase in IR hearts were attenuated by pretreatment with antioxidants (superoxide dismutase plus catalase, and N-acetylcysteine) and leupeptin, an inhibitor of Ca2+-dependent protease. The levels of mRNA for myosin...
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
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.
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 ...
Physiological Reports, 2016
Cardiac glycosides (CG) are traditionally known as positive cardiac inotropes that inhibit Na + /K +-ATPase-dependent ion transport. CG also trigger-specific signaling pathways through the cardiac Na + /K +-ATPase, with beneficial effects in ischemia/reperfusion (I/R) injury (e.g., ouabain preconditioning, known as OPC) and hypertrophy. Our current understanding of hypersensitivity to CG and subsequent toxicity in the ischemic heart is mostly based on specific I/Rinduced alterations of the Na + /K +-ATPase enzymatic function and has remained incomplete. The primary goal of this study was to investigate and compare the impact of I/R on Na + /K +-ATPase enzymatic and signaling functions. Second, we assessed the impact of OPC on both functions. Langendorff-perfused rat hearts were exposed to 30 min of ischemia and 30 min of reperfusion. At the inotropic concentration of 50 lmol/L, ouabain increased ERK and Akt phosphorylation in control hearts. In I/R hearts, this concentration did not induced positive inotropy and failed to induce Akt or ERK phosphorylation. The inotropic response to dobutamine as well as insulin signaling persisted, suggesting specific alterations of Na + /K +-ATPase. Indeed, Na + /K +-ATPase protein expression was intact, but the enzyme activity was decreased by 60% and the enzymatic function of the isoform with high affinity for ouabain was abolished following I/R. Strikingly, OPC prevented all I/R-induced alterations of the receptor. Further studies are needed to reveal the respective roles of I/R-induced modulations of Na + /K +-ATPase enzymatic and signaling functions in cardiomyocyte death.
Ischemia–reperfusion alters gene expression of Na +–K + ATPase isoforms in rat heart
Biochemical and Biophysical Research Communications, 2003
The present study investigated whether oxidative stress plays a role in ischemia-reperfusion-induced changes in cardiac gene expression of Na þ -K þ ATPase isoforms. The levels of mRNA for Na þ -K þ ATPase isoforms were assessed in the isolated rat heart subjected to global ischemia (30 min) followed by reperfusion (60 min) in the presence or absence of superoxide dismutase (5 Â 10 4 U/L) plus catalase (7.5 Â 10 4 U/L), an antioxidant mixture. The levels of mRNA for the a 2 , a 3 , and b 1 isoforms of Na þ -K þ ATPase were significantly reduced in the ischemia-reperfusion hearts, unlike the a 1 isoform. Pretreatment with superoxide dismutase + catalase preserved the ischemia-reperfusion-induced changes in a 2 , a 3 , and b 1 isoform mRNA levels of the Na þ -K þ ATPase, whereas the a 1 mRNA levels were unaffected. In order to test if oxidative stress produced effects similar to those seen with ischemia-reperfusion, hearts were perfused with an oxidant, H 2 O 2 (300 lM), or a free radical generator, xanthine (2 mM) plus xanthine oxidase (0.03 U/ml) for 20 min. Perfusion of hearts with H 2 O 2 or xanthine/xanthine oxidase depressed the a 2 , a 3 , and b 1 isoform mRNA levels of the Na þ -K þ ATPase, but had lesser effects on a 1 mRNA levels. These results indicate that Na þ -K þ ATPase isoform gene expression is altered differentially in the ischemia-reperfusion hearts and that antioxidant treatment appears to attenuate these changes. It is suggested that alterations in Na þ -K þ ATPase isoform gene expression by ischemia-reperfusion may be mediated by oxidative stress.
Calpain, calpastatin activities and ratios during myocardial ischemia-reperfusion
2002
The purpose of this study was to test the hypothesis that myocardial ischemia-reperfusion (I/R) is accompanied by an early burst in calpain activity, resulting in decreased calpastatin activity and an increased calpain/calpastatin ratio, thereby promoting increased protein release. To determine the possibility of a ‘calpain burst’ impacting cardiac calpastatin inhibitory activity, rat hearts were subjected (Langendorff) to either 45 or
AJP: Heart and Circulatory Physiology, 2004
We hypothesized that calpain inhibitor-1 protected left ventricular (LV) function from ischemia-reperfusion injury by inhibiting the proteolysis of α-fodrin. To test this hypothesis, we investigated the effect of calpain inhibitor-1 on LV mechanical work and energetics in the cross-circulated rat hearts that underwent 15-min global ischemia and 60-min reperfusion ( n = 9). After ischemia-reperfusion with calpain inhibitor-1, mean end-systolic pressure at midrange LV volume and systolic pressure-volume area (PVA) at midrange LV volume (total mechanical energy per beat) were hardly changed, although they were significantly ( P < 0.01) decreased after ischemia-reperfusion without calpain inhibitor-1. Mean myocardial oxygen consumption per beat (Vo2) intercepts (PVA-independent Vo2; Vo2 for the total Ca2+ handling in excitation-contraction coupling and basal metabolism) of Vo2-PVA linear relations were also unchanged after ischemia-reperfusion with calpain inhibitor-1, although they ...
General Physiology and Biophysics, 2010
Matrix metalloproteinases (MMPs) are enzymes that play an important role in degradation and remodeling of extracellular matrix and MMP-2 has been also shown as a primary mediator of the acute mechanical dysfunction of the heart immediately after ischemia/reperfusion (I/R). The aims of the study were to investigate the influence of I/R on MMP-2 and to study the effects of wortmannin on modulation of MMP-2 activities after cycle of short I/R procedures (ischemic preconditioning, IP). Wortmannin is a specific inhibitor of PI3K/Akt kinase pathway activation of which was found to play a role in infarct size limiting mechanisms in the rat heart. In the study isolated Langendorff-perfused rat hearts subjected to protocols of prolonged (test) I/R and/or IP were used. Wortmannin was infused before and during the reperfusion phase of IP. The levels and activation of proteins were determined by immunoblot assay. The MMP-2 activities were measured by zymography. We found that ischemia induced time-dependent activation of tissue pro-MMP-2. Strong activation occurred after 15 min ischemia, during prolonged ischemia and following reperfusion the activities of this form of MMP-2 declined. The specific activities of both 72 and 63 kDa forms of MMP-2 were increased in perfusates collected during reperfusion after 30 min ischemia and these activities peaked in the first minute of reperfusion. Cycle of short ischemia and reperfusion that led to increased cardiac tolerance against prolonged I/R reduced 72 kDa MMP-2 activities and induced also an activation of Akt kinase. The application of wortmannin was connected with inhibition of IP-mediated Akt kinase activation. Moreover, the actions of wortmannin were linked with modulation of MMP-2 activities. Our results suggest that MMP-2 may be involved in the responses of rat hearts to ischemia and point to possible relationship between Akt kinase and modulation of MMP-2 activities in rat hearts.
The Journal of Physiological Sciences, 2012
We have previously indicated that a new soluble calpain inhibitor, SNJ-1945 (SNJ), attenuates cardiac dysfunction after cardioplegia arrest-reperfusion by inhibiting the proteolysis of a-fodrin in in vitro study. Nevertheless, the in vivo study design is indispensable to explore realistic therapeutic approaches for clinical use. The aim of the present in situ study was to investigate whether SNJ attenuated left ventricular (LV) dysfunction (stunning) after mild ischemic-reperfusion (mI-R) in rat hearts. SNJ (60 lmol/l, 5 ml i.p.) was injected 30 min before gradual and partial coronary occlusion at proximal left anterior descending artery. To investigate LV function, we obtained curvilinear end-systolic pressure-volume relationship by increasing afterload 60 min after reperfusion. In the mI-R group, specific LV functional indices at midrange LV volume (mLVV), end-systolic pressure (ESP mLVV), and pressure-volume area (PVA mLVV : a total mechanical energy per beat, linearly related to oxygen consumption) significantly decreased, but SNJ reversed these decreases to time control level. Furthermore, SNJ prevented the a-fodrin degradation and attenuated degradation of Ca 2? handling proteins after mI-R. Our results indicate that improvements in LV function following mI-R injury are associated with inhibition of the proteolysis of a-fodrin in in situ rat hearts. In conclusion, SNJ should be a promising tool to protect the heart from the stunning.