Contribution of Calpains to Myocardial Ischaemia/Reperfusion Injury

Cardiovascular Research (2012) 96,23–31 SPOTLIGHT REVIEW doi:10.1093/cvr/cvs232

Contribution of calpains to myocardial ischaemia/reperfusion injury

Javier Inserte*, Victor Hernando, and David Garcia-Dorado

Laboratory of Experimental Cardiology, Department of Cardiology, Vall d’Hebron University Hospital and Research Institute, Universitat Auto`noma de Barcelona, Barcelona, Spain Downloaded from https://academic.oup.com/cardiovascres/article/96/1/23/540293 by guest on 28 September 2021

Received 10 May 2012; revised 3 July 2012; accepted 6 July 2012; online publish-ahead-of-print 10 July 2012

+ Abstract Loss of calcium (Ca2 ) 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 Ca2 -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 Ca2 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. ------Keywords Ischaemia † Reperfusion † Calpains † Calcium † Necrosis ------This article is part of the Review Focus on: The Calpain Family in the Cardiovascular System

1. Introduction provide evidence supporting the value of calpain inhibition as a cardioprotective therapy. Calpains form a large family of non-lysosomal neutral cysteine pro- + teases that require Ca2 for activity. Tightly regulated by their en- + dogenous inhibitor calpastatin, calpains participate in Ca2 -regulated 2. Ischaemia/reperfusion-mediated processes such as cell proliferation and differentiation, migration, 21 signal transduction, and platelet activation. However, deregulated Ca overload + calpain activity has been associated with a variety of pathophysiologic- Regulation of Ca2 handling is an active cellular process that requires a + + al conditions which have in common the loss of intracellular Ca2 large energy expenditure. The measurement of Ca2 kinetics, either control.1 A large amount of experimental data demonstrate that alter- in isolated cardiomyocytes3,4 or in isolated perfused hearts,5,6 demon- + ation in cellular Ca2 homeostasis plays a central role in the processes strates that energy depletion during ischaemia results in a sustained + leading to reperfusion-induced cell death, and many studies have iden- rise in intracellular Ca2 concentration, largely mediated by the + + + + tified different potential therapeutic targets directed at Ca2 overload Ca2 influx through the reverse mode of sarcolemmal Na /Ca2 ex- during reperfusion and its consequences.2 Among the mechanisms of changer (NCX).7– 11 The direction of NCX operation depends on the + Ca2 -mediated injury, the activation of different enzymes, and in par- difference between transmembrane potential and the reversal poten- ticular of calpains, has been demonstrated to contribute to myocardial tial which is determined by the intra- and extracellular concentrations + + + cell death by different mechanisms. In this review, we will briefly de- of Na and Ca2 .7 During ischaemia, a net Ca2 influx through + scribe the events leading to Ca2 overload during myocardial ischae- reverse NCX transport is predicted due to membrane depolarization + + mia/reperfusion and then summarize the process of calpain activation, and reduced Na gradient. The latter is a consequence of Na i rise + + the available data supporting their contribution to cell death, and the attributed to the inhibition of Na /K -ATPase and to an increased + mechanisms by which calpains mediate this injury. Finally, we will Na -influx associated with the activation of proton extrusion

* Corresponding author. Servicio de Cardiologia, Hospital Universitari Vall d’Hebron, Passeig Vall d’Hebron, 119-129, 08035 Barcelona, Spain. Tel: +34 93 4894038; fax: +34 93 4894032, Email: [email protected] Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2012. For permissions please email: [email protected]. 24 J. Inserte et al.

+ + + – mechanisms like Na /H exchanger and Na /HCO3 cotranspor- + 3.1 Kinetics of activation ter,12,13 as well as to persistent (non-inactivating) Na channels.14,15 + + Until recently, the kinetics of calpain activation during ischaemia/ In addition to NCX, Ca2 entry via the L-type Ca2 channel has + reperfusion and the contribution of the different factors that regulate been proposed to contribute to the rise in Ca2 during ischaemia.16,17 + calpain activity were poorly defined. While in some studies, the activ- During the first minutes of reperfusion, intracellular Na overload ity of calpains was detected in isolated cardiomyocytes submitted to associated with pHi correction and the still depolarized membrane 42,43 + hypoxia or metabolic inhibition without reoxygenation, early provides a large driving force for additional Ca2 influx through the 2+ studies performed in the intact heart did not observe proteolytic reverse mode of NCX. The magnitude of Ca entry during reperfu- 35,44 + + activity during myocardial ischaemia. Since low pHi inhibits cal- sion largely depends on the ability of Na /K -ATPase to restore its 45,46 + 18 pains in vitro, the absence of a significant intracellular acidosis in activity and normalize Na i. the cell models compared with the intact heart could explain these The activation of the calcium-dependent enzymes calcineurin and 2+ discrepancies. In agreement with this notion, recently our group has Ca /calmodulin kinase II (CaMKII) may result in further cytosolic 2+ + demonstrated that during ischaemia Ca overload induces the trans- Downloaded from https://academic.oup.com/cardiovascres/article/96/1/23/540293 by guest on 28 September 2021 Ca2 overload. Recently, it has been proposed that calcineurin acti- 2+ location of m-calpain to the sarcolemma, but ischaemic acidosis pre- vates during ischaemia and contributes to increase cytosolic Ca vents the enzyme activation. Calpain activation occurs exclusively concentration during early reperfusion through dephosphorylation during reperfusion, after pHi normalization (Figure 1).40 of phospholamban which inhibits sarcoplasmic reticulum Ca2+- + ATPase (SERCA) activity and sarcoplasmic reticulum Ca2 uptake.19 The activity of CaMKII is increased during ischaemia/reperfusion and 3.2 Translocation to the sarcolemma its inhibition reduces cell death.20 It has been suggested that CaMKII Whether the calpain translocation process that already occurs during accelerates the rate of pHi recovery at the onset of reperfusion by ischaemia is a necessary step for its activation during reperfusion is + + phosphorylation of the C-terminal domain of Na /H exchanger.21 relevant as it could generate an irreversible preactivation state, only + This effect modulates Ca2 entry into the cell but also activates transiently blocked by ischaemic intracellular acidosis. However, in a mechanisms involved in reperfusion injury that are repressed by low recent study, a reduction in the calpain fraction bound to the mem- pHi.22 branes by the disruption of lipid rafts demonstrated that calpain translocation during ischaemia is not mandatory for its activation at reperfusion.40 These results are in accordance with those obtained 3. Calpain activation during in other pathological models47,48 and in contrast with the studies that analyse calpain activation under physiological conditions and + ischaemia/reperfusion suggest that in those situations leading to severe Ca2 overload as The most abundant calpain isoforms, m-calpain and m-calpain, are occurs during ischaemia, calpain translocation is not a necessary + expressed in the heart.23 –25 Both isoforms differ in the Ca2 concen- step for their activation. + tration required for their activation in vitro, and although these Ca2 requirements are far above the concentrations reported in normal 3.3 Calpain phosphorylation beating cardiomyocytes, it has been demonstrated in vivo that add- Calpains have been reported to present several residues phosphory- itional mechanisms contribute to reduce the minimal concentration 49 + lated under physiological conditions. Some residues can be specific- of Ca2 required (see the review by Sorimachi in this focus issue ally phosphorylated by kinases such as protein kinase A (PKA), which for a detailed description of calpain activity regulation under physio- inhibits m-calpain activity,50 or extracellular signal-regulated kinases, logical conditions). In brief, the most accepted model proposes that which can activate m-calpain even in the absence of an increase in calpains are predominantly localized in the cytosol as inactive + the Ca2 concentration,51 and CaMKII.52 Although the involvement enzymes, binding in a substrate competitive manner to their endogen- + of these kinases in the mechanisms of ischaemia/reperfusion injury ous inhibitor calpastatin. In response to an increase in Ca2 concen- and cardioprotection has been largely reported in the bibliography,53 tration, calpains translocate to the membranes where the association + the regulation of calpain activity by phosphorylation during ischaemia/ to phospholipids reduces the Ca2 concentration needed for activa- + reperfusion has not been analysed. Only a relation between PKA action or places the enzyme near Ca2 channels.26,27 Once activated, tivity during ischaemic preconditioning and inhibition of calpain activ- calpains hydrolyse substrate proteins at membranes or at the ity after ischaemia/reperfusion has been described, although no clues cytosol after release from membranes. Other mechanisms that have 38 + about the nature of this interaction were given. been proposed to reduce the activation threshold by Ca2 in vivo include calpain phosphorylation and the involvement of activator proteins.28,29 In agreement with this model, the activation of calpains has 3.4 Calpastatin been observed in isolated cardiomyocytes with a moderate increase The ubiquitous calpains, m and m, have a specific endogenous inhibi- + in Ca2 concentration (500 nM),30 much lower than the concentra- tor, calpastatin, which is also expressed in the heart.40,54 –59 The pro- tion reached during ischaemia.31 –33 The determination of calpain ac- posed role for calpastatin is to prevent excessive activation of calpains + tivity by assessing the proteolysis of known calpain substrates in tissue in the case of increased Ca2 concentration. However, calpastatin has homogenates or of synthetic peptides in intact cells consistently con- been shown to be largely down-regulated during reperfusion due to firms that calpains are activated during ischaemia/reperfusion.34 – 40 the proteolytic activity of calpains40,58 or the proteasome.60 Research However, the main isoform responsible for the observed proteolytic in other possible mechanisms regulating calpain/calpastatin inter- + activity has not yet been elucidated as there are no isoform-specific action, such as Ca2 concentration, calpastatin phosphorylation, or inhibitors available41 and no genetic approaches have been used for translocation to membrane, is scarce in the heart and absent for is- this purpose. chaemia/reperfusion.56,57,59 Calpains and reperfusion injury 25 Downloaded from https://academic.oup.com/cardiovascres/article/96/1/23/540293 by guest on 28 September 2021

Figure 1 Calpain activation during myocardial ischaemia and reperfusion. Western blots show m-calpain translocation to the membranes and calpain activation (145/150 kDa calpain-dependent cleavage fragments of a-fodrin) after ischaemia with or without reperfusion in the isolated rat heart. 60MDL: hearts were perfused with the calpain inhibitor MDL-28170 before 60 min of ischaemia. Reperfusion lasted 15 min. Results are expressed in percentage with respect to normoxically perfused hearts (Nx ¼ 0) and demonstrate that calpain translocates to the membrane during ischaemia but activates during reperfusion. Modiﬁed from Hernando et al.40 with permission from Elsevier.

3.5 Modifications related to oxidative stress regulatory mechanisms compared with the activation induced by an 2+ It is well accepted that a burst of oxygen free radicals is generated excessive Ca overload during reperfusion deserves further study. during the first minutes of reperfusion and several works have demonstrated that oxidative stress can modify the activity of calpains, although the overall effect of free radicals on calpain activity during 4. Contribution of calpains myocardial reperfusion is unclear. On the one hand, S-nitrosylation to ischaemia/reperfusion injury can inhibit calpain activity in vitro and in neutrophils in a pH-dependent The use of calpain inhibitors35,37,38,40,44,70,71 and transgenic models way,61,62 and it has also been suggested to regulate calpain activity in overexpressing calpastatin72,73 by several groups in different models the reperfused heart. In agreement with these studies, L-arginine ad- of ischaemia/reperfusion have consistently demonstrated that ministration before and after ischaemia without preventing the forma- calpain activation plays an important role in reperfusion-induced con- tion of oxygen free radicals reduced calpain activity while augmenting tractile dysfunction and cell death due to the proteolysis of a wide their nitrosylation.63 On the other hand, carbonylation has been variety of proteins (Figure 2). Several mechanisms have been proposed shown to increase substrate susceptibility to calpain-dependent pro- by which calpains may contribute to reperfusion injury (Figure 3). teolysis in neurons and other cellular types, but the importance of this modification has not been studied in myocardial tissue nor in ischaemia/reperfusion.64,65 Ischaemia/reperfusion has been shown to 4.1 Increased sarcolemmal fragility increase protein nitrosylation66 and carbonylation67 and that S- Studies showing reduced tolerance of cardiomyocytes to osmotic nitrosylation has been implied in cardioprotection.68,69 Whether or stress demonstrate that ischaemia/reperfusion increases the fragility not these post-translational modifications of calpains are relevant of the sarcolemma.74,75 Sarcolemmal fragility has been shown to 26 J. Inserte et al. play an important role in sarcolemmal rupture occurring during the and cell swelling.74,76 It has been proposed that the sarcolemmal initial minutes of reperfusion by reducing the cell tolerance to the weakening during ischaemia/reperfusion is a consequence of an mechanical stress generated by the development of hypercontracture altered lipidic composition77 and the degradation of structural proteins.78 Although the mechanisms underlying these alterations remain incompletely defined, evidence indicates that the proteolytic activity of calpains plays a critical role in cell fragility, mainly through the cleavage of a-fodrin. a-Fodrin forms the backbone of the membrane cytoskeleton, and its degradation during ischaemia/reperfusion has been shown to correlate linearly with increased membrane fragility.75 It is well established that the uncontrolled activation of calpains leads to the degradation of fodrin79 into two fragments of 150 and 145 kDa and, in fact, this specific pattern of cleavage is widely used Downloaded from https://academic.oup.com/cardiovascres/article/96/1/23/540293 by guest on 28 September 2021 as an index of calpain activity.80 Other calpain substrates whose proteolysis could contribute to increase cell fragility are the subsarcolemmal dystrophin81 and the cytoskeleton-associated proteins paxillin, vinculin, and talin which locate within the focal adhesion complex and mediate the anchorage of the plasma membrane to the cytoskeleton.82

4.2 Impairment of Ca21 handling Once activated, calpains may also contribute to further impairment of + Ca2 handling occurring during reperfusion through different mechanisms. The sarcolemmal protein ankyrin has a central domain that binds Figure 2 Reperfusion-induced cell death correlates with calpain ac- to a-fodrin and an N-terminal domain that interacts with several + + tivation. Linear correlation between cell death (expressed as units of receptors and channels including the a-subunit of Na /K -ATPase.83 + + lactate dehydrogenase released per gram of dry weight) and calpain Binding to ankyrin connects the Na /K -ATPase to the fodrin-based a activity (145/150 kDa -fodrin fragments) in the isolated heart membrane cytoskeleton and determines its speciﬁc location in the model subjected to the ischaemia/reperfusion protocol. Symbols cor- sarcolemma and its correct function.84 During reperfusion, it has respond to different durations of myocardial ischaemia. Modiﬁed from 121 been demonstrated that calpains proteolyse both fodrin and ankyrin Inserte et al. by permission of the European Society of Cardiology. + + producing the detachment of the a-subunit of the Na /K -ATPase

Figure 3 Schematic diagram showing the proposed mechanisms by which calpains participate in reperfusion injury and in the cardioprotective + 2+ + 2 + + effects of preconditioning and postconditioning. NCX, Na /Ca exchanger; NBC, Na /HCO3 cotransporter; NHE, Na /H exchanger. Calpains and reperfusion injury 27 from its membrane anchorage and causing its dysfunction. Loss of concentration through the inhibition of SERCA,19 and CaMKII by ac- + Na -pump activity at the onset of reperfusion prevents a fast normal- celerating pHi recovery at the onset of reperfusion through the phos- + + + + ization of cytosolic Na concentration and results in further Ca2 phorylation of the Na /H exchanger21 may favour calpain activation. influx through the reverse mode of NCX.85 In addition, it has been The 60 kDa subunit of calcineurin has been identified as a calpain sub- proposed that calpains could reduce NCX activity by detaching the strate,104 and its calpain-mediated proteolysis has been suggested to transporter from its binding to ankyrin86 but also by direct cleavage increase its phosphatase activity in reperfused rat hearts and the of the protein.87 Loss of NCX activity after cellular repolarization human ischaemic myocardium.105 Studies in vitro demonstrate the + and normalization of Na gradient may impair the recovery of proteolysis of CaMKs by calpains.106 It has been shown the existence + Ca2 i through its forward mode and be detrimental.88 Finally, it has of a nuclear m-calpain acting as a regulator of CaMKIV and associated + + been suggested that calpains can also modulate Ca2 handling by signalling molecules under conditions of sustained Ca2 influx in cleaving RyR60,89 and SERCA2a,89,90 resulting in impaired sarcoplasmic neurons.107 In addition, neuronal CaMKII is proteolysed and activated + reticulum control of the cytosolic Ca2 concentration. by calpains.108,109 However, although these enzymes have important Downloaded from https://academic.oup.com/cardiovascres/article/96/1/23/540293 by guest on 28 September 2021 roles in different signalling pathways, the effect of their calpain- 4.3 Mitochondria dependent cleavage on reperfusion injury remains unknown. The effects of calpains on mitochondria are the subject of a review included in this issue. It has been proposed that calpain activation during myocardial ischaemia/reperfusion is responsible for initiating a mitochondrial-dependent apoptotic programme independent of cas- 5. Calpain inhibition pases91 that is related to the calpain-dependent activation of Bid.92,93 In addition, although considered cytoplasmic enzymes, recent studies 5.1 Pharmacological inhibition of calpains suggest the existence of different calpain isoforms in the mitochon- during ischaemia/reperfusion 94,95 dria. The activation of mitochondrial calpains during reperfusion Many studies have evaluated the effectiveness of calpain inhibition as has been proposed to be responsible of the degradation of the mito- cardioprotective therapy against myocardial reperfusion injury 87 chondrial inner membrane NCX and the activation of (Table 1). These studies have been performed in both in vivo and ex 71 apoptosis-inducing factor. vivo experimental models and using diverse inhibitors which differ in their pharmacological characteristics. Leupeptin was the first drug 4.4 Degradation of myofibrillar proteins used as calpain inhibitor in the context of ischaemia/reperfusion. Calpain-dependent degradation of proteins involved in the contractile However, although leupeptin preserved contractile function in iso- machinery has been involved in post-ischaemic myocardial dysfunc- lated guinea pig hearts subjected to transient ischaemia,110 this drug tion. Analysis of in vitro protein degradation by m-calpain in human inhibits other proteases like cathepsins, plasmin, or trypsin.111 myocardial tissue samples demonstrated that the myofibrillar proteins N-Ac-Leu-Leu-norleucinal (ALLN or calpain inhibitor I), a derivative titin, desmin, cTnT, and cTnI are calpain substrates, suggesting that the from leupeptin, and MDL-28170 (calpain inhibitor III), although uncontrolled intracellular activation of calpains alters the structure more specific for calpains, may also inhibit cathepsins.111 They have and the regulation of the contractile machinery in the human heart been used in several studies, most of them in isolated hearts, and in vivo.96 It has been demonstrated that overexpression of calpastatin demonstrated protective effects against reperfusion-induced cell induced by gene transfer prevents cTnI degradation and ameliorates death and contractile dysfunction.37,112,113 However, a problem contractile dysfunction in rat hearts subjected to ischaemia/reperfu- shared by these inhibitors that limits their applicability in vivo is their sion.73 Another troponin, cTnT, has been shown to be truncated by poor aqueous solubility. More recently, new water-soluble and cell- calpains after ischaemia/reperfusion and the transgenic expression of permeable calpain inhibitors were tested. Among them, A-705239 this truncated cTnT changed the kinetics of myocardial contraction and A-790523 reduced infarct size in isolated rabbit hearts70 and in in healthy hearts.97,98 In addition to troponin, the cleavage of an in vivo pig model of ischaemia/reperfusion,114 and SNJ-1945 pre- desmin by calpains detected in reperfused hearts38,44,99,100 was pro- served contractile function in hearts submitted to cardioplegia.40 posed to contribute to cardiac dysfunction.44,99 The overexpression Although calpains are activated after pHi correction during early of Hsp27 has been shown to improve post-ischaemic contractile func- reperfusion,40,113 in most of these studies, the drug was administered tion by reducing the interaction between m-calpain, cTnI, cTnT, and before ischaemia. Only two studies, using A-705253 and MDL-28170, desmin during ischaemia/reperfusion.100,101 have demonstrated the cardioprotective potential of calpain inhibitors when administered at the onset of reperfusion in ex vivo and in vivo 4.5 Proteolysis of regulatory enzymes models.115 Through the proteolysis of regulatory enzymes, calpains may contrib- While the beneficial effects of calpain inhibition in the context of ute indirectly to reperfusion injury by altering different signalling path- acute reperfusion injury are well supported, the consequences of pro- ways. Calpain-mediated degradation of protein kinase C (PKC) into longed calpain inhibition are less clear. Recently, chronic m-calpain in- protein kinase M was described years ago,102 and recently, it has hibition has been associated with the development of dilated been demonstrated that the proteolytic processing of PKCa by cal- cardiomyopathy due to the abnormal accumulation of protein aggre- pains activates pathological cardiac signalling through the generation gates, suggesting that m-calpain is necessary for ubiquitination and of a C-terminal fragment with unregulated kinase properties.103 Calci- proteasomal-dependent degradation.38,92,116 These latter results high- neurin and CaMKII have been proposed to be both activators and light the importance of developing inhibitors with calpain isoform + substrates of calpains. Calcineurin, by increasing cytosolic Ca2 specificity. 28 J. Inserte et al.

Table 1 Studies that have assessed the cardioprotective effects of calpain inhibition against myocardial ischaemia/reperfusion injury

Study Species Model Protocol Inhibitor Administration Effect on functional Effect on cell death recovery ...... Matsumura Guinea pig Ex vivo GI15′/R20′ Leupeptin PreI & PostI +16.2% LVDP NA et al.110 Yoshida et al.35 Rat Ex vivo GI20′/R30′ ALLN PreI & PostI +44% LVDP NA Matsumura et al.44 Guinea pig Ex vivo GI20′/R20′ Leupeptin PreI & PostI +21% LVDP NA Matsumura et al.44 Guinea pig Ex vivo GI20′/R20′ ALLN PreI & PostI +15.5% LVDP NA Chen et al.91 Rabbit Ex vivo GI30′/R120′ MDL-28170 PreI & PostI NA 280 CK/250% IS

Maekawa et al.73 Rat Ex vivo GI30′/R60′ Calpastatin over — +31% LVDP NA Downloaded from https://academic.oup.com/cardiovascres/article/96/1/23/540293 by guest on 28 September 2021 Neuhof et al.112 Rabbit Ex vivo GI45′/R60′ A-705239 PreI & PostI +60% LVDP 246.6 LDH, 273.5% CK Inserte et al.38 Rat Ex vivo GI60′/R30′ MDL-28170 PreI & PostI +18% LVDP 247.6% LDH Neuhof et al.37 Rabbit Ex vivo RI60′/R120′ A-705239 PreI & PostI ¼LVDP ¼LDH, ¼CK, ¼IS Neuhof et al.37 Rabbit Ex vivo RI60′/R120′ A-705253 PreI & PostI ¼LVDP 216% IS, ¼LDH, ¼CK Singh et al.89 Rat Ex vivo GI30′/R60′ Leupeptin PreI & PostI +50% LVDP NA Inserte et al.85 Rat Ex vivo GI60′/R30′ MDL-28170 PreI & PostI NA 235.4% LDH 70 ′ ′ Khalil et al. Pig In vivo RI45 /R360 A-705253 PreI & PostI +22% +dP/dtmax 233% IS Inserte et al.116 Rat Ex vivo GI60′/R30′ MDL-28170 PreI & PostI +26.2% LVDP 253.6 LDH, 224.4% IS Neuhof et al.112 Rabbit Ex vivo RI60′/R120′ A-705253 PostI ¼LVDP 224.4% IS, ¼LDH, ¼CK Gilchrist et al.39 Rat Ex vivo GI27′/R60′ ALLN PreI & PostI Earlier recovery in LVDP NA Gilchrist et al.39 Rat Ex vivo GI27′/R60′ ALLM PreI & PostI ¼LVDP NA Hernando et al.40 Rat In vivo RI30′/R120′ MDL-28170 PostI NA 219.8% IS 72 ′ ′ Shan et al. Mouse Ex vivo GI45 /R30 Calpastatin over — +22.1% + dP/dtmax 242.3% LDH Singh et al.126 Rat Ex vivo GI30′/R60′ Leupeptin PreI & PostI +48.2% LVDP NA Singh et al.126 Rat Ex vivo GI30′/R60 MDL-28170 PreI & PostI +43.2% LVDP NA Chen et al.71 Mouse Ex vivo GI30′/R30′ MDL-28170 PreI & PostI NA 237.6% LDH

Protocol: GI, global ischaemia; RI, regional ischaemia; R, reperfusion. Administration: PreI, before ischaemia; PostI, during reperfusion. Functional recovery: LVDP, left ventricle developed

pressure at the end of reperfusion; +dP/dtmax, the maximal value of the derivative of the left ventricle pressure. cell death: LDH and CK, accumulated release of lactate dehydrogenase or creatine kinase, respectively, during whole reperfusion; IS, infarct size; NA, data not available.

5.2 Ischaemic preconditioning the action of other proteins related to the pathway triggered by Ischaemic preconditioning has been shown to prevent calpain activa- PKA activation remains to be elucidated. tion during myocardial reperfusion in several studies.38 The further observation that calpain inhibitors can partially reproduce the protective effects of preconditioning and that calpain activity is modiﬁed 5.3 Ischaemic postconditioning by treatments aimed to mimic or blunt the effects of preconditioning Different groups including ours have demonstrated that the effective- strongly suggests that calpain inhibition mediates at least in part the ness of a postconditioning protocol in limiting infarct size depends on protection afforded by ischaemic preconditioning.92 its ability to delay normalization of intracellular acidosis during the Although calpain inhibition by preconditioning was proposed to be initial minutes of reperfusion.122 Recent results propose that + + + consequence of reduced Ca2 entry during index ischaemia in hearts protein kinase G-dependent inhibition of Na /H exchanger together previously subjected to preconditioning cycles,117 there is evidence with reduced lactate washout determine the observed delay in pH that preconditioning prevents calpain activity by phosphorylation. correction.22 The prolongation of acidosis may be protective by Freshly isolated calpains contain several conserved consensus sites modulating many mechanisms implicated in reperfusion injury121 for PKA phosphorylation,50 and it has been suggested that phosphor- and we have recently shown that slowing pH recovery during reper- ylation at serine 369 by PKA inhibits m-calpain.38,118,119 The precon- fusion by postconditioning or by acidic perfusion limits myocardial ditioning phase results in transient PKA activation, and the abolition of necrosis at least in part through the attenuation of calpain activa- this activation blunts the protective effects of ischaemic precondition- tion.40 These data support recent evidence demonstrating that cal- ing, whereas these effects, including calpain inhibition, are mimicked pains are inhibited by intracellular acidosis during ischaemia despite + by transient PKA stimulation.120,121 Whether PKA exerts its effect Ca2 overload and that activation occurs at reperfusion, after pH on calpain activity by direct phosphorylation of the proteases or by normalization.123 Calpains and reperfusion injury 29

+ 14. Williams IA, Xiao XH, Ju YK, Allen DG. The rise of [Na ]i during ischemia and 6. Perspectives reperfusion in the rat heart-underlying mechanisms. Pflugers Arch 2007;454: 903–912. Recent studies demonstrating that calpain activation occurs exclusive- 15. ten Hove M, Jansen MA, Nederhoff MG, Van Echteld CJ. Combined blockade of the + + + + ly during reperfusion, and the cardioprotective effects obtained pre- Na channel and the Na /H exchanger virtually prevents ischemic Na overload venting their activation by either postconditioning or by using in rat hearts. Mol Cell Biochem 2007;297:101–110. + 16. Chen X, Zhang X, Kubo H, Harris DM, Mills GD, Moyer J et al.Ca2 influx-induced pharmacological inhibitors applied at the onset of reperfusion, dem- + sarcoplasmic reticulum Ca2 overload causes mitochondrial-dependent apoptosis in onstrate that calpain inhibition is a potentially useful therapeutic strat- ventricular myocytes. 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