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 myo- cardial reperfusion, and subsequent proteolysis of a wide variety of proteins contributes to the development of con- tractile 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 in- hibition 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 cardi- oprotective 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 trans- location 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,