Received: 13 December 2019 | Revised: 8 January 2020 | Accepted: 31 January 2020 DOI: 10.1111/jcmm.15127 REVIEW Mitochondrial and mitochondrial-independent pathways of myocardial cell death during ischaemia and reperfusion injury Sean M. Davidson1 | Adriana Adameová2,3 | Lucio Barile4 | Hector Alejandro Cabrera-Fuentes5,6,7,8,9 | Antigone Lazou10 | 11,12 13 Pasquale Pagliaro | Kåre-Olav Stensløkken | David Garcia-Dorado14,15,16 | on behalf of the EU-CARDIOPROTECTION COST Action (CA16225) 1 The Hatter Cardiovascular Institute, University College London, London, UK 2 Faculty of Pharmacy, Comenius University Bratislava, Bratislava, Slovakia 3 Centre of Experimental Medicine SAS, Bratislava, Slovakia 4 Laboratory for Cardiovascular Theranostics, Cardiocentro Ticino Foundation and Faculty of Biomedical Sciences, Università Svizzera Italiana, Lugano, Switzerland 5 SingHealth Duke-NUS Cardiovascular Sciences Academic Clinical Programme and Cardiovascular and Metabolic Disorders Program, Duke-National University of Singapore Medical School, Singapore, Singapore 6 National Heart Research Institute Singapore, National Heart Centre Singapore, Singapore, Singapore 7 Tecnologico de Monterrey, Centro de Biotecnologia-FEMSA, Monterrey, Nuevo Leon, México 8 Institute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, Kazan, Russia 9 Institute of Physiology, Medical School, Justus-Liebig-University, Giessen, Germany 10 School of Biology, Aristotle University of Thessaloniki, Thessaloniki, Greece 11 Department of Biological and Clinical Sciences, University of Turin, Torino, Italy 12 National Institute for Cardiovascular Research, Bologna, Italy 13 Section of Physiology, Department of Molecular Medicine, Institute for Basic Medical Sciences, University of Oslo, Oslo, Norway 14 IIS-Fundación Jiménez Díaz University Hospital, Madrid, Spain 15 Department of Cardiology, Vascular Biology and Metabolism Area, Vall d’Hebron University Hospital and Research Institute (VHIR), Barcelona, Spain 16 Universitat Autónoma de Barcelona, Barcelona, Spain Correspondence Sean M. Davidson, The Hatter Abstract Cardiovascular Institute, University College Acute myocardial infarction causes lethal injury to cardiomyocytes during both is- London, 67 Chenies Mews, London WC1E 6HX, UK. chaemia and reperfusion (IR). It is important to define the precise mechanisms by Email: s.davidson@ucl.ac.uk which they die in order to develop strategies to protect the heart from IR injury. Funding information Necrosis is known to play a major role in myocardial IR injury. There is also evidence National Institute for Health Research for significant myocardial death by other pathways such as apoptosis, although this University College London Hospitals Biomedical Research Centre, Grant/ has been challenged. Mitochondria play a central role in both of these pathways of Award Number: BRC233/CM/SD/101320 cell death, as either a causal mechanism is the case of mitochondrial permeability ; Singapore Heart Foundation, Grant/ Award Number: SHF/FG657P/2017; transition leading to necrosis, or as part of the signalling pathway in mitochondrial Behring-Röntgen-Foundation (Marburg, cytochrome c release and apoptosis. Autophagy may impact this process by removing Germany); Agentúra na Podporu Výskumu a Vývoja, Grant/Award Number: APVV-15- *Prof Garcia-Dorado passed away 16th August 2019. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd J Cell Mol Med. 2020;24:3795–3806.  wileyonlinelibrary.com/journal/jcmm | 3795 3796 | DAVIDSON et al. 607, APVV-19-17052, VEGA-1/0271/16, 1/0016; British Heart Foundation, Grant/ dysfunctional proteins or even entire mitochondria through a process called mi- Award Number: PG/18/44/33790 ; Swiss tophagy. More recently, roles for other programmed mechanisms of cell death such National Science Foundation, Grant/Award Number: IZCOZ0_182948/1 ; Russian as necroptosis and pyroptosis have been described, and inhibitors of these pathways Government Program for competitive have been shown to be cardioprotective. In this review, we discuss both mitochon- growth of Kazan Federal University, Kazan (Russian Federation); University of drial and mitochondrial-independent pathways of the major modes of cell death, their Torino, Italy, Grant/Award Number: PAGP_ role in IR injury and their potential to be targeted as part of a cardioprotective strat- RILO_17_18_19 ; the Ministry of Education, Universities and Research, Grant/Award egy. This article is part of a special Issue entitled ‘Mitochondria as targets of acute Number: PAGP_FFABR_17_01 cardioprotection’ and emerged as part of the discussions of the European Union (EU)-CARDIOPROTECTION Cooperation in Science and Technology (COST) Action, CA16225. KEYWORDS apoptosis, autophagy, cardiac, cell death, ischaemia, myocardial infarction, necroptosis, necrosis, pyroptosis, reperfusion 1 | I NTRO D U C TI O N pathway may simply result in the cardiomyocyte dying by an alter- native pathway. This review aims to provide insight into the differ- Ischaemic heart disease remains a major cause of morbidity and mor- ent types of cell death which myocardial cells may undergo during tality throughout the world, and is responsible for ~20% of deaths in IR, with special emphasis on the role of mitochondria in those pro- the European Union in both men and women.1 Many of these deaths cesses, in order to understand how these processes can be targeted occur during an acute ischaemic event such as an ST-elevation to protect the heart.6 myocardial infarction (STEMI). Although fatality rates immediately It is important to note that the initial description and definition of following acute myocardial infarction have decreased in most coun- several of the cell death pathways (apoptosis, pyroptosis, etc) were tries,1 infarct size remains a major determinant of outcome and is based on experimental observations in leucocytes, and they may strongly associated with all-cause mortality and hospitalization for have different manifestations in cardiomyocytes or other non-in- heart failure within the following year. 2 Cardiomyocytes begin to die flammatory cell types in the heart. In this review, we focus on cell during exposure to prolonged ischaemia, and while reperfusion is death pathways occurring in cardiomyocytes. Clearly, mitochondria necessary to limit this process, it causes a spike of further cell death are central to the function of cardiomyocytes, occupying nearly 40% that contributes to final infarct size.3 Thus, finding ways to limit car- of the cytosolic volume,7 and providing the bulk of the ATP neces- diomyocyte death during ischaemia and reperfusion (IR) has been sary for contraction as well as ion pumps and metabolic processes 3 the focus of extensive studies over the past 30 years. Myocardial essential for survival. It is therefore not surprising that mitochon- IR is a complex process during which the ability of physiological pro- dria appear to have a central place in the process of cardiomyocyte cesses to return the cardiac cells to homeostasis is overwhelmed. A death. major cause of this is calcium overload which damages cellular com- ponents and drains energy (ATP) as ion pumps in the sarcolemma and sarcoplasmic reticulum (SR) are engaged to return cytosolic cal- 2 | O N COS I S A N D N EC ROS I S cium back to appropriate levels.4,5 Mitochondrial calcium overload causes mitochondrial damage and further depletes ATP as it is uti- During myocardial ischaemia, oxygen is rapidly depleted, causing lized to maintain mitochondrial membrane potential. In combination mitochondrial respiration to cease. Anaerobic metabolism is ac- with oxidative stress and calcium overload, ATP levels may decrease tivated within seconds of flow cessation, but is unable to provide to a critical level at which the ability of the cardiac cell to remain sufficient ATP for maintaining sarcolemmal ion gradients and mito- viable becomes compromised, and the cell undergoes uncontrolled chondrial membrane potential (ΔΨm). ATP is further depleted by the death through a process of oncosis and necrosis, which is described F0F1ATPase running in reverse, expending ATP in a futile attempt to in detail below. However, even before this step, programmed cell maintain ΔΨm. Eventually, the sarcolemmal ion pumps fail and the death pathways may be activated including apoptosis, necroptosis or cell swells in a process called ‘oncosis’, which is defined as a pre- pyroptosis. Although ultimately each of these pathways still results lethal stage following cellular injury.8 Shortly afterwards, oncosis in the death of the cell, they can have profoundly different effects on leads inevitably to necrosis, defined by the physical, chaotic disrup- the heart, for example in terms of the activation of an inflammatory tion of the cell membrane. Many techniques for identification of response. Furthermore, as many of the pathways appear to overlap dead (necrotic) cells rely on this permeability of the cell membrane, or utilize common cellular signalling components, modulation of one which allows dyes such as propodium iodide or trypan blue to enter DAVIDSON et al. | 3797 Non-cardiomyocyte cell-death pathways Cardiomyocyte death pathways Healthy cell Apoptosis Oncosis (early) (early) Oncosis (early) Oncosis (late) Necrosis/ necroptosis Pyroptosis Cell nucleus TUNEL+ve Cell nucleus PI+ve and TUNEL+ve Cell nucleus F I G U R E 1 The major pathways of cell death that contribute to myocardial ischaemia and reperfusion injury. During initial oncosis, cells swell—this is reversible but can proceed to necrosis. Non-cardiomyocytes can die via a processes of apoptosis or necrosis/necroptosis, in addition to other types of cell death described herein. Cardiomyocytes die primarily via a process of necrosis/necroptosis in addition to other cell-death processes such as pyroptosis, but there is a little evidence for any contribution of apoptosis. Plasma membrane rupture is the terminal event, and this is mediated either by MLKL channels during necroptosis or by GSDMD pores during pyroptosis. Multiple cell- death pathways can eventuate in plasma membrane permeabilization, as detected by dyes such as propodium iodide (resulting in red nuclei as shown) or trypan blue P DRP1 PGAM5 RIP3 RIP3 Mitochondrial DRP1 fission RIP1 RIP3 CaMKII MLKL Mitochondrial MPTP dysfuncon Necroptoc opening Cell death F I G U R E 2 Possible mitochondrial pathways of necroptosis. Ischaemia and reperfusion causes an increase in levels or phosphorylation and activation of RIP1 and RIP3 (receptor-interacting serine/threonine-protein kinase 1 and 3), MLKL (mixed-lineage kinase domain-like pseudokinase) and PGAM5 (phosphoglycerate mutase family member 5). RIP3 translocates to the mitochondria and interacts with PGAM5 and DRP1. PGAM5 dephosphorylates and activates DRP1 leading to mitochondrial fission and dysfunction. Additionally, RIP3-mediated activation of CaMKII (Ca2+-calmodulin-dependent protein kinase) may trigger necroptosis via the MPTP. Mitochondrial dysfunction leads ultimately the formation of the necroptosome consisting of activated RIP1, RIP3 and MLKL, and necroptotic cell death ensues. and stain the cell (Figure 1). However, it is important to note that apoptosis. Likewise, it should also be noted that various cell death membrane permeabilization, and hence necrosis, can occur second- modalities with the features of ruptured plasma membrane can arily to cell death by any mechanism, including the later phase of occur in parallel. These facts can make it difficult to ascertain the 3798 | DAVIDSON et al. TA B L E 1 The key characteristics of Mode of Characteristics known to occur in the main cell death processes discussed cell death Key characteristics cardiomyocytes in this review, and their manifestation in Necrosis Plasma membrane permeability; MPTP MPTP opening13; Plasma cardiomyocytes (if known) opening13 membrane permeability; Hypercontraction/contraction band necrosis10 Apoptosis Mitochondrial outer membrane Controversial; Mitochondrial permeabilization (MOMP), fission41 Mitochondrial cytochrome c release, caspase activation, DNA fragmentation cell shrinkage, Chromatin condensation, Plasma membrane blebbing21 Autophagy LC3-II; Pink1/Parkin accumulation at Similar to the process in other cell mitochondria44; BNIP3 up-regulation49; types58,59 FUNDC1-ser13 dephosphorylation51 Necroptosis Recruitment of cytosolic adaptor proteins Similar to the process in other to complex I62; Plasma membrane cell types 62; RIP3-mediated permeability; relocalization of pMLKL to activation of CaMKII71 the plasma membrane62; RIP3 activation Pyroptosis NLRP3 inflammasome formation; Believed to be similar to the Cleavage of GSDMD, caspase-1, process in other cell types87,91 pro-IL-18 and pro-IL-1β82-84 Parthanatos Activation of poly(ADP-ribose) Believed to be similar to the polymerase-1 (PARP-1)117 process in other cell types Ferroptosis Bid translocation to the mitochondria121; Believed to be similar to the Loss of glutathione peroxidase 4122 process in other cell types Note: As some processes can overlap, not all features are necessarily diagnostic of the type of cell death and may depend on the time-point being examined (eg: most forms of cell death will ultimately result in plasma membrane permeability). precise cause of myocardial cell death, particularly when examining ion pumps cease functioning, and the cells die through a process of a single time-point. oncosis and necrosis. Mitochondria play a critical role in the process of myocardial IR It may be possible to target necrosis during the early stages of injury. In particular, upon reperfusion, when the supply of oxygen infarction in order to protect the heart from IR injury. Mice lack- to the cardiac cells is re-introduced and mitochondrial respiration ing either the MPTP or the MCU have smaller infarct sizes follow- recommences. The mitochondrial substrate succinate, having accu- ing IR.12-14 Cardioprotection against IR injury can also be achieved mulated during ischaemia, provides a powerful source of electrons experimentally by activation of the MAPK/ERK1/2 or PI3K/AKT which produce oxygen radicals by reverse electron transport via signalling pathways.15,16 Activation of the reperfusion injury salvage complex I, resulting in oxidative stress (excess reactive oxygen spe- kinase (RISK) pathway protects the heart by delaying opening of the 9 cies or ROS). The rapid replenishment of oxygen and ATP to the MPTP.17 At least in the isolated, perfused heart, blocking hypercon- cells is a double-edged sword. ATP is necessary to restore ionic ho- traction by lowering pH or administering inhibitors of the contractile meostasis but it also reactivates the sarcoplasmic reticulum ATPASE machinery reduces infarct size, as does blocking of reverse electron 2A (SERCA2A) allowing it to pump Ca2+ back into the SR. However, flow by providing malonate.18-20 However, as will be discussed later, 2+ hyperactivation of the SR Ca release channel, RyR2, results in it cannot be ruled out that such previously reported MPTP opening rapid cycles of SR Ca2+ uptake and spontaneous SR Ca2+ release. associated with necrosis17 can be also a mechanism of other necro- 2+ The restoration of ATP levels while cytosolic Ca overload is still sis-like cell death modes, which have been identified more recently.13 current also leads to hypercontraction of cardiomyocytes, which can be detected during the first few minutes of reperfusion by the appearance of ‘contraction band necrosis’ in haematoxylin and eo- 3 | A P O P TOS I S sin-stained myocardial histological sections.10 The excessive SR Ca2+ release contributes to mitochondrial Ca2+ uptake via the mitochon- Apoptosis is a form of cell death that can be distinguished micro- 2+ drial calcium uniporter (MCU), leading to mitochondrial Ca over- scopically from oncosis by cell shrinkage, chromatin condensa- load and opening of the mitochondrial permeability transition pore tion and distinctive blebbing (budding) of the plasma membrane. (MPTP).11 With the MPTP open, the mitochondria are no longer able Apoptosis can occur via intrinsic or extrinsic mechanisms but both to maintain ΔΨm, and shortly afterwards, ATP stores are depleted, result in mitochondrial outer membrane permeabilization (MOMP), DAVIDSON et al. | 3799 mitochondrial cytochrome c release, caspase activation, DNA frag- factors such as cytochrome c and SMAC/Diablo from the intermem- 21 mentation and cell blebbing. brane space into the cytosol.38 Oxidative stress and Ca2+ overload Early studies detected evidence of apoptosis along with necrotic leading to MPTP opening may contribute to apoptosis by increasing cell death following myocardial IR. 22 However, the relative contri- MOMP and cytochrome c release. bution of apoptosis to the extent of cardiac damage is still debated Another important mechanism for mitochondrial quality control due to the large differences in its magnitude as reported by different is mitochondrial fusion and fission. Mitochondrial fission is consid- investigators. DNA laddering, one of the hallmarks of apoptosis, was ered to be the prerequisite for the occurrence of mitophagy and sev- not detected in myocardium subjected to ischaemia alone, but was eral studies revealed the causal relationship between mitochondrial only observed after reperfusion, suggesting that the apoptotic com- fission and the induction of apoptosis.39,40 In this respect, it has been ponent of cell death in the myocardium is triggered at the time of demonstrated that increased mitochondrial fission in the ischaemic reperfusion and does not manifest during the ischaemic period. 23,24 heart contributes to apoptosis induction and infarct generation, In contrast, other studies have shown that apoptosis begins either while inhibiting mitochondrial fission reduces myocardial injury and after prolonged myocardial ischaemia without reperfusion or after improves cardiac function following myocardial infarction.41 25,26 a brief period of ischaemia followed by reperfusion. Detection of pro-apoptotic factors and caspase activation during ischaemia in the absence of DNA fragmentation followed by a more massive in- 4 | AU TO PH AG Y crease during reperfusion indicates that the apoptotic cascade is ini- tiated during ischaemia, but is fully executed during reperfusion. 27,28 Mitochondrial damage negatively affects cardiomyocyte function More supportive evidence for the acceleration of apoptosis during via disruption of oxidative phosphorylation, Ca2+ dyshomeostasis, reperfusion comes from studies showing a reduction in infarct size increased oxidative stress and incomplete digestion of mitochondrial using inhibitors of pro-apoptotic mediators at early reperfusion. 29,30 DNA. These events play a role in triggering the formation of inflam- Studies in humans have also demonstrated the detection of apop- masomes within cells.42 Thus, a process that contributes to the elim- totic cardiomyocytes in the border zone of the infarcted myocar- ination of toxic mitochondrial contents and mitochondrial quality dium within hours to days of infarction.31 control is crucial to the maintenance overall cardiomyocyte health. In contrast to the above, other studies have argued against the Mechanisms of mitochondrial degradation include mitochondrial au- significant role of apoptosis in IR-induced cell death, based on the tophagy, also known as mitophagy, and micromitophagy, whereby fact that there is minimal expression of most proteins required for lysosomes directly fuse with and degrade mitochondria.43 A well- 32,33 the apoptotic program in adult cardiomyocytes. In addition, established mechanism of mitophagy in cardiomyocytes requires using cardiac-specific knockout mice, it was conclusively shown that the accumulation of mitochondrial PTEN-induced putative kinase-1 the executioner caspase-3 and caspase-7 do not significantly con- (PINK1) at depolarized mitochondrial outer membranes, which pro- tribute to the acute effects of myocardial IR injury.34 Even forced motes translocation of the E3 ubiquitin ligase Parkin from the cytosol overexpression of caspase in cardiomyocytes is not able to trigger to the damaged mitochondria.44 PINK1-mediated phosphorylation a full apoptotic response in cardiomyocytes during IR, although it of Parkin induces a conformational change in the active form which 35 does result in increased infarct sizes. This raises the possibility ubiquitinates mitochondrial surface proteins such as the voltage- that the previous observations of apoptosis in the heart are likely to dependent anion channel-1 (VDAC1) and Mitofusin 1, 2 (MFN1, 2), have been due to apoptosis of non-cardiomyocytes. 21,32 Notably, as leading to mitochondrial autophagic removal.45,46 Another E3 ubiq- non-cardiomyocytes significantly outnumber cardiomyocytes in the uitin ligase, TNF-receptor-associated factor 2 (TRAF2), has also been heart, their apoptosis could easily account for the observed DNA shown to promote the removal of ubiquitin-tagged and damaged mi- laddering. Similarly, TUNEL staining is weak evidence for cardio- tochondria during IR injury, but in a Parkin-independent manner.47 myocyte apoptosis because ultrastructural studies have shown that Two further Parkin-independent mechanisms of mitophagy involve TUNEL staining is present only in cardiomyocytes that have already mitochondrial outer membrane proteins: the mitochondrial pro- 36 died by necrosis. Of note, apoptosis of endothelial cells and leuco- apoptotic BH3 domain-only protein BNIP3 and FUNDC1.48 BNIP3 cytes would also be expected to indirectly affect cardiomyocyte cell is up-regulated in the myocardium during hypoxia49 and promotes 32 survival and cardiac performance. Thus, anti-apoptotic strategies mitophagy that does not require ubiquitination. Under hypoxic con- may still be cardioprotective. 21 For example, a recently developed ditions, FUNDC1 is dephosphorylated by phosphatases (eg PGAM5) peptide targeting the FAS-dependent apoptotic signal during IR in- at Serine 13, which induces protein interaction with microtubule- jury decreased infarct size in mice, even when administration was associated proteins 1A/1B light chain 3 (LC3), thereby enhancing delayed 30 minutes into reperfusion.37 mitophagy.50,51 Mitochondria play an important role in the execution of apop- Mitophagy can be considered a beneficial cellular process that tosis. These organelles are the major contributors of ROS and the enhances cell viability following stressful stimuli by eliminating dys- major target for ROS-induced damage. The mitochondrial apoptosis functional mitochondria. Mitophagy is therefore essential for cardio- pathway is triggered by mitochondrial swelling and outer mitochon- myocyte survival.52 Accumulated evidence suggests that IR causes drial membrane rupture, thus favouring the release of pro-apoptotic an imbalance in the mitophagy process, and one could easily imagine 3800 | DAVIDSON et al. how this would allow dysfunctional mitochondria to accumulate in MLKL.72 An increase was also seen in the mitochondrial membrane the cell, causing further cytotoxic damage and potentially leading to protein PGAM5 (phosphoglycerate mutase family member 5), an cell death. However, it remains controversial whether it is excessive atypical Ser/Thr phosphatase that dephosphorylates and activates mitophagy or large-scale accumulation of autophagosomes that is the mitochondrial fission protein DRP1.72 Furthermore, PGAM5 the main mechanism underlying ‘autophagic cell death’.40,53,54 The knockdown suppressed the generation of ROS and preserved ΔΨm, emerging consensus is that cellular insults induce changes consistent implicating mitochondria in the process of necroptosis.72 In a similar with autophagosome formation and the initiation of autophagy in model of H9c2 subjected to HR, siRNA targeting RIP3 was shown to cardiac cells, and that these processes can lead to cell death. prevent mitochondrial fragmentation and necroptosis, by preventing The results of early studies of the role of autophagy in IR- activation of DRP1, again suggesting a link between necroptosis and induced cardiomyocyte death were somewhat contradictory, indi- mitochondria.73 cating that autophagy could be cyto-protective, but could also direct Subcellular analyses have further revealed that RIP3 co-local- cells towards apoptosis.55 These observations are in contrast with izes with mitochondria69 or translocates to mitochondria during HR the notion that autophagy may trigger cell death in a caspase-inde- and interacts with DRP1, but not with RIP1 and MLKL.73 This pro- 56 57 pendent way as assessed in vitro and in vivo, where it has been vides evidence for necroptosis execution being independent of the shown that impairing the expression of ATG genes leads to reduc- canonical pathways these proteins are involved in.71 These findings tion of cell death. However, rigorous kinetic analyses are required to suggest that RIP3-PGAM5-DRP1 cause mitochondrial dysfunction establish whether autophagic cell death is independent from apop- that promotes necroptosis. However, in contrast, Lu et al found that totic or necrotic processes and whether it represents a step through PGAM5 ablation was deleterious because it increased infarct size which these processes culminate with cell disruption.53 Evidence and exacerbated necroptosis.74 Promotion of mitophagy, which can from Sadoshima's group suggested that autophagy is beneficial prevent necroptosis by clearing ROS-producing unhealthy mito- during ischaemia but harmful during reperfusion.58 However, more chondria,75 can underlie such a protective regulatory function of this recently, the balance of evidence favours a beneficial role for auto- protein phosphatase in necroptosis-associated conditions. phagy in the heart under most conditions.59 From the above discussion, it is evident that the extent of mi- tochondrial participation in necroptosis in cardiomyocytes or other cell types is not clear. It has been suggested that the contribution 5 | N EC RO P TOS I S of mitochondria to necroptosis may be secondary or context-de- pendent.76 For instance, MLKL, the ultimate effector of necropto- Necroptosis, a regulated mode of cell death with a necrotic appear- sis, is unlikely to directly assemble at mitochondrial membranes, as ance, has been identified in various cardiac pathologies, including shown for the much higher efficiency of the MLKL N-terminal do- 60,61 myocardial IR (reviewed in ). The precise cytotoxic mechanisms main to permeabilize liposomes with a composition resembling the of necroptosis are not fully understood; however, the activation of plasma membrane, in comparison with cardiolipin-rich liposomes.77 RIP1 and RIP3 (receptor-interacting serine/threonine-protein ki- Nonetheless, at early stages of SMAC-mimetic induced necroptosis nase 1 and 3) is essential for necroptotic cell membrane rupture- in various cancer cell types, a disruption of mitochondrial membrane inducing events that occur as a consequence of the relocalization potential is observed that presumably depends on BAK-BAX activ- to the plasma membrane of phosphorylated mixed-lineage kinase ity.78 In accordance with this, the pro-apoptotic protein PUMA could 62 domain-like pseudokinase (MLKL) (Figure 2). Accordingly, the ma- act as an amplifier of necroptosis by exposing mitochondrial DNA jority of studies of both acute63-67 and chronic myocardial IR injury to the cytosolic sensors, which further stimulates the necrosome 68-70 progressing into heart failure indicate that membrane-asso- formation.79 Moreover, at the inner mitochondrial membrane, MPTP ciated RIP1-RIP3-MLKL axis is a key player in necroptotic damage response could be an important mediator of this kind of cell death, leading to worsening of heart function and adverse cardiac remodel- as either cyclosporine A treatment or cyclophilin D deficiency confer ling. However, only a very limited number of experiments have in- resistance to necroptosis prompted by TNFα exposure of endothelial vestigated the importance of mitochondria in this process. By using cells.80 In summary, therefore, while specific mitochondrial mecha- a pharmacological inhibitor of RIP1, necrostatin-1 (Nec-1), which re- nisms in the membrane canonical pathway of necroptosis cannot be duced infarct size in wild-type but not in CypD−/− mice, it has been ruled out, the details remain to be elucidated. suggested that cyclophilin D, an important regulatory component of the MPTP, might be involved in necroptosis signalling66 due to promotion of MPTP opening.67 RIP3-mediated activation of CaMKII 6 | PY RO P TOS I S (Ca2+-calmodulin-dependent protein kinase), serving as an upstream regulator of the MPTP, is likely to trigger this critical event in IR- Pyroptosis, meaning ‘fire’ and ‘falling’, is a pro-inflammatory cell- 71 induced necro(pto)sis. death program occurring after cytosolic receptor-mediated rec- Necroptosis has been seen to occur in both H9c2 cardiomyo- ognition of pathogen-associated molecular patterns (PAMPs), or blasts subjected to hypoxia/reoxygenation (HR) and in vivo rat hearts host-derived, danger signals such as damage-associated molecu- subject to IR, as evidenced by increased levels of RIP1, RIP3 and lar patterns (DAMPs). Some well-characterized DAMPs include DAVIDSON et al. | 3801 glucose-regulated proteins (GRPs), high-mobility group box 1 Despite the fact that IR-damaged myocardium releases a com- (HMGB-1), IL-1β, S100 family proteins and some heat shock proteins bination of priming and triggering factors of the NLRP3 inflam- (HSPs). Interaction of these proteins with cellular pattern recognition masome, it has been proposed that the NLRP3 inflammasome in the receptors can lead to the assembly of the intracellular NLRP3 inflam- heart is not sufficient to respond to a trigger signal in the absence masome complex. The assembled NLRP3 inflammasome elicits auto- of a priming.98 Actually, after IR, the size of the infarct is found to proteolytic cleavage and activation of caspase-1, which mediates the increase more in the presence of an active NLRP3,93,96,99 especially cleavage/maturation of the pro-inflammatory cytokines, pro-IL-18 if metabolic syndrome has primed the inflammasome.99 and pro-IL-1β, and cleaves/activates gasdermin-D (GSDMD), releas- Although little is known about the role of mitochondria in ing its N-terminal fragment.81 Activated GSDMD-N, as well as ac- pyroptosis occurring in the myocardium specifically, there is ev- tive caspase-1, may induce the formation of membrane pores thus idence for mitochondrial involvement in other cell types. In fact, inducing cell lysis, called pyroptotic cell death.82-84 The subsequent mitochondrial dysfunction, leading to oxidative stress and mito- immune response triggered by DAMPs can lead to various forms of chondrial DNA (mtDNA) release, is emerging as a key mechanism cell death, including apoptosis, pyroptosis, necrosis and necroptosis. in triggering NLRP3 assembly and activation in several conditions Of note, bidirectional crosstalk between pyroptotic and apoptotic and indeed has been proposed as the universal trigger for NLRP3 cell death mechanisms has been described: caspase-1 can cleave and activation.100,101 Mitochondrial ROS induce NLRP3-dependent activate caspase-3 and caspase-7 to start apoptosis, which in turn lysosomal damage and further inflammasome activation.95,102 can cleave and inactivate GSDMD to limit pyroptosis.85 Likewise, a ROS favour the mitochondrial localization of NLRP3 and ASC for crosstalk between pyroptosis and necroptosis being associated with further NLRP3 activation. VDAC1-induced ROS formation and RIP3 and MLKL activation has also been suggested.86 BAX/BAK activation can also trigger NLRP3 activation. Calcium The DAMPs released after cell death can induce further injury, overload and calcium entry into mitochondria may favour MPTP thus causing a vicious cycle that expands the region of ischaemic opening thus generating ROS, which promote the deubiquitylation damage. Evidence for a role of inflammasome/pyroptosis in acute of NLRP3 and inflammasome activation, thus facilitating mtDNA myocardial IR injury comes from several studies. 87 In 2001, pre- release to further activate NLRP3.103 TLR4-IRF1 mediates tran- ceding the notion of the inflammasome, it was reported that the scriptional up-regulation of UMP-CMPK2 (uridine/cytidine mo- secretion of IL-1β and IL-18 was increased in an IR model of iso- nophosphate kinase-2), which favours mtDNA synthesis and the 88 lated human atrial myocardium. In 2003, targeted deletion of formation of oxidized short strands of mtDNA that exit the mi- caspase-1 was shown to reduce early mortality and left ventricular tochondria.104 Also, exposed mitochondrial cardiolipin can tether dilation following cardiac infarction in mice, thus supporting a link NLRP3 to the mitochondria for activation in a ROS-dependent and between inflammasome activation, apoptosis and cardiovascular ROS-independent fashion.105 Released cardiolipin in combination 89 90 diseases. In 2011, Kawaguchi et al reported an essential role with ineffective mitophagy lead to NLRP3-mediated activation of for cardiac fibroblasts inflammasome activation in myocardial IR caspase-1 and subsequent production of IL-1.105 Loss of BCL-XL 91 injury. Simultaneously, Mezzaroma et al, using a gene silencing and MCL-1 activity can activate BAX/BAK, which may favour NLRP3 model, suggested that NLRP3 overexpression occurs not NLRP3 activation, via downstream effectors (activated caspase-3 only in cardiac fibroblasts, but also in infiltrating cells and, impor- and caspase-7), in a K+ efflux-dependent way. Furthermore, the tantly, in cardiomyocytes of the border zone of the infarct area. In serine threonine kinase NIMA-related kinase 7 (NEK7) is a pro- 2013, Sandanger et al reported a reduced infarct size in Nlrp3−/− moter of NLRP3 inflammasome assembly downstream of ROS and mice in an ex vivo Langendorff perfused heart model of IR.92 Also K+ efflux.106,107 In fact, the catalytic domain of NEK7 interacts using isolated hearts, it was confirmed that pre-treatment with the with the NACHT/LRR domain to favour NLRP3 inflammasome NLRP3 inhibitor, INF4E, reduced infarct size and improved ven- activation.106,107 Intriguingly, this interaction may be disrupted by 93 tricular developed pressure after IR. These findings were con- cytochrome c, an intrinsic/mitochondrial regulator of apoptosis, firmed by Luo et al in a type 2 diabetes rat model.94 to limit pyroptotic cell death, in a sort of yin yang, bidirectional The mechanisms of myocardial IR damage by DAMPs may in- process between apoptosis and pyroptosis. 85,107 Finally, it has also volve binding to receptor for AGE (advanced glycation end prod- been proposed that mitophagy may dampen NLRP3 activation by ucts, RAGE) or to TLR4 (toll-like receptor 4), thus activating NFκB removing injured mitochondria. Actually, NLRP3 activity and met- and exacerbating myocardial damage. Indeed, among pro-inflam- abolic disease progression have been associated with low levels of matory genes, NFκB promotes the transcription of components of mitochondrial mitofusin proteins and elevated levels of DRP1, es- the NLRP3 inflammasome in cardiac cells.95 Therefore, the NLRP3 pecially in hyperglycaemic conditions.108,109 Yet, deletion of DRP1 inflammasome may be considered a sensor that links myocardial may lead to an increase in NLRP3-dependent caspase-1 activation damage to inflammation, thereby contributing to the progression of and IL-1 secretion.110 96,97 the wavefront of IR injury. After activation, the NLRP3 inflam- Recent evidence has shown that autophagy is necessary to re- masome promotes cardiomyocyte death and infarct size progression duce myocardial damage after acute myocardial infarction, thus con- in the first hours of reperfusion likely through pyroptosis and then firming that the autophagic process can limit the activation of the through production of IL-1β and inflammation.91,96 NLRP3 inflammasome by removing damaged mitochondria and that 3802 | DAVIDSON et al. impaired mitophagy may contribute to adverse cardiac remodelling 8 | TR A N S L ATI O N A L PE R S PEC TI V E S 111,112 in myocardial infarction. Arguing against an important role for NLRP3 inflammasome in Although chemical inhibitors of specific cell death pathways are very cardiac injury is a work of Sandanger et al who reported that NLRP3 effective in limiting infarct size after IR in the experimental setting, inflammasome activation is cardioprotective during myocardial few studies have investigated these drugs in patients experiencing 113 IR. Moreover, Hermansson et al found that NLRP3 levels were cardiac IR.3,6,16 The paucity of studies is partly because available low in human ischaemic myocardial tissue compared with non-isch- cell death inhibitors may not be suitable for use in patients, due to aemic control cardiac tissue.114 However, gene analysis showed mu- insufficient specificity or incompletely determined pharmacokinet- tations in NLRP3 in human cardiac tissues from ischaemic patients, ics or pharmacodynamics. One exception is the CIRCUS trial which but not in those from non-ischaemic controls; the authors suggested investigated whether CsA would prevent MPTP and mitochondrial- that genetic defects in the inflammasome and related proteins may mediated necrosis in STEMI patients; however, this unfortunately represent a background for promoting ischaemic cardiac disease. failed to provide evidence of benefit.125 There are many potential Yet, Zuurbier et al,115 testing the hypothesis that NLRP3 inflam- reasons for this, including the high prevalence of co-morbidities masome, plays a role either in preconditioning (IPC) or in acute IR and the pharmacological background of patients, which may modify injury, observed that NLRP3 deletion did not affect cell death, but cell death pathways. This is discussed further in the accompanying exacerbated IR-induced mechanical dysfunction. Moreover, NLRP3 review in this series, ‘Translational issues and mitoprotection’. One deletion abrogated the protective effects of IPC against IR damage. further explanation may be that cardiomyocytes that escape early The authors proposed that the observed effects are due to an al- MPTP-mediated death can go on to die via other pathways.126 If so, tered IL-6/STAT3 dependent mechanism. Therefore, it is possible this may mean that such a single-target approach will always be des- that some concomitant modifications of protective pathways may tined to fail, and an approach targeting multiple cell death and/or cell explain discordant results.116 survival pathways may be necessary.4 7 | OTH E R T Y PE S O F C E LL D E ATH 9 | CO N C LU S I O N S Although the involvement of mitochondria in extrinsic and intrinsic An important limitation in all investigations of cell death pathways apoptotic pathways is undeniable, and their role in necroptosis and is the methodology used to evaluate cell death in cardiomyocytes pyroptosis is being elucidated, the degree to which mitochondria are (Table 1). TUNEL staining can be misleading as it alone does not involved in other, more newly recognized, forms of cell death has just distinguish between cells undergoing apoptosis, necrosis or DNA begun to be disentangled. In this respect, ‘parthanatos’ entails cell repair.62 Caspase activity can be measured but is also not specific death characterized by excessive activation of poly(ADP-ribose) pol- to apoptosis as caspases can be involved in other pathways of cell ymerase-1 (PARP-1).117 PARP-1 transfers ADP-ribose to apoptosis- death. Cell-based assays for more recently described forms of cell inducing factor (AIF), which in turn translocates from mitochondria death such as pyroptosis and necroptosis have not yet been devel- to the nucleus where it triggers large-scale DNA fragmentation and oped and many used assays are rather nonspecific being limited to thus precipitates cell death.118,119 Hence, mitochondria are central the detection of cell damage due to membrane rupture and impair- to PARP-1-mediated cell death. Genetic disruption of this pathway ment of cell metabolic activity. Distinguishing between different protects mice against IR injury.120 forms of cell death is further complicated by the potential overlap ‘Ferroptosis’ is a form of programmed cell death dependent on between them, and sharing of common signalling components. In iron. Ferroptosis seems to converge on the BCL2-family protein this regard, recently published guidelines offer useful advice to the member BID, promoting its translocation to the mitochondria where use of different assays of cell death.62 However, complicating this it causes profound mitochondrial fragmentation and dysfunction.121 analysis is the fact that different types of cell can exhibit different This suggests a major role for mitochondria during ferroptotic death. characteristics during cell death, and cardiomyocytes may exhibit However, integrity of the anaplerotic mitochondrial glutaminolysis some particular differences to other cell types. Using observations and the tricarboxylic acid cycle is indispensable for ferroptosis pro- taken from immune cells to interpret the response of cardiomyo- duced by erastin or cysteine starvation but not by glutathione per- cytes to injury may not always lead to the correct interpretation and 122 oxidase 4 deficiency. Besides, coenzyme Q, an essential electron must be performed with care. carrier of the mitochondrial electron transport chain, ought to be In summary, mitochondria seem to be a convergent point be- able to suppress ferroptosis caused by glutathione peroxidase 4 deg- tween various regulated cell death processes. In some cases, such radation by an as-yet-unknown mechanism, as the hydrophilic coen- as MPTP-mediated necrosis, apoptosis and parthanatos, their par- 123 zyme Q analogue, idebenone, counteracts FIN56-induced death. ticipation is clear, whereas in other cases, such as necroptosis, the Furthermore, the outer membrane protein CDGSH iron sulphur extent of their involvement might be context-dependent. In either domain 1 (CISD1) negatively regulates mitochondrial iron accumula- case, increasing evidence points to a crosstalk between the diverse tion, lipid peroxidation and erastin-induced ferroptosis.124 pathways of death, with mitochondria likely to be a central node of DAVIDSON et al. | 3803 all such pathways. They are therefore a key target for maintaining constitutive phosphorylation of ryanodine receptors. Cardiovasc Res. 2019;115:556-569. the health of cardiomyocytes during IR and thereby protecting the 6. Hausenloy DJ, Garcia-Dorado D, Botker HE, et al. Novel targets heart from injury. and future strategies for acute cardioprotection: position Paper of the European Society of Cardiology Working Group on Cellular AC K N OW L E D G E M E N T S Biology of the Heart. Cardiovasc Res. 2017;113:564-585. 7. Laguens RP, Weinschelbaun R, Favaloro R. Ultrastructural and This work was supported by grants from the Swiss National morphometric study of the human heart muscle cell in acute coro- Science Foundation (IZCOZ0_182948/1 to LB) (APVV-15-607, nary insufficiency. Hum Pathol. 1979;10:695-705. APVV-19-17052, VEGA-1/0271/16, 1/0016/20 to AA) from the 8. Majno G, Joris I. Apoptosis, oncosis, and necrosis. An overview of National Institute for Health Research University College London cell death. Am J Pathol. 1995;146:3-15. Hospitals Biomedical Research Centre (BRC233/CM/SD/101320 9. Chouchani ET, Pell VR, Gaude E, et al. Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS. to SMD) from the British Heart Foundation (PG/18/44/33790 to Nature. 2014;515:431-435. SMD); from the University of Torino, Italy (PAGP_RILO_17_18_19 10. Rodriguez-Sinovas A, Abdallah Y, Piper HM, et al. Reperfusion in- to PP); and from the Ministry of Education, Universities and jury as a therapeutic challenge in patients with acute myocardial Research (PAGP_FFABR_17_01). HAC-F was supported by the infarction. Heart Fail Rev. 2007;12:207-216. 11. Harisseh R, Abrial M, Chiari P, et al. A modified calcium retention Russian Government Program for competitive growth of Kazan capacity assay clarifies the roles of extra-and intracellular calcium Federal University, Kazan (Russian Federation), by the Singapore pools in mitochondrial permeability transition pore opening. J Biol Heart Foundation (SHF/FG657P/2017), and by the von Behring- Chem. 2019;294(42):15282–15292. Röntgen-Foundation (Marburg, Germany). All authors contribut- 12. Luongo TS, Lambert JP, Yuan A, et al. The mitochondrial cal- cium uniporter matches energetic supply with cardiac workload ing the writing and revision of the paper. This article/publication during stress and modulates permeability transition. Cell Rep. is based upon work from COST Action EU-CARDIOPROTECTION 2015;12:23-34. CA16225 supported by COST (European Cooperation in Science 13. Baines CP, Kaiser RA, Purcell NH, et al. Loss of cyclophilin D re- and Technology). veals a critical role for mitochondrial permeability transition in cell death. Nature. 2005;434:658-662. 14. Lim SY, Davidson SM, Hausenloy DJ, et al. Preconditioning and C O N FL I C T O F I N T E R E S T postconditioning: the essential role of the mitochondrial permea- The authors confirm that there are no conflicts of interest. bility transition pore. Cardiovasc Res. 2007;75:530-535. 15. Schulman D, Latchman DS, Yellon DM. 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