Received: 6 November 2019 | Revised: 26 November 2019 | Accepted: 27 November 2019 DOI: 10.1111/jcmm.14953 REVIEW Translational issues for mitoprotective agents as adjunct to reperfusion therapy in patients with ST-segment elevation myocardial infarction Hans Erik Bøtker1 | Hector Alejandro Cabrera-Fuentes2,3,4,5,6 | Marisol Ruiz-Meana7,8 | Gerd Heusch9 | Michel Ovize10 1 Department of Cardiology, Aarhus University Hospital, Aarhus N, Denmark 2 SingHealth Duke-NUS Cardiovascular Sciences Academic Clinical Programme and Cardiovascular and Metabolic Disorders Program, Duke-National University of Singapore Medical School, Singapore, Singapore 3 National Heart Research Institute Singapore, National Heart Centre, Singapore, Singapore 4 Institute of Biochemistry, Medical School, Justus-Liebig University, Giessen, Germany 5 Tecnologico de Monterrey, Centro de Biotecnologia-FEMSA, Monterrey, Mexico 6 Institute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, Kazan, Russian Federation 7 Vall d'Hebron Institut de Recerca, University Hospital Vall d'Hebron-Universitat Autònoma, Barcelona, Spain 8 Centro de Investigación Biomédica en Red-CV, CIBER-CV, Spain 9 Institute for Pathophysiology, West German Heart and Vascular Center, University of Essen. Medical School, Essen, Germany 10 CarMeN Laboratory, Hôpital Louis Pradel, Hospices Civils de Lyon, Université de Lyon and Explorations Fonctionnelles Cardiovasculaires, INSERM U1060, Lyon, France Correspondence Hans Erik Bøtker, Department of Cardiology, Abstract Aarhus University Hospital, Palle Juul- Pre-clinical studies have indicated that mitoprotective drugs may add cardioprotec- Jensens Boulevard 99, DK-8200 Aarhus N, Denmark. tion beyond rapid revascularization, antiplatelet therapy and risk modification. We Email: haboet@rm.dk review the clinical efficacy of mitoprotective drugs that have progressed to clinical Funding information testing comprising cyclosporine A, KAI-9803, MTP131 and TRO 40303. Whereas HEB is supported by the Novo cyclosporine may reduce infarct size in patients undergoing primary angioplasty as Nordisk Foundation (grant numbers NNF14OC0013337 and evaluated by release of myocardial ischaemic biomarkers and infarct size imaging, NNF15OC0016674); HACF is supported the other drugs were not capable of demonstrating this effect in the clinical setting. by the Russian Government Program for competitive growth of Kazan Federal The absent effect leaves the role of the mitochondrial permeability transition pore University, Kazan (Russian Federation), for reperfusion injury in humans unanswered and indicates that targeting one single by the Singapore Heart Foundation (SHF/ FG657P/2017) and by the von Behring- mechanism to provide mitoprotection may not be efficient. Moreover, the lack of ef- Rӧntgen-Foundation (Marburg, Germany). fect may relate to favourable outcome with current optimal therapy, but conditions MRM is funded by ISCIII (PI19-01196), CIBER-CV, Fundació MTV3-122/C/2015, such as age, sex, diabetes, dyslipidaemia and concurrent medications may also alter SEC-2016 and the European Regional mitochondrial function. However, as long as the molecular structure of the pore re- Development Fundings (ERDF-FEDER); and GH is supported by the German Research mains unknown and specific inhibitors of its opening are lacking, the mitochondrial Foundation (SFB 1116 B8). This article is permeability transition pore remains a target for alleviation of reperfusion injury. based on the work of COST Action EU- CARDIOPROTECTION (CA16225) and Nevertheless, taking conditions such as ageing, sex, comorbidities and co-medication supported by COST (European Cooperation in Science and Technology). 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;24:2717–2729.  wileyonlinelibrary.com/journal/jcmm | 2717 2718 | BØTKER et al. into account may be of paramount importance during the design of pre-clinical and clinical studies testing mitoprotective drugs. KEYWORDS cyclosporine A, ischaemia, mitochondria, myocardial infarction, reperfusion 1 | I NTRO D U C TI O N whether pharmacological mitoprotection is a useful way to pursue for improving outcome in STEMI patients undergoing reperfusion Modern reperfusion therapy has improved outcome for patients with therapy. ST-elevation myocardial infarction (STEMI) tremendously.1 Over the past 5 years, however, mortality reduction has levelled out1 and the decline in the incidence of post-MI heart failure is modest. 2 So, there 2 | PATH O PH YS I O LO G I C A L BAC KG RO U N D still may be a need to reduce infarct size to further improve outcome. FO R I NTE RV E NTI O N AG A I N S T Because infarct size depends on ischaemia time, the most im- M ITO C H O N D R I A L DYS FU N C TI O N I N portant way to diminish it and improve outcome remains a reduction I S C H A E M I A- R E PE R FU S I O N I N J U RY in the ischaemic time by reducing any delay and insuring rapid revas- cularization in STEMI patients. Beyond this focus, a major target may In a clinical context, mitochondrial dysfunction has been reported be an attempt to reduce infarct size by addressing the reperfusion in cardiac diseases including ischaemia-reperfusion injury as well as injury that occurs, when injuring mechanisms are activated upon in comorbidities associated with ischaemic cardiomyopathies such opening of the coronary artery.3,4 as diabetes or obesity. Under aerobic conditions, mitochondria are Mitochondria in the heart are crucial for the generation of ade- indispensable for cell function and viability primarily through ATP nosine triphosphate (ATP) necessary to sustain contractile function production, regulation of cellular redox potential and control of ap- and for the dynamic adjustment of the cardiomyocytes' metabolic optosis. Because of their close functional and anatomical association demand and ionic homeostasis. Hence, the organelle is considered with the sarcoplasmic reticulum, mitochondria play an active role in an important target for cardioprotection of the myocardium exposed calcium uptake, which in turn is a critical regulator of the Krebs cycle to an acute ischaemia-reperfusion injury. In particular, acute opening and therefore of mitochondrial respiration and NAPDH-dependent of the mitochondrial permeability transition pore (MPTP) has been antioxidant regeneration. 24-26 Mitochondrial calcium uptake is 5,6 involved in ischaemia-reperfusion injury because of its disruptive driven by the mitochondrial calcium uniporter, whose low affinity is role in mitochondrial respiratory coupling and ATP production.7,8 counteracted by the high calcium concentration present at the sar- Experimental studies indicate that pharmacological approaches coplasmic reticulum–mitochondria interface. 27 aimed at preventing MPTP have cardioprotective effects in the con- Mitochondrial dysfunction can be of various origins and may text of myocardial ischaemia reperfusion.9,10 However, translation of alter cell homeostasis or viability through several mechanisms in- this concept into the clinic has been disappointing,11-17 suggesting cluding reduction in ATP production, enhanced oxidative stress and that targeting a single intracellular molecule, such as the MPTP or release of pro-apoptotic molecules. Following prolonged ischaemia 18 dynamin-related protein 1 (Drp1), may not be sufficient to create reperfusion, the accumulation of calcium within the mitochondrial cardioprotection.19,20 It emphasizes that more mechanistic insight matrix and increased reactive oxygen species (ROS) production fa- about the mode of action of cardioprotective modalities is needed. vour the opening of the MPTP. The opening of this non-selective Lack of efficacy might also reflect that clinical outcome in STEMI pa- mega-channel dissipates the mitochondrial electrochemical gradient tients undergoing primary percutaneous coronary intervention (PCI) necessary for ATP production and precipitates energy exhaustion is excellent by modern reperfusion therapy, such that ischaemia and mitochondrial matrix swelling. Rupture of the outer mitochon- reperfusion as a target for protection has diminished. Median infarct drial membrane favours the release of pro-apoptotic factors. During size with current reperfusion therapy is small—in the order of magni- ischaemia, MPTP remains inhibited because of the acidic pH, but 21 tude of 7% and in anterior infarcts 16% of the left ventricle. Infarct upon restoration of blood flow, rapid normalization of pH in the sizes up to 17% rarely translate into clinical symptoms manifesting presence of calcium overload and excessive ROS triggers MPTP as cardiac death and hospitalization for heart failure, 22 which are the and exacerbates reperfusion-induced cell injury.5 Various proteins most appropriate clinical end-points for evaluating the efficacy of have been reported to contribute in the formation and function of 23 cardioprotective pharmacological agents. MPTP, including adenine nucleotide translocase (ANT), voltage-de- The aims of the present review were to provide an overview pendent anion channel (VDAC) and the phosphate carrier, whereas of the pharmacological agents that have advanced to clinical test- others, including cyclophilin D (CypD), appear as key regulators. Its ing and to identify obstacles for a clinical benefit in order to clarify molecular identity remains, however, not fully elucidated. Bernardi BØTKER et al. | 2719 et al have proposed that mitochondrial ATP synthase could be the administered during reperfusion fails to restore cardioprotection true molecular entity of the MPTP, although this hypothesis remains in pre-diabetic Zucker obese rats in vivo. 39 Overall, CsA variably 28,29 controversial. and inconsistently seems to reduce infarct size across species in experimental models of reperfused myocardial infarction.40 3 | M EC H A N I S M S U N D E R LY I N G AT TE N UATI O N O F M ITO C H O N D R I A L 3.2 | KAI-9803 DYS FU N C TI O N I N I S C H A E M I A- R E PE R FU S I O N I N J U RY BY Although contentious,41 the protein kinase C (PKC) family of iso- PH A R M ACO LO G I C A L A N D M EC H A N I C A L enzymes has been involved in pre-conditioning protection against CO N D ITI O N I N G A PPROAC H E S ischaemia-reperfusion injury.42 The KAI-9803 peptide (delcasertib) inhibits δ-PKC activity and prevents translocation of δ-PKC to the Based on the absence of a definite molecular structure of the mitochondria during prolonged ischaemia reperfusion. Whereas ad- MPTP and the existence of multiple modulators of its mechanics, a ministration of KAI-9803 may preserve mitochondrial function, it precise therapeutic strategy to prevent MPTP opening is difficult has no direct action on MPTP opening, but would rather prevent to establish. Pharmacological or non-pharmacological (eg condi- apoptosis by limiting the accumulation and dephosphorylation of the tioning) interventions either aim at a particular target site of a pu- pro-apoptotic Bcl-2–associated death promoter.43 In the in vivo pig tative component or modulator of the MPTP or try to prevent the model, intracoronary administration of KAI-9803 immediately prior conditions of its opening (eg calcium accumulation into the matrix) to reperfusion reduced infarct size and improved contractile func- or to attenuate its consequences (eg altered oxidative phospho- tion recovery.44 rylation). Based on encouraging in vitro and in vivo results, the ability of some pharmacological agents supposed to prevent MPTP opening has recently been tested in humans. Studies in isolated 3.3 | TRO 40303 cardiac mitochondria from patients undergoing coronary artery bypass surgery had suggested improved mitochondrial func- TRO 40303 was initially presented as an inhibitor of MPTP open- tion along with reduced troponin release with remote ischaemic ing.10 In vitro experiments suggested that this compound might 30 pre-conditioning. reduce oxidative stress and subsequently prevent opening of the MPTP. Experimental evidence suggests that TRO40303 acts through its binding to the translocator protein (TSPO) located in the 3.1 | Cyclosporine A outer mitochondrial membrane (Figure 1). Importantly, Sileikyte et al demonstrated using calcium retention capacity measurements in Apart from the immunosuppressive action related to its binding isolated mitochondria that TRO40303 has no direct effect on the to the cytosolic calcineurin and subsequent inhibition of the tran- MPTP.45 However, it is important to emphasize that no targets and scription factor nuclear factor of activated T cells (NFAT), cyclo- mechanisms for TRO40303 have been defined. Intravenous admin- sporine A (CsA) inhibits MPTP opening following binding to the istration of 2.5 mg/kg of TRO40303 immediately prior to reperfu- mitochondrial matrix chaperone CypD (Figure 1). CypD, which sion reduced infarct size by 38% in the in vivo rat model of ischaemia is known to bind to the oligomycin sensitivity conferral protein reperfusion.10 However, in saline-perfused rat hearts and in anaes- (OSCP), acts on the MPTP by facilitating the removal of the F1 thetized pigs, TRO40303 did not reduce infarct size when given at domain from the c subunit in a CsA-sensitive manner during pore reperfusion.46 31 opening. The concept of a putative cardioprotection by CsA was also based on the observation that CypD-deficient mice de- velop significantly smaller infarcts after a prolonged ischaemic in- 3.4 | MTP131 sult. 32,33 Hausenloy et al first reported that administration of CsA at the time of reperfusion could reduce infarct size in the isolated MTP-131 (Bendavia) may reduce infarct size, when administered rat heart model. 34 In vivo administration of NIM811, a non-immu- at the time of reflow in various animal models. In isolated cardiac nosuppressive CsA derivative, was able to inhibit MPTP opening mitochondria, Zhao et al suggested that MTP-131 was able to limit in mitochondria isolated from reperfused rabbit myocardium and ROS production.47 Alternatively, an enhanced ROS scavenger 35 limit infarct size when administered at the time of reperfusion. capacity of MTP-131 during ischaemia reperfusion has been dis- CsA also reduced infarct size in anaesthetized pigs with 90-min- cussed, but no specific target or mechanisms have been defined. utes regional ischaemia, when given intravenously just before rep- In a rat model of acute kidney injury, MTP-131 binds to cardiolipin, erfusion. 36 However, CsA failed to induce robust cardioprotection prevents its peroxidation by cytochrome c, thereby protecting in other studies with efficacy dependency on the experimental mitochondrial cristae membranes during renal ischaemia reper- conditions, such as duration of ischaemic period. 37,38 Also, CsA fusion.48 Conflicting results regarding infarct size reduction have 2720 | BØTKER et al. F I G U R E 1 Cyclosporine A and TRO40303 inhibit opening of mitochondrial permeability transition pores (MPTP). Proteins implicated in MPTP formation include the matrix cyclophilin D (CyD), the inner membrane (IMM) and the outer mitochondrial membrane (OMM). Additional proteins such as the translocator protein 18 kDa (TSPO), located in the OMM, interact with proteins implicated in MPTP formation. Under pathophysiological conditions, such as high Ca2+ concentration and increased oxidative stress, the complex forms an open pore between the inner and outer membranes that ultimately result in mitochondrial swelling, mitochondrial Ca2+ efflux and the release of apoptogenic proteins. Cyclosporine A targets matrix CyD, where Ca2+ overload triggers MPTP opening. TRO40303 binds to TSPO in the outer membrane been reported suggesting a not so clear putative cardioprotection ventricular [LV] remodelling) was demonstrated within 1 year after 49 potential. reperfused acute anterior STEMI by 2.5 mg/kg CsA intravenously before primary PCI in 971 patients.12 Because the study demon- strated a significant interaction between Killip class and treatment 4 | C LI N I C A L S T U D I E S O F D RU G S effect (P = .009), suggesting a beneficial effect of CsA in the sub- TA RG E TI N G M ITO C H O N D R I A L FU N C TI O N group of patients with acute myocardial infarction (AMI) compli- A S A N A DJ U N C T TO R E PE R FU S I O N I N cated by heart failure, the authors recently presented a post hoc S T-S EG M E NT E LE VATI O N M YO C A R D I A L subgroup analysis of 97 patients with large anterior MI and Killip I N FA RC TI O N class ≥2. Although the composite end-point at 1 year occurred less frequently in the CsA arm than in the control arm (35% vs 58%, An overview of clinical studies of mitoprotective drugs is given in P = .02), CsA was not associated with improved clinical outcome the Table 1. after adjustment for baseline characteristics (adjusted HR: 0.68 [0.3-1.4]). 52 Of note, the initial proof-of-concept studies and the CIRCUS trial used different solvents for CsA, and the solvent in 4.1 | Cyclosporine A the CIRCUS trial may have induced protection per se and thus have obscured differences between CsA and placebo. 53 The first seminal clinical proof of concept by Piot et al demon- An absent effect of CsA was subsequently confirmed in the strated that CsA 2.5 mg/kg iv <10 minutes before primary PCI CYCLE (CYCLosporinE A in Reperfused Acute Myocardial Infarction) with direct stenting yielded a 40% reduction in creatine kinase trial.13 A single intravenous CsA bolus (2.5 mg/kg) before primary 50 (CK) release over 72 hours in 58 patients with reperfused STEMI. PCI had no effect on ST-segment resolution or high-sensitive cardiac The reduction on infarct size persisted at 6 months and was as- troponin T, and it did not improve clinical outcome or LV remodel- 51 sociated with less detrimental remodelling. In a subgroup of 27 ling up to 6 months.13 In patients undergoing reperfusion therapy patients, the absolute mass of the area of hyperenhancement on with thrombolysis, CsA did not have any effect on troponin T and cardiac magnetic resonance imaging (MRI) was significantly re- CK myocardial band (CK-MB) release or percentage ST resolution at duced in the CsA group corresponding to a reduction in infarct 90 minutes after treatment.11 50 size from 18% to 15% of the left ventricle. In the subsequent A more recent meta-analysis did not demonstrate any significant CIRCUS phase III trial, no benefit in clinical outcome (composite differences between the CsA and placebo in terms of all-cause death of all-cause mortality, worsening of heart failure during the initial (OR: 1.21, 95% CI: 0.78-1.87) and cardiovascular death (OR: 1.05, hospitalization, hospitalization for heart failure and adverse left 95% CI: 0.66-2.49).54 BØTKER et al. | 2721 TA B L E 1 Clinical trials investigating pharmacological agents for the treatment of ischaemia-reperfusion injury in patients with ST- elevation myocardial infarction undergoing primary percutaneous coronary intervention Study design and number of Author Year patients Treatment and follow-up Results 50 Piot et al 2008 Multi-centre, single-blinded RCT CsA (iv bolus 2.5 mg/kg, prior to PCI), infarct 44% reduction in CK (P = .04) (58 patients with STEMI and size quantification by CK and TnI release 13% reduction in TnI (P = .15) TIMI flow grade 0 undergoing and CMR at 5 d post-MI in a subgroup of 27 20% infarct size reduction by primary PCI) patients CMR (P = .04) Mewton et al51 2010 Multi-centre, single-blinded RCT CsA (iv bolus 2.5 mg/kg, prior to PCI), infarct 24% infarct size reduction (28 patients with STEMI and size quantification and remodelling by CMR (P = .04) TIMI flow grade 0 undergoing at 6 mo post-MI primary PCI) Ghaffari et al11 2013 Double-blinded RCT (101 patients CsA (iv bolus 2.5 mg/kg immediately before No significant effect with anterior STEMI undergoing thrombolysis), infarct size evaluation by thrombolytic therapy) peak CK-MB and TnT release, ST-segment resolution and 6-month clinical follow-up Cung et al12 2015 Multi-centre, double-blind, RCT CsA (iv bolus 2.5 mg/kg, prior to PCI), No significant effect (791 patients with anterior follow-up at 12 mo by a composite end- STEMI and TIMI flow grade 0 point (all-cause mortality, worsening of undergoing primary PCI) heart failure during initial hospitalization, rehospitalization for heart failure or adverse left ventricular remodelling) Ottani et al13 2016 Multi-centre, open-label RCT CsA (iv bolus 2.5 mg/kg, prior to PCI), No significant effect (410 patients with large STEMI end-points and TIMI flow grades 0 to 1 ≥70% ST-segment resolution 60 min after undergoing primary PCI) TIMI flow grade 3, TnT release and clinical outcome at 6 mo Bates et al15 2008 Dose-escalation safety study (154 KAI-9803 (delcasertib) 0.05 mg; 0.5 mg; No safety and tolerability patients with anterior MI 1.25 mg; 5.0 mg vs placebo i.c.), primary concerns end-point serious adverse event, but Non-significant reductions in CK-MB AUC, ST-recovery AUC and infarct CK-MB release, improvements size by 99M-technetium sestamibi also of ST recovery and (99m) monitored technetium sestamibi infarct size Linkoff et al16 2014 Multi-centre, double-blind RCT KAI-9803 (delcasertib) (50, 150 or 450 mg/h) No significant effect (1010 patient with anterior by iv infusion prior to PCI and continued STEMI and 166 with inferior for approximately 2.5 h, infarct size STEMI (an exploratory cohort) quantification by CK (AUC). undergoing primary PCI) Atar et al14 2015 Double-blind, randomized trial TRO40303 (iv bolus 6 mg/kg prior to PCI), No significant effect. A larger (163 patients large STEMI infarct size quantification by CK, TnT and number of adjudicated safety and TIMI flow grades 0 to 1 CMR events in the TRO40303 undergoing primary PCI) group Gibson et al17 2016 Multi-centre, double-blind phase MTP-131 vs. placebo infused at a rate of No significant effect 2A safety and efficacy RCT 0.05 mg/kg/h for 1 h, infarct size by CK-MB (118 first-time anterior STEMI AUC, CMR and clinical outcome at 6 mo patients primary PCI) Abbreviations: AUC, area under the curve; CK, creatine kinase; CK-MB, creatine kinase myocardial band; CsA, cyclosporine A; PCI, percutaneous coronary intervention; RCT, randomized clinical trial; STEMI, ST-elevation myocardial infarction; TIMI, thrombolysis in myocardial infarction; TnT, troponin T. Whereas the lack of translation may reflect that improvement failed to show a clear reduction in infarct size with biochemical in treatment and outcome has diminished ischaemia reperfusion markers despite improved myocardial salvage by SPECT or cardiac as a target for protection, it was surprising in both studies that it MRI. Also, the RIC-STEMI trial on remote ischaemic conditioning56 was not possible to confirm the immediate reduction in biochem- and the NHLBI-sponsored trial on ischaemic post-conditioning57 ical myocardial necrosis marker release observed in the original reported better clinical outcome in the absence of significant in- proof-of-concept study by Piot et al. 50 However, this finding is farct size reduction. Biochemical markers may not be sufficiently not unique in trials evaluating cardioprotection in humans. The accurate to evaluate reduction within the range of infarct sizes CONDI-1 study 21 and the LIPSIA CONDITIONING trial55 also seen in humans with contemporary reperfusion therapy. Also, 2722 | BØTKER et al. our focus on infarct size reduction to improve clinical outcome not different in patients treated with KAI-9803 vs placebo. Other 58 may have been too narrow. We must pay more attention to also safety end-points, including changes in QT intervals and standard lab- reduce coronary microvascular obstruction. 59-61 In fact, in the oratory values after study drug administration, also did not differ be- NHLBI-sponsored trial on ischaemic post-conditioning, coronary tween treatment groups. The study was not powered to demonstrate microvascular obstruction on MRI was reduced along with better efficacy by biochemical myocardial necrosis marker release. However, 57 clinical outcome. Pharmacological agents that specifically tar- it suggested evidence for drug activity by trends for consistent, non- get microvascular obstruction would be of interest, administered significant reductions in CK-MB area under the curve and ST-recovery alone or in association with infarct size reducing agents. The so far area under the curve values across all dosages of KAI-9803 compared mostly disappointing data do not exclude that ischaemia-reper- with placebo. A similar trend was demonstrated for improvements 99 fusion injury can be a target for modification. We must possibly in M-technetium sestamibi infarct size with the active study drug address more targets to translate protection into a clinical benefit. at all doses of intracoronary KAI-9803.15 However, the subsequent As for mitochondria, we must also direct our attention not only PROTECTION AMI international, multi-centre, double-blind trial, to cardiomyocyte, but also to endothelial fibroblast, and smooth which also used a dose-response approach (50, 150 or 450 mg/h vs 62 muscle cell mitochondria. placebo intravenously) as soon as possible after randomization, before Modern reperfusion therapy also includes treatment with the first contrast injection during PCI, and continued until all study platelet inhibiting P2Y12 antagonists, which possess inherent cardi- drug had been administered (∼2.5 hours), in patients presenting within oprotective capacity63,64 such that the efficacy of additional cardi- 6 hours and undergoing primary PCI for anterior (the primary analysis oprotection may be increasingly difficult to demonstrate.65 In the cohort, n = 1010 patients) or inferior (an exploratory cohort that was CIRCUS trial, more than 90% of the patients were treated with P2Y12 capped after inclusion of 166 patients) STEMI, demonstrated no re- antagonists.12 duction in circulating biochemical markers of myocardial injury meas- During cardiac surgery under cardiopulmonary bypass with isch- ured by CK-MB fraction area under the curve.16 Similarly, there were aemic cardioplegic arrest, 2.5 mg/kg CsA intravenously reduced no treatment-related differences in secondary end-points of infarct cumulative troponin I release in patients undergoing aortic valve size, electrocardiographic ST-segment recovery measured as area surgery.66 Similarly, CsA also reduced cumulative troponin T and under the curve or time to stable ST recovery, or LV ejection fraction CK-MB release in patients with the longest cardiopulmonary bypass at 3 months. Adjudicated clinical end-points (death, heart failure or time of 120 (range: 85-120 minutes) as compared with those with a serious ventricular arrhythmias) also did not differ between the study shorter bypass time in the rage of 50-85 minutes during aortocoro- groups. The doses used in the PROTECTION AMI trial had been cal- nary bypass surgery.67 The same dose of CsA did not reduce release culated to provide steady-state blood concentrations similar to or ex- of cardiac troponin I or CK-MB or incidence of arrhythmias by ad- ceeding the levels that prevented reperfusion injury in animal models ministration before thrombolysis with streptokinase in 101 patients and to deliver cardiac tissue doses similar to those with intracoronary with acute anterior STEMI.11 administration in the original DELTA-MI trial. Circulating levels of KAI- Noteworthy, there has been no evidence from the above-men- 9803 were not measured in PROTECTION AMI. Even the highest dose tioned trials that in these clinical conditions of STEMI, CsA had any of KAI-9803 used in PROTECTION AMI may have been insufficient detectable biological effect, thereby questioning our ability to deliver by intravenous administration, as the dose may not have secured an it in due time at the appropriate dose to the right molecular target. equivalent tissue concentration as the intracoronary administration Although the magnitude of reperfusion injury may be too small for used in the DELTA-MI study. A lack of any side effect leaves the ques- a modification that translates into a clinically important benefit, the tion unanswered whether higher doses may have been feasible and emerging evidence that CsA may not have effect on infarct size leaves effective. Differences in the design of the DELTA-MI study and the the role of the MPTP in reperfusion injury in humans unanswered PROTECTION AMI trial studies may have been relevant, particularly because no MPTP inhibitors are available. As long as its molecular because patients with totally occluded vessels were included in the structure remains unknown and specific inhibitors of its opening are DELTA-MI study while approximately one-third of patients in the lacking, MPTP remains a candidate to alleviate reperfusion injury. PROTECTION AMI trial had spontaneous reperfusion prior to PCI. Finally, the favourable trends in the original DELTA-MI may have been merely incidental in the small trial with multiple dosing arms. Delta- 4.2 | KAI-9803 PKC may not be of sufficient significance as a target for prevention of reperfusion injury despite some experimental mechanistic evidence, The selective inhibitor of delta-protein kinase C (delta-PKC), KAI- which remains somewhat controversial.41 9803, was first studied in a phase I dose-escalation safety study of 154 patients with acute STEMI (DELTA-MI trial).15 Doses were 0.05 mg, 0.5 mg, 1.25 mg and 5.0 mg vs placebo administrated in two divided 4.3 | TRO 40303 doses via intracoronary injection before and after reestablishment of antegrade epicardial flow by primary PCI in patients with documented The single clinical study of TRO 40303 was the multi-centre totally occluded vessels. The incidence of serious adverse events was proof-of-concept MITOCARE study,14 in 163 patients with STEMI, BØTKER et al. | 2723 totally occluded culprit vessels and large area at risk. The patients 5 | C H A LLE N G E S I N TR A N S L ATI N G received TRO40303 (6 mg/kg), or placebo intravenously as a M ITO PROTEC TI O N I NTO TH E C LI N I C A L slow bolus (35 mL/min; given over approximately 1 minute) be- S E T TI N G fore guide wire passage, 15 minutes prior to balloon inflation and stenting. The primary end-point was infarct size expressed as area The lack of clinical efficacy by agents that target mitochondria to under the curve for CK and for troponin I over three days. The confer cardioprotection might be understood in terms of redun- result was neutral. In a subgroup of 93 patients, there were no dancy of mitochondrial pathways, insufficient pathophysiological significant differences in CMR-assessed myocardial salvage index knowledge on the role of mitochondria in cell death/survival path- (1—infarct size/myocardium at risk) (mean 52% vs 58% with pla- ways, limited information about their cellular origin (cardiomyocyte cebo, P = .1), mean CMR-assessed infarct size (21.9 g vs 20.0 g, vs coronary vascular), their structures and functions, and the com- corresponding to 17% vs 15% of LV mass) or LVEF (46% vs 48%), or plex interplay of the conditions closely related to cardiovascular dis- in the mean 30-day echocardiographic LVEF (51.5% vs 52.2%) be- ease that affects mitochondria, such as ageing, comorbidities and tween TRO40303 and placebo. One of the reasons for the neutral co-medication.69-71 result might be that the experimental background to justify a pro- ceeding to a proof-of-concept clinical study was not sufficiently solid in relation to the choice of dose. Rat studies have shown a 5.1 | Ageing dose-response–dependent infarct reduction in TRO40303.10,68 A closer look on the data underlying this conclusion demonstrates As lifespan has extended, age-related conditions accumulate in elder uncertainties. The area at risk varied unexpectedly, and infarct people. Some of these conditions disrupt mitochondrial functions. sizes were modest in the model. Importantly, infarct size related Ageing by itself induces a progressive impairment of mitochondrial to area at risk did not decrease with the highest TRO40303 dose respiratory efficiency as well as changes in mitochondrial morphol- but increased significantly in the vehicle group—not because of ogy and mitochondrial pool72 that collectively account for the exer- a change in infarct size but because of a change in area at risk. cise intolerance seen in elderly patients.73 The aerobic capacity of As a consequence, the relative reduction in infarct size compared the human myocardium is depressed in elderly people (>75 years) with vehicle did not seem convincing. Concentrations in rats were because of an excessive mitochondrial calcium accumulation that lower than those achieved in humans. In saline-perfused rat hearts eventually leads to less number of respiring mitochondria.74 This and in anaesthetized pigs, TRO40303 did not reduce infarct size mechanism may facilitate the transition from a healthy to a failing when given at reperfusion.46 Circulating levels of the drug were cardiomyocyte and could underlie the reduced tolerance to ischae- not measured in the MITOCARE study, and adjudicated side ef- mic damage observed in elderly patients74 and aged animals.75 The fects were frequent in the treatment group perhaps indicating that interfibrillar mitochondria are particularly sensitive to the effects of the dose might not have been optimal. However, the lack of effect ageing, whereas subsarcolemmal mitochondria remain rather pre- and demonstration of side effects have prevented further testing served.76,77 During ageing, mitochondria also experience structural of TRO40303. changes, including reduced inner membrane surface and disarrange- ment of cristae.78 Furthermore, impairment of the electron transfer chain complexes III and IV may be the cause of the disorganized 4.4 | MTP131 ‘respirosomes’ observed in the aged heart.79 Other functional altera- tions of mitochondria of aged cardiac cells are excessive ROS produc- 17 The EMBRACE STEMI was undertaken to study whether the clini- tion that favours mitochondrial DNA (mtDNA) damage,80 diminished cal efficacy of the cell-permeable peptide that preserves the integ- mitophagy81 and an increased susceptibility to undergo MPTP open- rity of cardiolipin, MTP-131, enhances mitochondrial energetics and ing upon reperfusion.75 Telomerase is not only present in the nu- improves myocyte survival during reperfusion. The study included cleus, but also in mitochondria, and telomerase abundance typically 118 patients with first-time anterior STEMI because of a proximal or decreases with ageing and pre-disposes to myocardial ischaemia- middle LAD lesion, who underwent successful PCI. Administration reperfusion injury.82 Telomerase-deficient rat hearts have increased of MTP-131 was safe and well-tolerated. Treatment with MTP-131 infarct size.83 As a result of the wide range of altered mitochondrial was not associated with a decrease in myocardial infarct size as as- mechanisms because of the natural process of ageing, pharmacologi- sessed by area under the curve of CK-MB within 0-72 hours. No ef- cal agents aimed at promoting mitoprotection might fail. fects were observed on secondary end-points including troponin I, volume of infarcted myocardium by cardiac MRI, measures of myo- cardial structure and function TIMI flow grade, ST-segment eleva- 5.2 | Sex differences 17 tion resolution or clinical outcome. As such, MTP-131 adds to the number of potential mitoprotective pharmacological compounds Experimental observations have confirmed the results of epidemio- that do not translate into a clinical benefit in patients with STEMI logical studies investigating sex-specific differences in cardiac toler- undergoing reperfusion therapy. ance to ischaemia.84 2724 | BØTKER et al. Female gender appears to favourably influence cardiac remod- Dyslipidaemia affects the myocardium directly, obscuring and elling after ischaemia-reperfusion injury. Detailed mechanisms of preventing cardioprotection provided by ischaemic conditioning sex-related differences remain unknown and may involve genomic and pharmacological agents.98 This might be consequence of MPTP and non-genomic effects of sex steroid hormones, particularly the opening secondary to decreased levels of antioxidant enzyme ex- oestrogens, which have been the most extensively studied, but also pression99 or to reduced glycogen synthase kinase (GSK)-3β phos- by influences during the early-phases of ontogenetic development. phorylation.100 The MPTP opening inhibitor CsA cannot by its own Experimental studies of the mitochondrial proteome have identified offer cardioprotection in hypercholesterolaemic animals.101 Another a number of mitochondrial proteins that have male/female differ- target of mitoprotection is the mitochondrial potassium-ATP (K ATP) ences in post-translational modification.85 Specifically, males have in- channel located at the inner membrane. In hyperlipidaemic rats, creased phosphorylation of the pyruvate dehydrogenase (PDH)-E1α neither cromakalim nor diazoxide, two K ATP channel activators, subunit, whereas females have increased phosphorylation of mito- maintain their cardioprotective effects in the context of ischaemia chondrial aldehyde dehydrogenase-2 (ALDH2), an enzyme involved in reperfusion.102 post-ischaemic reperfusion and remote conditioning pathways,86,87 and the E2 subunit of α-ketoglutarate dehydrogenase.85 Therefore, females exhibited reduced ROS generation on reoxygenation. Similar 5.4 | Co-medication to CsA, oestrogen may contribute to maintaining mitochondrial func- tion during ischaemia reperfusion by stabilizing the mitochondrial At the same time as comorbidities may influence the efficacy of 88 membrane potential and inhibiting MPTP opening. Also, the pro- mitoprotective interventions, drugs used as treatments may alter tection during conditions of increased contractility seems to involve mitochondrial processes by interference with the mitochondrial res- an increase in nitric oxide signalling that leads to S-nitrosylation of piratory chain (eg uncoupling) or inhibition of important mitochon- the L-type calcium channel. The nitrosylation reduces calcium loading drial processes including oxidative phosphorylation, mitochondrial during ischaemia and early reperfusion, and hence moderates isch- DNA replication and ADP/ATP translocation. aemia-reperfusion injury.89 Hence, sex-related differences in cardiac sensitivity to ischaemia-reperfusion injury may influence mitoprotec- tive strategies in patients with acute coronary syndrome. 5.4.1 | Antidiabetic drugs The antidiabetic compounds glibenclamide and other sulphonylu- 5.3 | Comorbidity and risk factors reas promote MPTP opening, causing calcium efflux to the cytosol and resulting in swollen mitochondria.103 Conversely, metformin Diabetes seems to attenuate the cardioprotective effects of phar- lowers mitochondrial oxidative stress in cultured human endothelial macological and ischaemic conditioning manoeuvers.90 This may cells subjected to low glucose concentrations.104 Metformin may be partly be because of dysregulation of mitochondrial homeostasis, by a pharmacological modulator of MPTP opening,105 but it failed to impairing autophagy, overproduction of ROS, lipotoxicity and acti- improve LVEF at four months after STEMI in patients without dia- 91,92 vation of calpain that has been linked to F-ATPase proteolysis. betes undergoing primary PCI.106 Continuous delivery of metformin After an AMI, cardiomyocytes of type 2 diabetes mellitus rats pre- to a murine model of dilated cardiomyopathy increases mitophagy sent a significant down-regulation of genes involved in mitochondrial through AMPK phosphorylation and results in milder fibrosis and fusion and autophagy when compared with non-diabetic animals, reduced LV hypertrophy.107 The selective sodium-glucose cotrans- manifesting as accumulation of incompletely degraded mitochon- porter (SGLT2) inhibitor, canagliflozin, and the glucagon-like pep- dria and enhanced apoptosis of cardiomyocytes. These findings cor- tide-1 (GLP-1) receptor antagonist, exenatide, attenuate myocardial relate with a severe form of heart failure and increased mortality infarction size.108,109 Mechanisms may involve improvement of among diabetic rats.93 Accordingly, cardiac cells of type 2 diabetic mitochondrial function through the GLP-1 receptor/cAMP/PKA mice exhibit degenerated swollen mitochondria with disintegrated pathway.109 Hence, type 2 diabetes mellitus per se, along with its cristae and sparse autophagosomes, linked with the reduction in medication, could be major confounders in clinical trials aimed to the phosphorylated state of the 5' adenosine monophosphate-ac- prove mitoprotective agents. tivated protein kinase (AMPK).94 In non-diabetic animals, two-week administration of resveratrol reverses heart remodelling after myo- cardial infarction via up-regulated autophagy mediated by AMPK 5.4.2 | Lipid-lowering drugs activation.95 Additionally, resveratrol improved LV diastolic and en- dothelial function in patients with a history of myocardial infarction, Statins inhibit mitochondrial respiration, stimulate ROS production 96 among whom nearly one-third were diabetic. To what extent these and may facilitate MPTP opening.110,111 A proprotein convertase last findings are generally applicable to diabetic patients remains to subtilisin/kexin 9 (PCSK9) inhibitor as well as high doses of ator- be proven. Loss of cardiomyocyte-restricted insulin signalling de- vastatin lowers the ratio between phosphorylated and total Drp- creases the mitochondrial capacity of fatty acid catabolism.97 1, a key factor in mitochondrial fission, whereas only the PCSK9 BØTKER et al. | 2725 inhibitor restores mitochondrial ROS levels in insulin-resistant, 5.4.4 | Non-steroidal anti-inflammatory drugs 112 dyslipidaemic rats. Expression of PCSK9 is increased in the vi- able reperfused myocardium and its pharmacological inhibition Non-steroidal anti-inflammatory drugs (NSAIDs) such as naproxen, di- can reduce infarct size, possible through autophagy, in the in vivo clofenac and celecoxib have been associated with an increased risk of mouse model of myocardial infarction.113 Paradoxically, although cardiovascular disease, predominantly coronary thrombosis, because some statins such as pravastatin seem to facilitate MPTP opening, of their variable affinity to the cyclooxygenase 1 and cyclooxygenase atorvastatin reduces infarct size in the isolated mouse-perfused 2 (COX-1 and COX-2) enzymes that may alter thrombogenicity.128 114 hearts by activating the Akt-eNOS pathway, suggesting that Recent experimental studies on isolated rat heart mitochondria have MTPT modulation may vary between statins. Mitochondrial dam- demonstrated that NSAIDs may also increase ROS formation, mito- age is not restricted to cardiomyocytes, but it is also encountered chondria membrane collapse, mitochondria swelling, lipid peroxida- in other cell types. In pigs with metabolic syndrome, mitochondrial tion, and glutathione and ATP depletion,129 which may play important density is diminished in LV endothelial cells, and accordingly, en- roles in developing cardiotoxicity. MPTP sealing agents and antioxi- dothelium-dependent vasorelaxation of coronary arteries is com- dants may prevent mitochondrial toxicity.129 However, the clinical promised. Of note, regular exercise seems to exert mitoprotective implications of these observations remain unknown. effects even in dyslipidaemic conditions.99 6 | PE R S PEC TI V E A N D CO N C LU S I O N 5.4.3 | Chemotherapeutics Despite pre-clinical evidence for a cardioprotective effect, most Widely prescribed chemotherapeutics display a plethora of ad- firmly established for CsA, neither of the clinically tested mitopro- verse side effects mediated by mitochondria damage with cardio- tective drugs has demonstrated protective capability on clinical 115,116 toxicity as the most feared. This is the case for the inhibitor outcome beyond that provided by rapid revascularization alone. of topoisomerase II, doxorubicin and other anthracyclines, which Whereas it may relate to favourable outcome with current optimal stand as the main cause of chemotherapy-related cardiotox- therapy, risk factors, comorbidity and concurrent medications may icity.117 Doxorubicin diverts electrons from respiratory chain also alter mitochondrial function, sometimes in an irreversible way complex I and other dehydrogenases that induce ROS overproduc- and often by multiple mechanisms. In consequence, targeting one tion.118 Despite its intended effect is preventing DNA replication single mechanism to provide mitoprotection may be ineffectual. at the nucleus of cancer cells, doxorubicin also reduces mtDNA However, as long as the molecular structure of the MPTP remains levels and thus mitochondrial biogenesis in all tissues, including unknown and specific inhibitors of its opening are lacking, it re- 119 the myocardium. Indirectly, doxorubicin triggers the irreversible mains a candidate to alleviate reperfusion injury. Nevertheless, tak- accumulation of mtDNA adducts through ROS production in a car- ing conditions such as ageing, sex, comorbidities and co-medication dioselective manner.120 The modes of action of mitoxantrone and into account may be of paramount importance during the design of doxorubicin present a high degree of similarity.121 Mitoxantrone pre-clinical and clinical studies testing mitoprotective drugs. hampers oxidative phosphorylation, whereas its associated ROS production is relatively limited and secondary to ATP depletion.122 C O N FL I C T O F I N T E R E S T Nevertheless, it mitoxantrone inhibits the mitochondrial calcium None. uniporter by a direct interaction.123 The tyrosine kinase inhibitors, trastuzumab, is an antibody directed against the human epider- AU T H O R C O N T R I B U T I O N S mal growth factor receptor 2 that has as an adverse side effect All authors contributed equally to the manuscript. of the heightened risk of congestive heart failure.124 Trastuzumab cardiomyopathy could be because of the disruption of critical sig- DATA AVA I L A B I L I T Y S TAT E M E N T 125 nalling that sustains cardiomyocyte survival, but mitochondrial Data sharing is not applicable to this article as no new data were cre- dysfunction and ROS imbalance cannot be dismissed.126 Other an- ated or analysed in this study. ticancer drugs, including tamoxifen, flutamide, alkylating agents and taxanes, produce considerable alterations in essential mito- ORCID chondrial functions. 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