There are numerous reviews on the signal transduction of

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1 Review Molecular Basis of Cardioprotection Signal Transduction in Ischemic Pre-, Post-, and Remote Conditioning Gerd Heusch Abstract: Reperfusion is mandatory to salvage ischemic myocardium from infarction, but reperfusion per se contributes to injury and ultimate infarct size. Therefore, cardioprotection beyond that by timely reperfusion is needed to reduce infarct size and improve the prognosis of patients with acute myocardial infarction. The conditioning phenomena provide such cardioprotection, insofar as brief episodes of coronary occlusion/ reperfusion preceding (ischemic preconditioning) or following (ischemic postconditioning) sustained myocardial ischemia with reperfusion reduce infarct size. Even ischemia/reperfusion in organs remote from the heart provides cardioprotection (remote ischemic conditioning). The present review characterizes the signal transduction underlying the conditioning phenomena, including their physical and chemical triggers, intracellular signal transduction, and effector mechanisms, notably in the mitochondria. Cardioprotective signal transduction appears as a highly concerted spatiotemporal program. Although the translation of ischemic postconditioning and remote ischemic conditioning protocols to patients with acute myocardial infarction has been fairly successful, the pharmacological recruitment of cardioprotective signaling has been largely disappointing to date. (Circ Res. 2015;116: DOI: /CIRCRESAHA ) Key Words: acute myocardial infarction ischemic postconditioning ischemic preconditioning myocardial ischemia reperfusion There are numerous reviews on the signal transduction of the conditioning phenomena, and some of them are excellent, so why this one now? The present review attempts to (1) briefly summarize and update the existing knowledge, and such update is practically useful from time to time, (2) focus on novel and emerging signaling modules, (3) identify potential common signaling patterns for the conditioning phenomena that are robust, translatable, and therefore potential drug targets, and (4) highlight failure and success in the translation of signaling events from reductionist experimental preparations and models to humans with ischemic heart disease (IHD) and suggest future strategies. Reperfusion is mandatory to salvage ischemic myocardium from infarction; however, reperfusion also contributes to irreversible myocardial injury. 1 4 The infarct size (IS) resulting from both ischemia- and reperfusion-induced injury is a major determinant of the prognosis of patients who have survived their acute myocardial infarction (AMI) event. 5 9 Now, given that (1) the prevalence of IHD is increasing not only in developed but also in developing countries, 10,11 (2) an increasing number of patients survive their AMI, largely secondary to timely reperfusion and better medical treatment options, 12,13 and (3) many of these patients have progressive myocardial remodeling and eventual heart failure, 14 cardioprotection beyond that by reperfusion is urgently needed with the aim to minimize IS as the main determinant of evolving heart failure. There is currently no stronger cardioprotection than that by the conditioning phenomena. Conditioning Phenomena After the pioneering observations that reperfusion indeed salvages ischemic myocardium from infarction 15,16 and that infarction spreads in a wavefront during ongoing ischemia but leaves salvageable myocardium up to 2 to 3 hours of ischemia, 17,18 the prime cardioprotective paradigm of ischemic preconditioning (IPC) was established by Murry et al, 19 who reported that 4 cycles of 5 minutes coronary occlusion/5 minutes reperfusion immediately before a sustained 40 minutes coronary occlusion with reperfusion reduced IS (Figure 1); however, IS was not reduced when the sustained coronary occlusion was of 3 hours duration, emphasizing the need for timely reperfusion. The observed IS reduction was greater than anything that could be achieved by even combined pharmacological treatment. Initially, this cardioprotective phenotype was attributed to Original received October 28, 2014; revision received November 20, 2014; accepted November 25, In December 2014, the average time from submission to first decision for all original research papers submitted to Circulation Research was days. From the Institute for Pathophysiology, West German Heart and Vascular Centre, University of Essen Medical School, Essen, Germany. Correspondence to Gerd Heusch, FRCP, Director, Institute for Pathophysiology, West German Heart and Vascular Centre, University of Essen Medical School, Hufelandstr. 55, Essen, Germany. gerd.heusch@uk-essen.de 2015 American Heart Association, Inc. Circulation Research is available at DOI: /CIRCRESAHA

2 Heusch Signal Transduction of Conditioning 675 Nonstandard Abbreviations and Acronyms Akt protein kinase B AMI acute myocardial infarction ATF activating transcription factor COX cyclooxygenase Cx 43 connexin 43 ERK extracellular regulated kinase GSK 3β glycogen synthase kinase 3β HIF1α hypoxia-inducible factor 1α IHD ischemic heart disease IPC ischemic preconditioning IS infarct size K ATP ATP-dependent potassium channel MPTP mitochondrial permeability transition pore NO nitric oxide NOS nitric oxide synthase PI3K phosphatidylinositol (4,5)-bisphosphate 3-kinase PKA protein kinase A PKC protein kinase C PKG protein kinase G POC ischemic postconditioning RIC remote ischemic conditioning RIPC remote ischemic preconditioning RISK reperfusion injury salvage kinase ROS reactive oxygen species SAFE survival activating factor enhancement STAT signal transducer and activator of transcription TNFα tumor necrosis factor α metabolic adjustments, notably slowed energy metabolism. 21 It was the landmark study by Downey and collaborators 22 who identified the action of adenosine as a signaling event in such IPC and opened the avenue for all subsequent studies on cardioprotective signaling. IPC comes in 2 different temporal forms, that is, an acute form which confers immediate protection but vanishes after an interval of 2 hours between the preconditioning stimulus and the event which needs protection, 19 and a delayed form which reappears after 24 to 48 hours, lasts longer but is less protective in strength. 23,24 The acute form of IPC relies on the recruitment of acutely available signaling modules, whereas the delayed form involves increased expression of protective proteins in response to an acute signal. 25 IPC has been successfully translated to humans with IHD, but because of its nature ( pre ) can only be used in elective settings, such as percutaneous coronary interventions and coronary artery bypass grafting and not in AMI. 4,26 Ischemic postconditioning (POC) was established by the group of Vinten-Johansen 27 who first reported that 3 cycles of 30 seconds reperfusion/30 seconds reocclusion at the immediate onset of reperfusion after 60 minutes coronary occlusion reduced IS to an equivalent amount as IPC. This seminal study once and forever terminated the long-lasting and controversial debate whether reperfusion injury contributed to ultimate IS; 28 moreover, this study unequivocally demonstrated that reduction of IS with modified reperfusion was possible 1 and thus revitalized prior debate on gentle reperfusion. 29 POC Figure 1. Typical examples of triphenyl tetrazolium staining of an unprotected pig heart with large anterior infarction and a protected heart with small infarction. Infarct size (IS) or, even better, IS as a function of residual blood flow is the most robust and relevant end point of cardioprotection. Schematic diagram of typical protocols of ischemic preconditioning (IPC), late IPC, ischemic postconditioning (POC), and remote ischemic conditioning (RIC). Modified from Ref. 20. is largely limited to the early minutes of reperfusion. As IPC, POC has been successfully translated to humans with IHD, and as an intervention that is performed at immediate reperfusion, it can be used in patients undergoing interventional reperfusion of AMI. 4 Remote ischemic preconditioning (RIPC) was derived from mathematical model calculations on the spatial transfer of potential signaling molecules and subsequently demonstrated as IS reduction after 60 minutes left anterior descending coronary artery occlusion with reperfusion by 4 prior cycles of 5 minutes left circumflex coronary artery occlusion/5 minutes reperfusion. 30,31 This RIPC phenomenon was initially regarded as a laboratory curiosity and somewhat neglected, but gained popularity when it was also elicited from organs remote from the heart 32,33 and later successfully translated to humans where it protects not only the heart, but also the vasculature and various other parenchymal organs from ischemia/reperfusion injury, 4 and where it may even improve clinical outcome and prognosis With respect to the temporal relation between the remote stimulus and the myocardial ischemic event, remote ischemic conditioning (RIC) comes, also clinically, in the forms of pre (before)-, per (during evolving infarction)-, and post (after)-conditioning. 37 The conditioning phenomena exert powerful cardioprotection but still use ischemia/reperfusion which as such is injurious, and in IPC and POC, they also involve manipulation of the atherosclerotic culprit lesion with the risk of coronary microembolization, 38 particularly in POC. 39 Therefore, a better understanding of the signal transduction underlying the conditioning phenomena may help to recruit cardioprotection without the inevitable injury associated with ischemia/reperfusion and also without manipulation of the culprit coronary lesion. Understanding the signal transduction may also help to account for and attenuate interference from confounding risk factors, comorbidities, and comedications. 20,40

3 676 Circulation Research February 13, 2015 Signaling Molecules and Mechanisms in Conditioning There are now thousands of studies which reported >100 different signaling molecules and mechanisms of conditioning in a wide range of experimental preparations, from isolated subcellular structures (eg, mitochondria) to isolated cardiomyocytes with exposure to hypoxia/reoxygenation, from cultured cardiomyoblast cell lines to freshly isolated neonatal or adult cardiomyocytes, from buffer-perfused isolated hearts with global or regional ischemia and reperfusion to in vivo and in situ preparations with coronary artery occlusion and reperfusion, using different species and anesthetic regimes, using different methodologies from biochemical to immunoblotting techniques, from pharmacological agonist to antagonist approaches to transgenes and transfections. The ultimate end point of protection is reduction of IS or its surrogate in more reductionist models. The present review cannot be comprehensive because of the multitude of these studies but must select the most essential ones; focus will therefore be on studies which use IS reduction as the most robust end point of cardioprotection. This review must also use a structuring scheme to present the important information in an organized fashion. Downey et al proposed a classification of signaling originally for IPC, which uses a logical/causal sequence of events which coincides with the temporal sequence of protocol steps. 41,42 A trigger is released during the preconditioning ischemia/reperfusion cycle(s) and acts as the stimulus to activate a mediator to transmit the cardioprotective signal during the sustained ischemia onto an effector which ultimately attenuates irreversible injury during the sustained ischemia or early reperfusion. It never has become clear where, in which molecule, and in which modification of such molecule exactly the memory resides, which remembers the trigger during the preconditioning cycle(s) of ischemia/reperfusion and activates mediator and effector during the sustained ischemia/reperfusion. Such causal sequence of events must also be operative in POC but is more difficult to associate with different protocol steps as trigger, mediator, and effector must all become immediately and almost simultaneously active during early reperfusion. The causal/temporal classification is often used in parallel to a spatial classification where a hypothetical cardiomyocyte at risk is exposed to extracellular trigger molecules which originate from various cell types, including cardiomyocytes, endothelium, neurons, inflammatory cells and so on, and activate a cytosolic mediator cascade of small molecules and enzymes which, in turn, ultimately converge on subcellular effector structures, such as mitochondria or cytoskeleton (Figure 2). Stimuli/Triggers of Conditioning Physical Stimuli Myocardial stretch activates gadolinium-sensitive sarcolemmal channels, activates downstream signaling (adenosine, protein kinase C (PKC), and ATP-dependent potassium (K ATP ) channels), and reduces IS, 43 but it is unclear whether and to what extent stretch of dyskinetic myocardium 44 contributes to the protection of conditioning. Even mild hypothermia during ongoing ischemia provides protection from infarction, not only by slowing energy metabolism but also by activation of protective signaling, notably of extracellular regulated kinases (ERK). 45 Hyperthermia provides delayed protection through upregulation of heat shock proteins. 23 Neither hyponor hyperthermia, however, are involved in the protection by conditioning. Chemical Stimuli Endogenously released chemical stimuli which elicit cardioprotection include small molecules, such as calcium ions, reactive oxygen species (ROS), reactive nitrogen species, hydrogen sulfide, and more classical ligands which activate sarcolemmal receptors. Extracellular calcium ions can precondition through adenosine and PKC activation, but their role in ischemic conditioning phenomena is probably minor. Accordingly, calcium antagonists do not interfere with IPC. 50 ROS have an ambivalent role in the conditioning phenomena: whereas excess formation of ROS contributes to irreversible injury, small amounts of ROS, for example, in response to mitochondrial K ATP activation or mitochondrial permeability transition pore (MPTP, see below) opening contribute to protection, possibly through oxidation of protective cytosolic kinases ROS share the same paradox with the conditioning phenomena per se, in that a little ischemia/reperfusion or ROS protects, whereas more profound ischemia/reperfusion or ROS formation induces injury. Reactive nitrogen species, notably nitric oxide (NO), also share this dose-dependent paradox. 54 Small concentrations of NO improve ventricular function 55 and the matching of oxygen consumption to contractile function, 56 whereas high concentrations of NO depress contractile function. 57 NO is not only synthetized from specific enzymes, but also formed nonenzymatically during ischemia, 58 such that the lack of effect of a specific NO synthase (NOS) blocker does not really rule out a role for NO in cardioprotection. Exogenous NO can trigger preconditioning, but endogenous NO seems to be no requisite for IPC in its acute form. 59,60 In contrast, endogenous NO generated from endothelial NOS not only triggers, but also generated from inducible NOS mediates delayed IPC. 61,62 Endogenous NO is also causally involved in POC 63 and in RIPC. 64 More recently, gaseous molecules other than NO have also received attention for their cardioprotective actions, that is, hydrogen sulfide and carbon monoxide, both of which reduce IS on exogenous administration. 65 Hydrogen sulfide is synthetized by cystathionine-β-synthase, cystathionine-γ-lyase, and 3-mercaptopyruvate-sulfurtransferase and targets different signaling proteins of cardioprotection Hydrogen sulfide contributes to IS reduction by IPC 69 and POC. 68,70 Carbon monoxide is synthetized by heme oxygenase, targets several cardioprotective signaling proteins, and reduces IS on exogenous administration; however, to date, there is no evidence for its role in the conditioning phenomena. 65 Autacoids Autacoids, such as adenosine and bradykinin, are released from cardiomyocytes, endothelium, and interstitial cells during the preconditioning ischemia/reperfusion cycle(s). 71 Interstitial adenosine concentrations are increased; 72 further increase of interstitial adenosine concentration with uptake inhibition by dipyridamole enhances 73 and enzymatic

4 Heusch Signal Transduction of Conditioning 677 Figure 2. Simplified scheme of cardioprotective signal transduction. Please note, this scheme does not entail the dimension of time, and details of mitochondrial signaling are displayed in Figure 2. Akt indicates protein kinase B; AMPK, cyclic adenosine monophosphate activated kinase; BNP, brain natriuretic peptide; camp, cyclic adenosine monophosphate; cgmp, cyclic guanosine monophosphate; COX, cyclooxygenase; Cx 43, connexin 43; DAG, diacylglycerol; EGFR, epidermal growth factor receptor; ERK, extracellular regulated kinase; FGF, fibroblast growth factor; G s /G i/q, stimulatory/inhibitory G protein; GPCR, G protein-coupled receptor; gp130, glycoprotein 130; GSK3β, glycogen synthase kinase 3 β; H 2 S, hydrogen sulfide; H11K, H11 kinase; HIF1α, hypoxiainducible factor 1α; IGF, insulin-like growth factor; inos, inducible NO synthase; IP 3, inositoltrisphosphate; JAK, Janus kinase; K ATP, ATP-dependent potassium channel; Na + /H +, sodium/proton-exchanger; NPR, natriuretic peptide receptor; pgc, particulate guanylate cyclase; p38, mitogen-activated protein kinase p38; NO, nitric oxide; enos, endothelial nitric oxide synthase; PI3K, phosphatidylinositol (4,5)-bisphosphate 3-kinase; PKC, protein kinase C; PKG, protein kinase G; PLC, phospholipase C; PTEN, phosphatase and tensin homolog; PTK, protein tyrosin kinase; ROS, reactive oxygen species; sgc, soluble guanylate cyclase; SR, sarcoplasmic reticulum; STAT, signal transducer and activator of transcription; and TNFα, tumor necrosis factor α. The NO/PKG pathway is displayed in green, the RISK pathway in yellow, and the SAFE pathway in red. catabolism by adenosine deaminase attenuates cardioprotection. 72 Cardiomyocytes express adenosine A 1, A 2A, A 2B, and A 3 receptors on their sarcolemma; 74 for the A 2B receptor, there seems to be also a mitochondrial localization. 75 A 2A receptors couple to G s proteins, which activate adenylate cyclase and through increased camp protein kinase A (PKA), whereas A 1 and A 3 receptors couple to G i and G q proteins which inhibit adenylate cyclase. Different from other G i -coupled receptors, adenosine receptor activation does not rely on ROS formation or mitochondrial K ATP activation to induce protection. 76 Adenosine also activates PKC directly, 77 and it activates the downstream reperfusion injury salvage kinase (RISK, see below) and endothelial NOS/protein kinase G (PKG) pathways. 78 A 1 and A 3 receptors are essential for IPC, and their blockade abrogates protection. 22,79,80 In contrast, A 2A and A 2B receptors are essential for POC, and they must be activated during the early minutes of reperfusion to achieve protection. 81 The major action of adenosine and its subsequent RISK activation is at reperfusion because an adenosine receptor antagonist and RISK blockade, when given just before reperfusion, abrogate IS reduction by IPC. 82 Blockade of adenosine receptors does not abrogate the protection by RIPC. 83 There seem to be species differences, in that in rats, adenosine is not important for IPC. 84 Also, the reduction of IS by exogenous adenosine is still controversial, among rodents and larger mammals. 85 Bradykinin is cleaved from kininogen precursors in the interstitium and catabolized through the angiotensin-converting enzyme in the vasculature but also neutral endopeptidase in the interstitium. 86,87 During preconditioning ischemia/reperfusion cycle(s), the interstitial bradykinin concentration is rapidly increased, 71 and it activates the bradykinin receptor 2 subtype on cardiomyocytes, which couples to G i proteins and activates the downstream endothelial NOS/PKG and RISK pathways. 78 Bradykinin also activates cyclo-oxygenase (COX) and prostacyclin synthesis to attenuate IS. 88,89 Bradykinin receptor 2 blockade abrogates protection by IPC, POC, and RIPC. 71,90 92 Angiotensin-converting enzyme inhibition and angiotensin

5 678 Circulation Research February 13, 2015 receptor 1 blockade both increase the bradykinin concentration and exert additive cardioprotection. 93 Neurohormones Neurotransmitters and hormones, such as acetylcholine, 76,94 angiotensin, 95,96 catecholamines, endothelin, 97,98 and opioids, can induce cardioprotection on exogenous administration and activation of their respective receptors; catecholamines act through α and β-adrenoceptor 104 activation. However, only α-adrenoceptor activation is causally involved in the conditioning phenomena through formation of adenosine and activation of PKC in IPC and upregulation of inducible NOS and COX in delayed IPC, and their blockade abrogates protection. 100,103 Opioids are released acutely from nerve endings but also synthetized in cardiomyocytes. In adult cardiomyocytes, 105,106 opioids activate δ and k receptors which couple to G i proteins and thus share part of their downstream signaling with adenosine and bradykinin, notably also ERK activation. 107,108 The δ receptor is most important in the conditioning phenomena, that is, not only nonspecific opioid receptor blockade with naloxone abrogates protection by IPC 109,110 and RIPC, 108 but also specific δ receptor blockade largely abrogates protection by IPC, 111,112 POC, 113,114 and RIPC. 115 The above autacoids and neurohormones not only share parts of their downstream signaling but act additively. 71,90 Peptide Hormones Several peptide hormones, such as adrenomedullin, 116 the natriuretic peptides, which also have specific receptors, the urocortins 120,121 and leptin, 122,123 protect from myocardial ischemia/reperfusion injury on exogenous administration. These peptide hormones act in a postconditioning mode and involve signal transduction elements, such as NO, 116,119 cgmp, ,121 mitochondrial K ATP -channels, and RISK 118,120 or survival activating factor enhancement (SAFE), 123 but there is no evidence that they are causally involved in IPC, POC, or RIC. 124,125 Exogenous glucagon-like peptide 1 and 2 activate their G s -coupled receptor and subsequently PKA and the RISK pathway to reduce IS, but they are not involved in endogenous conditioning. Lipid Molecules Several lipid molecules which activate G protein-coupled receptors have been implicated in the conditioning phenomena; however, their role as trigger or mediator is not really clear. Prostaglandins are causally involved in IPC and POC, and COX inhibition abrogates protection. 89,129 However, stronger IPC stimuli can overcome COX inhibition. 129 Epoxyeicosanotrienoic acids elicit IS reduction on exogenous administration, 130 and selective epoxyeicosanotrienoic acid antagonists abrogate the protection by IPC, POC, 131 and remote preconditioning by trauma or capsaicin. 132 Sphingosine- 1-phosphate (S1P) is produced from the sphingosine kinase 1 isoform during IPC 133 and POC 134 and reduces IS during IPC and POC by activation of S1P 1 and S1P 3 receptors 135 and subsequent activation of the RISK pathway. 134 The sphingosine kinase isoform 2 is also important for cardioprotection by IPC, as their genetic deficiency abrogates protection, 136,137 possibly secondary to increased susceptibility to MPTP opening. 136 Sphingosine and S1P also interact with the RISK 138,139 and SAFE pathways, involving tumor necrosis factor α (TNFα) and signal transducer and activator of transcription 3 (STAT 3) Growth Factors Growth factors, such as insulin-like growth factor or fibroblast growth factor and fibroblast growth factor 2, 145 reduce IS on exogenous administration, and fibroblast growth factor 2 acts in part through connexin 43 (Cx 43) phosphorylation, 145 but there is no evidence that growth factors are causally involved in IPC, POC, or RIC. Cytokines/Chemokines In contrast to peptide hormones and growth factors, cytokines/ chemokines have a causal role in IPC and POC. The prototypic cytokine TNFα reduces not only IS on exogenous administration, 146 but is mandatory for IPC 147 and POC. 148 The protection is mediated through activation of TNF receptor 2, STAT 3, and mitochondrial K ATP -channels TNFα is also required for IS reduction by delayed IPC, 149 and both TNF receptor 1 and 2 are activated. 150 Interleukin 6 with subsequent STAT 3 signaling is mandatory for IPC, 151 as is interleukin 10 for RIPC. 152 The stromal cell derived factor-1α activates chemokine receptor 4 and reduces IS on exogenous administration, 153 and stromal cell derived factor-1α is causally involved in IS reduction by RIPC. 154 Exogenous erythropoietin reduces IS when given before ischemia, 155 but also when given before reperfusion 156,157 with involvement of phosphatidylinositol (4,5)-bisphosphate 3-kinase (PI3K), ERK, p38, and heme oxy genase, but there is no evidence that erythropoietin is causally involved in the conditioning phenomena. Intracellular Mediators of Conditioning It is certainly somewhat simplistic to discuss the cytosolic mediators in an upstream downstream scheme, and there is good evidence for a spatially and temporally concerted action, notably for differences between ischemia and reperfusion Nevertheless, I will first discuss the different mediators as such because information on their spatial and temporal mode of action is not available from many studies, and I will address patterns of signaling further below. Protein Kinase C The first cytosolic mediator was identified by Downey and collaborators who demonstrated abrogation of protection by IPC with 3 different PKC inhibitors, 161,162 and shortly thereafter a similar finding was reported for delayed IPC. 163 The concept was proposed that after pertussis-sensitive G protein coupled receptor activation, phospholipase C was activated to form inositoltrisphosphate and diacylglycerol as second messengers, and diacylglycerol then causes phosphorylation, activation, and translocation of PKC. 164 Upstream activation of PI3K generates phosphatidylinositol (3,4,5)-trisphosphate and activates phosphoinositide-dependent kinase to then also activate PKC. 165,166 PKC is not only causal for IPC in isolated human right atrial trabeculae, but PKC-activated protection is abrogated by a K ATP antagonist. 167 This straightforward, but somewhat naïve, concept was quickly challenged by controversial data in different species. 168,169 The emerging controversy was subsequently solved by the notion that (1) there is interaction

6 Heusch Signal Transduction of Conditioning 679 of PKC with other cytosolic kinases, notably protein tyrosine kinases, (2) different isoforms of PKC serve contrasting functions, 173,174 and (3) intracellular translocation/colocalization of different isoforms is important. 173, In larger mammals, the PKCα isoform is most important for protection by IPC through activation of ecto 5 nucleotidase and adenosine formation 178 and interaction/colocalization with sarcolemmal Cx In rodents, the PKCε isoform is most important for protection by IPC, 180,181 and PKCε is translocated to the mitochondria through a heat shock protein dependent import, 182 where it activates mitochondrial K ATP channels during IPC 183 to increase ROS formation, which then further activate PKCε in a positive feedback loop. 184 PKCε is also translocated to the sarcoplasmic reticulum where it reduces calcium content. 185 The role of the PKCδ isoform in IPC is more controversial, and both protection 186 and injury 187 through PKCδ activation were reported. This controversy continued into a recent clinical trial, in which after promising preliminary data the PKCδ inhibitor delcasertib, when given before reperfusion, failed to protect patients with reperfused AMI in terms of IS reduction, as reflected by creatine kinase-muscle band release. 188 PKC activation, notably of its ε isoform, is also causally involved in POC, where it again interacts with mitochondrial K ATP channels and ROS formation. 189,190 Finally, PKCε and its translocation from cytosolic to the particulate fraction are also mandatory for late IPC. 191 Of note, there is a positive feedback loop between PKC activation and the trigger signal adenosine, in that PKC sensitizes the A 2B receptor during early reperfusion and thereby contributes to IPC. 192 PKCε expression and its mitochondrial localization are not involved in RIC. Mitogen-Activated Protein Kinases Among mitogen-activated protein kinases, 193 p38 is the one that plays a major role in IPC, although jun-activated kinase may also be involved. 194 The activity of p38 is increased after IPC, 195 and its inhibition abrogates IS reduction The protective function of p38 activation seems to occur during the preconditioning cycles, whereas during the sustained coronary occlusion, attenuated p38 activity is associated with protection. 199,200 The p38 isoforms α and β seem to serve opposing functions, 201 and they are activated at different times. The activation of p38α during the preconditioning cycles is causal to mediate IPC, 202 and an increased activity of p38ß during sustained ischemia is associated with reduced IS in pigs undergoing IPC. 179 p38 activation during IPC increases the phosphorylation of heat shock proteins. 203 I am not aware of studies on the role of p38 in late IPC or POC. Although there was no change in p38 expression with RIPC, 204 p38 inhibition abrogated the protection by RIPC. 205 Given the ambivalent effects of p38 isoforms, not surprisingly a clinical trial with an oral nonisoform selective inhibitor in patients with non-st segment elevation AMI was neutral in terms of IS reduction, as reflected by troponin I release. 206 NO and Protein Kinase G Endothelial NOS-derived NO, cgmp, and PKG will be considered one mediator system here. 207,208 Endothelial NOS is activated after G protein coupled receptor activation through a sequence of signals involving PI3K, phosphoinositide-dependent kinase, and protein kinase B (Akt), which then phosphorylates and activates endothelial NOS. 210 The resulting NO activates soluble guanylate cyclase to form cgmp, which then activates PKG. PKG is causally involved in IPC 211,212 and POC, 213 and the target of PKG is apparently the mitochondrial K ATP channel with subsequent ROS formation. 211,213 During POC, the sodium proton exchanger is another target of PKG, and PKG activation delays the normalization of acidosis. 214 NO signals protection by IPC not only through PKG, but also through nitrosylation, 215,216 and the PKG-independent action of NO seems to occur at reperfusion. 217 RISK Pathway The RISK pathway has become popular by the studies of Hausenloy, Yellon, and collaborators who not only created the catchy name, but more importantly demonstrated the activation of PI3K, phosphoinositide-dependent kinase, Akt, and ERK at early reperfusion in response to exogenous cardioprotective agonists. 120, In fact, the RISK pathway is also important in IPC. Activation of PI3K and its downstream targets Akt and glycogen synthase kinase 3β (GSK 3β) by IPC 166,222,223 and activation of ERK by the IPC trigger adenosine, and subsequent transactivation of the epidermal growth factor receptor 224 was demonstrated after the preconditioning cycles and found mandatory for IS reduction by IPC. Importantly, elegant studies by Hausenloy and Yellon then reconciled the studies on the role of RISK activation by IPC and by exogenous agonists during reperfusion; they demonstrated a biphasic RISK activation response during the preconditioning cycles and again at early reperfusion. 225 The importance of the RISK pathway was further supported by studies demonstrating a causal role of PI3K, Akt, and further downstream kinases, such as p70 S6 kinase and GSK 3β in IPC 226 and in POC, 227,228 including studies in isolated human right atrial trabeculae. 229 GSK 3β was proposed to be the downstream point of convergence of the RISK pathway, which when phosphorylated and thus inhibited acts to inhibit MPTP opening. 226,230 Recent studies also demonstrated morphological protection of mitochondria by Akt activation. 231 The phosphatase and tensin homolog dephosphorylates phosphatidylinositol (3,4,5)-trisphosphate and thus limits Akt activation and is a negative regulator of the RISK pathway, insofar as genetic haplodeficiency of phosphatase and tensin homolog reduces the threshold for IPC to reduce IS. 232 Collectively, from these studies, the intriguing concept emerged that RISK activation at early reperfusion is a unifying pattern of cardioprotective signaling. 158,218, Some caveats remain: Genetic ablation of GSK 3β did not abrogate protection by IPC or POC in one study, 236 but GSK 3β was mandatory for POC in another study using the same animal model, 237 such that the pivotal role of GSK 3β remains contentious. Also, although the RISK pathway as such is obviously mandatory for protection in rodents, it is not for POC in pigs. 238 SAFE Pathway The delineation of the SAFE pathway goes back to the group of Lecour who created the catchy name 239 and Opie. IS reduction by IPC and also in response to exogenous TNFα was

7 680 Circulation Research February 13, 2015 abrogated in STAT 3 deficient mouse hearts, 240 and STAT 3 activation in interaction with Akt activation 241 was mandatory for IPC. The coronary effluent from a preconditioned heart also activates STAT 3 in the acceptor heart and induces functional protection. 242 POC also relies on STAT 3 signaling in mice 148,243 and pigs. 244 Protection by POC also involves TNFα and its receptor 2 activation upstream of STAT Collectively, TNFα, its receptor subtype 2, and STAT 3 were then viewed as the SAFE system. 245 STAT 3 activation not only has a role in more long-term transcriptional upregulation of cardioprotective proteins, 246 but also an acute effect to improve mitochondrial respiration 247 notably during POC 244 and to attenuate apoptosis. There is indirect evidence for STAT 3 involvement in RIPC. Apolipoprotein A has been proposed as a humoral transfer signal of RIPC, 248 and IS reduction by RIPC and exogenous apolipoprotein A was reduced by blockade of PI3K, ERK, and STAT Interestingly, STAT 5 rather than STAT 3 activation was apparent in patients undergoing RIC before coronary artery bypass grafting. 250 STAT signaling, although acutely protective, is also involved in more longterm remodeling and heart failure. 251,252 Protein Kinases There is indirect evidence that other protein kinases are also involved in IPC. PKA activates endothelial NOS and increases NO formation during IPC; 209 PKA activation increases the phosphorylation of the camp response element binding protein and its inhibition abrogates IS reduction. 253 Also, PKA inhibition attenuates the reduction of lactate dehydrogenase release by IPC. 254 Transgenic overexpression of protein H 11 kinase results in largely reduced IS, along with activation of Akt. 255 AMP-activated protein kinase is activated by IPC and associated with sarcolemmal K ATP activation and action potential duration shortening, 256 and AMP-activated protein kinase activation contributes to glucose transporter 4 upregulation, improved glucose uptake, reduced IS in acute IPC, 257 and attenuation of stunning in late IPC. 258 Hypoxia-Inducible Factor-1α Hypoxia-inducible factor-1α (HIF1α) is the master regulator of cellular oxygen homeostasis and a requisite for mitochondrial ROS formation and reduction of IS by IPC, as evidenced in heterozygous HIF1α-deficient mice 259 and by gene silencing of HIF1α. 260 There seems to be a somewhat ambiguous interaction of HIF1α with adenosine in IPC 261 because exogenous adenosine still protects in heterozygous HIF1α-deficient mice, 259 but HIF1α-dependent protection is abrogated in A 2B receptor-deficient mice. 260 HIF1α seems to target the mitochondrial MPTP 262 and is, in turn, activated by small ubiquitinlike modifier-specific protease The role of HIF1α in RIPC is controversial because one study showed skeletal muscle HIF1α important for interleukin 10 formation and IS reduction by limb RIC after 24 hours, 264 whereas in another study, using the same heterozygous HIF1α-deficient mice limb RIC still reduced IS, however, without a 24 hours time lag. 265 MicroRNAs MicroRNAs are important signaling molecules not only in cardiovascular development and differentiation, 266 but also in myocardial ischemia/reperfusion. 267 Several different micro RNAs have been identified in protocols of IPC, POC, 270, and RIC 270, in various species, including humans. 276,277 Unless the microarray screening data were confirmed by RT- PCR, 269,270,272,275,277 it is difficult to decide whether the identified micrornas reflect robust data or circumstantial epiphenomena. Likewise, unless the identified micrornas were demonstrated to be causally related to the expression/activity of an established cardioprotective protein 268,270,274 or, even better, to IS reduction, for example, by abrogation of IS reduction by an antagonist 269,271,273,276 to an increased microrna or exogenous administration of a decreased microrna, 268,272 respectively, the functional relevance of the reported microrna in cardioprotective signaling must remain unclear. Given these criteria, several different micrornas seem to be involved in IPC, notably microrna-21, which seems to be upregulated and serve a cardioprotective function The microrna-144 cluster seems to be also upregulated and protective. 271 During POC, again microrna and also microrna-133 are upregulated and possibly involved in cardioprotection. 273,275 In RIC, microrna and microrna are upregulated and potentially protective. 276 The microrna-144 cluster upregulation is associated with increased RISK signaling. 274,276 The microrna-21 upregulation is associated with decreased programmed cell death protein 4 269,270 and increased endothelial NOS and heat shock protein expression. 268 The emerging field of micrornas in cardioprotective signaling is promising, but requires further robust data with cause- and -effect relationships and notably also in larger mammals. Protein Expression in Late IPC Increased cardioprotective protein expression is a hallmark of delayed IPC. 279 During the preconditioning cycles, trigger molecules, such as ROS, 280 NO, 61,281 adenosine, 282,283 and opioids, 284 are generated which initiate a complex signal transduction cascade, including activation of PKCε, 191 protein tyrosine kinases, 171 Janus kinases, 285,286 as well as transcription factors, such as nuclear factor k B, 287 STAT 1 and 3, 172,285 to ultimately induce gene transcription and increased expression of cardioprotective proteins. The major upregulated proteins, which mediate protection against infarction and, in contrast to the acute form of IPC, also against stunning 24 to 48 hours after the preconditioning cycles include the inducible NOS, 288,289 COX 2, 290,291 superoxide dismutase, 292 aldose reductase, 293 and heme oxygenase. 294 Gene transfer of these proteins also conferred protection against infarction. 279, The upregulation of cardioprotective proteins in late IPC shares a protective phenotype with hibernating myocardium, which is also characterized by lack of infarction, despite reduced coronary blood flow, 298,299 and has increased expression of inducible NOS, 57 stress proteins, such as αb crystallin, 300 H11 kinase, HIF1α, and heat shock protein Effectors of Cardioprotective Conditioning Mitochondria There is unequivocal consensus that mitochondria are the most important effector of conditioning s protection, where most, if not all, of the above signaling pathways converge (Figure 3). The mitochondria are decisive for cellular survival or death, respectively. They provide the ATP for the maintenance for

8 Heusch Signal Transduction of Conditioning 681 ionic gradients and, thus excitability and excitation-contraction coupling, for the contractile machinery, and for the maintenance of cellular integrity. Ischemia with its inherent lack of supply of oxygen as an electron acceptor inhibits the flow of electrons along the respiratory chain, induces inner mitochondrial membrane depolarization, and limits the formation of ATP. Opening of the MPTP on reperfusion initiates a deleterious chain of events. The mitochondria are, however, equipped with several modules, which inhibit MPTP opening. Mitochondrial Permeability Transition Pore The MPTP is a large-conductance megachannel in the inner mitochondrial membrane which when open for longer terms dissipates the inner mitochondrial membrane potential, results in matrix swelling, rupture of the outer mitochondrial membrane, and the release of cytochrome C from the intermembrane space into the cytosol where it activates proteolytic processes and initiates cellular desintegration. 302,303 The molecular identity of the MPTP is still unclear. Although MPTP was traditionally thought of as a multimeric complex of voltage-dependent anion channels of the outer membrane in interaction with adenine nucleotide translocase of the inner membrane, which was regulated by cyclophilin D in the matrix, 302 all of these individual constituents seem to be dispensable, and the MPTP is more recently considered to be formed from F-ATPase. 303, Inner membrane depolarization, high concentrations of inorganic phosphate, ROS, and reactive nitrogen species are all present during myocardial ischemia and more importantly during reperfusion 311 and favor MPTP opening. Transient MPTP opening may serve a physiological function 312,313 in ROS homeostasis 314 and calcium release, 315 and indeed transient MPTP opening is cardioprotective during IPC. 316 Thus, the genuine paradox of conditioning a little injury protects, whereas profound injury is deleterious characterizes also MPTP opening. No matter, what exactly the molecular composition of MPTP is, cyclophilin D regulates MPTP opening, and it decreases the threshold for MPTP opening in response to calcium and inorganic phosphate. 303,317 Cyclosporine A is a drug which inhibits cyclophilin D, can thus inhibit MPTP opening, and ultimately also reduce IS in experimental studies, but also in humans with reperfused AMI 323 and elective coronary artery bypass grafting. 324 The MPTP not only plays a central role in mitochondrial and cellular death or survival, but is also a point of convergence of cardioprotective signaling. In particular, inhibition of GSK 3β was proposed to integrate all upstream signals and exert an inhibitory effect on MPTP opening. 226,230 K ATP Channels The K ATP channel was first identified as a cardioprotective drug target 325,326 and only subsequently as a signaling element of conditioning Initially, the focus was on the K ATP channel in the sarcolemma, which contributes to action potential duration shortening 330 during myocardial ischemia and was therefore thought to alleviate cytosolic calcium overload Indeed, sarcolemmal K ATP channels contribute to cardioprotection in rodents where heart rate is high. 333,335,336 However, the predominant site of cardioprotective signaling by K ATP channels are the mitochondria, as first identified by Marban and collaborators, 337,338 notably K ATP channels in the inner mitochondrial membrane. 339 The molecular identity of mitochondrial K ATP channels is not clear in detail, but there seems to be a multiprotein complex consisting of a sulphonylurea receptor and a potassium channel unit; the potassium channel unit was recently characterized in detail and shares homology with the renal outer medullary potassium channel. 343 The mitochondrial K ATP channel is a target of NO, 344 PKC, 167, and PKG 350 and on its activation releases ROS which, in turn, activate the PKCε Figure 3. Simplified scheme of cardioprotective signaling at the mitochondria. I,II,III, IV indicates respiratory chain complexes I,II,III, IV; AlDH, aldehyde dehydrogenase; Cx 43, connexin 43; CypD, cyclophilin D; ER/SR, endoplasmic/sarcoplasmic reticulum; F, F-ATPase; GSK3β, glycogen synthase kinase 3 β; HIF1α, hypoxia-inducible factor 1α; HK-2, hexokinase 2; K +, potassium ion; K ATP, ATP-dependent potassium channel; MPTP, mitochondrial permeability transition pore; NO, nitric oxide; NOS, nitric oxide synthase; PKC, protein kinase C; PKG, protein kinase G; ROS, reactive oxygen species; and STAT, signal transducer and activator of transcription.

9 682 Circulation Research February 13, 2015 in a positive feedback loop. 184,351 There is also a close interaction of mitochondrial K ATP with mitochondrial Cx 43 in the release of ROS. Subsarcolemmal mitochondria may be more responsive to protection by K ATP channel activation than interfibrillar mitochondria, 352 and K ATP channels then share this feature with Cx It is not really known how the mitochondrial K ATP channel induced ROS release induces protection, other than further activating PKCε. Late IPC also involves K ATP channels; activation of the sarcolemmal K ATP channel seems to trigger protection by late IPC. 335 POC s cardioprotection depends on a signal transduction of PKC activation, mitochondrial K ATP activation, and ROS release, 189,213 very much alike IPC. Mitochondrial K ATP channels also effect the protection by RIPC. 354 Connexin 43 As for K ATP channels, Cx43 has a role in the sarcolemma and in the mitochondria. In the sarcolemma, Cx 43 is the constituent protein of gap junctions, and its inhibition prevents the spread of injury and extension of IS, 355 but whether this function is related to IPC is contentious However, with IPC, sarcolemmal Cx 43 phosphorylation and thus activity during the sustained ischemia is better preserved, 179 and this IPC-related Cx 43 activation may serve a protective function in attenuating cellular edema. 359 The predominant role of Cx 43 in cardioprotection, however, resides in the inner mitochondrial membrane. IPC translocates Cx 43 into the mitochondria through a heat shock protein dependent transport mechanism. 360,361 Activation of mitochondrial Cx 43 channels contributes to mitochondrial potassium uptake, 362,363 increases respiration at complex I and mitochondrial oxygen consumption and ATP formation, 364 and in close interaction with mitochondrial K ATP -channels releases ROS. 365 Cx 43 is expressed in subsarcolemmal mitochondria, presumably serving a signaling function, but not in interfibrillar mitochondria. 353 Mitochondrial Cx 43 phosphorylation is increased with IPC, 360 but RISK and SAFE are not involved in the protective role of Cx 43 in IPC. 366 Mitochondrial Cx 43 is also a target of nitrosation. 367 The loss of IPC s protection with aging is associated with loss of Cx 43 in both sarcolemma and mitochondria. 368 Of note, although Cx 43 is important for protection by IPC, 358,369 it is not for that by POC. 370 RIC is associated with better preservation Cx 43 phosphorylation. 371 STAT 3 STAT3 is a transcription factor which is involved in several physiological and pathological processes in the heart. 251 However, a mitochondrial location and function has also recently been identified. STAT 3 is located in the matrix of subsarcolemmal and interfibrillar mitochondria 321 and serves to increase complex I respiration 244,247 and attenuate MPTP opening 244,321 and ROS formation. 372,373 STAT 3 is causal for IS reduction by IPC 240,241 and POC. 243,244 Whether the cardioprotection is effected by the mitochondrial STAT 3 or a cytosolic STAT 3 is not exactly clear from most studies where STAT 3 or the SAFE system were involved. Clearly, in late IPC, it is the function of the transcription factor STAT 3 which is causal to upregulate cardioprotective proteins. 172,246 The exact function of STAT 3 in mitochondrial respiration is still unclear; based on its low abundance in relation to the respiratory chain proteins, protein protein interaction is rather unlikely. 374 Hexokinase Binding of hexokinase isoform 2 to the mitochondria is cardioprotective, whereas its dissociation from the mitochondria releases cytochrome C from the intermembrane space and initiates cell death Hexokinase 2 is the predominant isoform in the heart, and it plays a major role in cellular glucose metabolism; its mitochondrial localization favors glycolysis. 377 Hexokinase 2 localization to the mitochondria is facilitated by RISK activation, 377,379 but also by decreased cytosolic glucose-6-phosphate concentration, 380 and reduced glucose-6-phosphate concentration, in turn, is one consequence of IPC. 21 Mitochondrial hexokinase 2 stabilizes contact sites between the inner and outer mitochondrial membranes and thereby attenuates loss of cytochrome C from the intermembrane space to the cytosol during myocardial ischemia/ reperfusion. 381 Through inhibiting interaction with voltage-dependent anion channels in the outer membrane, hexokinase 2 also attenuates loss of adenine nucleotides. 382 Ultimately, the detailed mechanisms of mitochondrial hexokinase in cardioprotection are not clear, but they definitely include inhibition of MPTP opening, and hexokinase is clearly involved in IPC. 383,384 Aldehyde Dehydrogenase Aldehyde dehyrdogenase isoform 2 is located in the mitochondrial matrix and detoxifies aldehydes, 385 and it is a target of PKCε. 386 Inhibition of aldehyde dehydrogenase 2 by cyanamide abrogates IS reduction by RIPC in rabbits, and a single nucleotide polymorphism which renders this enzyme inactive abrogates the attenuation of endothelial dysfunction by RIPC in humans. 387 Protein Nitrosation/Nitrosylation NO is an essential molecule in IPC, POC, and RIC, as detailed earlier. Not all of NO s actions in cardioprotection are mediated through increased cgmp formation and consequent PKG activation. 217 In fact, NO directly targets several proteins and alters their structure and function through nitrosation or nitrosylation. 215,216 The nitrosylation is thought to shield cysteine residues from oxidation. 388 IPC, 215 POC, 389,390 and RIC 64 reduce IS independently from PKG, and several nitrosylated proteins were identified in preconditioned myocardium, notably also mitochondrial proteins. 215,388 Mitochondrial Cx 43 is a target of nitrosation during IPC. 367 Nitrosation of complex I along with reduced respiration and ROS formation contributes to RIPC. 64 Sarcoplasmic Reticulum Excessive calcium oscillations during early reperfusion, when sarcoplasmic reticulum and contractile machinery have been re-energized in the presence of cytosolic calcium overload, contribute to uncoordinated contracture and eventual cellular disruption, 391 rendering the sarcoplasmic reticulum a potential target of protection during early reperfusion. 392 There is also close interaction of the sarcoplasmic reticulum with MPTP during reperfusion, and calcium oscillations contribute to MPTP opening and vice versa IPC improved sarcoplasmic reticulum function, that is, increased calcium uptake and release along with increased phosphorylation of the ryanodine receptor, sarcoplasmic reticulum ATPase and phospholamban at the end of reperfusion, 396 whereas ryanodine-sensitive calcium release was decreased after the preconditioning cycles in another study, 397 or pharmacological elimination of

10 Heusch Signal Transduction of Conditioning 683 sarcoplasmic reticulum function did not at all affect protection of left ventricular function by IPC. 398 The sarcoplasmic reticulum is a target of PKCε 185 and of protein nitrosylation, 399 POC delays phosphorylation of phospholamban by PKG, and reduced sarcoplasmic reticulum ATPase activity in the presence of acidosis favors the extrusion of cytosolic calcium through the sodium calcium exchanger and reduces calcium oscillations, thus contributing to reduced IS. 400 Cytoskeleton, Cell Volume, and Ionic Balance Decreased cytoskeletal stability and increased cell volume contribute to membrane disruption and cellular fragility. IPC attenuates osmotic fragility through cytochalasin D sensitive stabilization of the actin cytoskeleton and a K ATP -dependent mechanism in isolated rabbit cardiomyocytes. 401 Also, IPC increases the phosphorylation and translocation of αb-crystallin. 402,403 The microtubular network is involved in activation and translocation of PKCε and p38 during IPC, and its depolymerization abrogates IS reduction. 404 Calpain activation during early reperfusion contributes to cellular digestion, 405 and IPC attenuates the calpain-mediated degradation of structural proteins. 254 IPC reduces the increases in sodium and calcium during myocardial ischemia by inhibition of sodium proton and of sodium calcium exchange. 406 IPC also attenuates the intracellular volume load which is associated with sodium and calcium overload. 407 The role of chloride channel activation in such attenuation of intracellular edema is contentious. 408,409 Potassium channels may also be involved in cell volume control during IPC to maintain electroneutrality along with chloride efflux. 410 Similar findings on chloride and potassium channels were also reported in cardiomyocytes subjected to plasma dialysate from a RIPC protocol. 411 Apart from chloride and potassium channels, Cx 43 is involved in intracellular volume regulation. Dephosphorylated Cx 43 contributes to intracellular edema during myocardial ischemia, whereas preservation of Cx 43 phosphorylation with IPC may attenuate such edema. 359 Endoplasmic Reticulum Stress Response, Autophagy Myocardial ischemia/reperfusion is associated with an increased abundance of structurally and functionally defective proteins, which jeopardize normal cellular function and must therefore be removed. The endoplasmic reticulum stress response and autophagy are characteristic processes which remove dysfunctional and defective proteins. The endoplasmic reticulum stress response involves sensing of unfolded proteins by specific proteins, among them notably the activating transcription factor 6 (ATF 6), which then induce the transcription of genes which encode for chaperones and degrading enzymes. 412 ATF 6 is activated in myocardial ischemia/ reperfusion and its gene silencing increases cell death. 413 ATF 6 induces increased expression of a protein-disulfide isomerase, which again reduces ischemia/reperfusion associated cell death. 414 Nuclear translocation of ATF 6 and ATF 3 abundance were increased by late IPC, and genetic ablation of ATF 3 abrogated IS reduction by late IPC. 294 The endoplasmic reticulum stress response was also enhanced by POC, but not in RIC, and a pharmacological endoplasmic reticulum stress response inhibitor abrogated the IS reduction. 415 Autophagy is activated during myocardial ischemia/reperfusion and involves conjugation of defective proteins with ubiquitin and sequestration in a double-membrane structure (autophagosome) which subsequently fuses with a lysosome where proteins are then degraded and recycled. 416 Several specific proteins are involved in this process, the autophagyrelated proteins, beclin 1, parkin, and light chain 3-phosphatidyl ethanolamine which are activated by ROS, reactive nitrogen species, calcium, and MPTP opening. Mitophagy is the specific autophagy of mitochondria which is preceded by mitochondrial fission. A dominant negative transgene of autophagy-related protein 5 abolished the IS reduction by IPC. 417 IPC induced protein translocation to mitochondria, and genetic parkin deficiency abrogated IS reduction. 418 Exogenous induction of autophagy by chloramphenicol in pigs reduced IS. 419 POC also increases the expression of autophagy-related proteins, and a pharmacological inhibitor of autophagy abrogates the IS reduction by POC. 420 Autophagy is also activated in repetitive myocardial ischemia/reperfusion over a more long-term time frame, 421 and here again, chronically preconditioned and hibernating myocardium share a protected phenotype. Autophagy is also involved in remote POC, 422 but apparently plays no role in patients undergoing coronary artery bypass grafting with RIPC. 423 Acidosis During Reperfusion Acidosis during early reperfusion inhibits MPTP opening but also the development of hypercontracture. 424,425 Acidosis at early reperfusion is mandatory for IPC s reduction of IS, and protection is abrogated by sodium bicarbonate infusion. 426 To maintain acidosis at early reperfusion, the sodium proton exchanger is inhibited by IPC. 427 Likewise, protection by POC requires maintenance of acidosis at early reperfusion, 428 and vice versa acidic reperfusion reduces IS. 429,430 Activation of PKG contributes to delayed recovery of acidosis during reperfusion through inhibition of the sodium proton exchanger. 214 Cardioprotective Signal Transfer During RIC The transfer of the cardioprotective signal from the peripheral organ (brain, mesentery, kidney, skeletal muscle, skin) to the heart is the result of a complex neurohumoral interaction. 37 Ischemia/reperfusion-, 32,64,92, trauma-, and chemically (adenosine, 433 bradykinin, 92,439 caspaicin) 434,437,440 or electrically 440,441 induced activation of sensory fibers in the peripheral organ (1) activate the autonomic cardiac innervation through the central nervous system, and (2) release an as yet unidentified humoral factor (Figure 4). The activation of the peripheral sensory fibers involves activation of the PKCγ isoform. 439 In some studies, activation of the autonomic nervous system through spinal and supraspinal reflexes ,442 was mandatory to reduce IS in the ischemic/reperfused myocardium, 32,92,434,435,437,443 in others it was not. 432,444 Of importance, humoral transfer of protection with plasma or dialysate obtained after the peripheral preconditioning cycles to another individual or individual heart, even across species, is possible and highlights the functional importance of the humoral transfer signal. 433,440,441 The humoral transfer signal appears to be of a molecular weight <15 kda and hydrophobic. There are data to support a role of NO, 64 stromal cell derived factor-1α, 154

11 684 Circulation Research February 13, 2015 interleukin 10, 152 and microrna-144, 276 but none of these factors alone sufficiently explains the protection observed with RIC. Data on RIC s signal transduction in the protected myocardium are relatively sparse. Adenosine, 445,446 bradykinin, 437 opioids, 431,432,447 interleukin 10, 152 and stromal cell derived factor-1α 154 have been implicated as local triggers, PKC, 432,437,448 the RISK system, 83,152,264,265,449,450 and endothelial NOS 64 and HIF-1α 264,265 as mediators and again the mitochondria 277,387 and their K ATP channels as effectors, 451 not different from the above signal transduction in local IPC or POC. The only myocardial signal established in human RIPC to date is STAT Comprehensive View on Spatiotemporal Patterns of Cardioprotective Signaling The standards for robust data on cardioprotective signaling have risen, and single dose blocker experiments as in the early landmark studies are no longer satisfactory to identify a signaling step. Unequivocal identification of a signaling step requires not only an appropriate conditioning protocol with IS as end point, but also biochemical or immunoblotting data for signal activation along with IS reduction and abrogation of both the signal and the IS reduction by genetic ablation or pharmacological inhibition of the signal molecule. There are several unresolved issues with the conditioning phenomena, including differences between species, between the different conditioning phenomena, steep dose response relationships, and the spatial and temporal organization of cardioprotective signals. Clearly, there are important species differences. It is not surprising that in smaller rodents with their high heart rate, the control of action potential duration by the sarcolemmal K ATP channel plays a greater role in cardioprotection 333,335,336,452 than in larger mammals where the mitochondrial K ATP channel is important for cardioprotection. In rats, opioids seem to be more important for cardioprotection than adenosine. 84 The RISK system plays a major role in cardioprotection in rodents, 221 but has no role at least in POC in pigs, 238 where the SAFE system is more important. 244 STAT 3 is an important signaling molecule in rodents and pigs, 78,221 but apparently STAT 5 was the only signaling molecule found activated during RIC in humans, 250 and interestingly STAT 5 seems also to play a major role in cardioprotection in rabbits. 453 Many signaling steps have not been addressed in the various conditioning phenomena, such that we largely do not know whether the same signaling steps are involved in IPC, POC, and RIC. There is at least one signal, that is, Cx43, which is important in IPC 369 and possibly also RIC, 371 but not in POC. 370 A major concern with most of the studies on cardioprotective signaling is that they focussed on one or few of the signaling steps and often reported abrogation of IS reduction with elimination of a single signaling step. Such all-or-none response is difficult to reconcile with the multitude of different signaling steps detailed above: why would elimination of a single signal abrogate protection, unless it is a/the downstream point of convergence? Indeed, the few studies which have looked at stimulus response relationships in conditioning have found them steep. 71,90,129,243 Also, cardioprotective signaling is highly interactive, at the level of the triggers 446,454 but also at the level of the mediators where, in particular, RISK and SAFE system closely interact. 139,158,455 Cardioprotective signaling is obviously a highly organized concerted action where localization and timing matter. As Figure 4. Signal transfer from the peripheral organ to the heart in remote ischemic conditioning (RIC). Akt indicates protein kinase B; Cx 43, connexin 43; enos, endothelial NO synthase; ERK, extracellular regulated kinase; GSK3β, glycogen synthase kinase 3 β; K ATP, ATP-dependent potassium channel; MPTP, mitochondrial permeability transition pore; NO, nitric oxide; PI3K, phosphatidylinositol (4,5)-bisphosphate 3-kinase; PKC, protein kinase C; SDF-1α, stromal-derived factor 1α; and STAT, signal transducer and activator of transcription. Modified from Ref. 37.

12 Heusch Signal Transduction of Conditioning 685 Figure 5. Forest plot of clinical studies on ischemic postconditioning (POC), remote ischemic conditioning (RIC), and drugs in patients with acute myocardial infarction (AMI) and with infarct size (IS) as end point. detailed earlier, activated PKCε translocates to the mitochondria 182,183,456 where it acts in a localized signalosome 457 involving also PKG, mitochondrial K ATP, and ROS in a positive feedback loop. 184,351 However, activated PKC is also required at the sarcolemma where it sensitizes the A 2B receptor for adenosine during early reperfusion. 192 Caveolae with their constituent proteins caveolin 1 and 3 form intracellular signalosomes which include endothelial NOS, translocate to the mitochondria 458 to induce nitrosylation of mitochondrial proteins, 459 and are causally involved in protection by IPC And clearly, mitochondria and ultimately inhibition of MPTP are a point of convergence for all cardioprotective signaling, that is, the holy grail of cardioprotection. 302 However, although only one mitochondrium is usually displayed schematically, there are in fact many mitochondria in each cardiomyocyte, and we do not know the dose response relationship between the number of mitochondria with MPTP opening and cell death. 463 It is obvious that for protection by POC, the early and immediate reperfusion phase is critical, 464 and only a single study reported still protection by POC when delayed up to 30 minutes reperfusion. 465 However, the early reperfusion phase after the sustained myocardial ischemia is obviously also critical

13 686 Circulation Research February 13, 2015 for the execution of IPC because it can still be abrogated then by inhibition of the RISK pathway 225,466 or blockade of adenosine receptors. 82 Therefore, the early reperfusion phase after the sustained ischemia has been emphasized as critical, 158,159,233 and this is obviously also the critical phase where additional cardioprotection beyond that by reperfusion per se would be needed in patients with AMI. Recruitment of Cardioprotective Signaling in Patients With AMI IPC by its very nature cannot be used in patients with AMI, but studies on preinfarction angina suggest that protection by IPC might nevertheless occur. 26,467,468 In contrast, POC and RIC can be used and have been used in patients with AMI, and the majority of studies demonstrated IS reduction, as reflected by biomarkers or imaging (Figure 5) Not all studies on POC were positive, and this may be related to lack of direct stenting with the consequence of coronary microembolization during the POC procedure 39,507 or to preexisting partial and gentle reperfusion. 508 For RIC, even an improved clinical outcome has been reported. 36 In contrast to the positive clinical studies with mechanical conditioning strategies, studies attempting to recruit cardioprotective signals pharmacologically have largely failed, including those on adenosine, on PKCδ inhibition, 188 and on intravenous nitrite which is bio-converted to NO. 499 Erythropoietin failed to reduce IS in 2 trials, 501,502 but there was no evidence before that erythropoietin participated in the conditioning phenomena. One study on atrial natriuretic peptide demonstrated modest IS reduction, but atrial natriuretic peptide is not really an endogenous trigger or mediator of conditioning. 500 Exenatide inhibits the catabolism of glucagon-like peptide 1, and it reduced IS in 2 recent trials in patients with reperfused AMI, 503,504 but, similarily to atrial natriuretic peptide, glucagon-like peptide 1 is not a trigger or mediator of conditioning. The only study which successfully recruited an established cardioprotective signal, that is, MPTP inhibition, is the study by Ovize and collaborators who demonstrated IS reduction by an intravenous bolus of cyclosporine A just before reperfusion. 323 However, another recent study using a different MPTP inhibitor failed to reduce IS in patients with reperfused AMI and even raised safety concerns. 505 Metoprolol 506 when given just before reperfusion of AMI reduced IS, but its relation to endogenous cardioprotective signaling is unclear. The reasons for the to date disappointing translation of cardioprotective signaling to pharmacological use in patients with AMI have been discussed in detail elsewhere 509,510 and include inconsistent prior experimental data, poor design of the trials, but also confounding variables which are often not accounted for in experimental studies, such as age, 511 comorbidities, and comedications. 40 To improve translation, we will need to (1) get more mechanistic insight into the conditioning phenomena, (2) place equal emphasis on the identification of novel signaling elements in potentially reductionist experimental models and on the translation of such novel, but reductionist findings into more complex and integrative models, (3) identify signaling elements of cardioprotection in human myocardium and retrospectively those experimental models which might have predicted these elements, (4) develop standards and infrastructures to identify robust signaling elements which can possibly serve as drug targets, such as the CAESAR consortium, 512 (5) organize interaction between basic scientists and clinician scientists to set up appropriate proof-of-concept clinical trials and eventually larger prospective, randomized multicenter trials, such as CIRCUS (NCT ) or ERICCA (NCT ). Sources of Funding G. Heusch was supported by the German Research Foundation (He 1320/18-1, 3 and SFB 1116), the Heinz Horst Deichmann Foundation, and the Hans und Gertie Fischer Foundation. None. Disclosure References 1. Heusch G. Postconditioning: old wine in a new bottle? J Am Coll Cardiol. 2004;44: doi: /j.jacc Yellon DM, Hausenloy DJ. Myocardial reperfusion injury. N Engl J Med. 2007;357: doi: /NEJMra Ovize M, Baxter GF, Di Lisa F, Ferdinandy P, Garcia-Dorado D, Hausenloy DJ, Heusch G, Vinten-Johansen J, Yellon DM, Schulz R; Working Group of Cellular Biology of Heart of European Society of Cardiology. Postconditioning and protection from reperfusion injury: where do we stand? Position paper from the Working Group of Cellular Biology of the Heart of the European Society of Cardiology. Cardiovasc Res. 2010;87: doi: /cvr/cvq Heusch G. Cardioprotection: chances and challenges of its translation to the clinic. 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27 Molecular Basis of Cardioprotection: Signal Transduction in Ischemic Pre-, Post-, and Remote Conditioning Gerd Heusch Circ Res. 2015;116: doi: /CIRCRESAHA Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX Copyright 2015 American Heart Association, Inc. All rights reserved. Print ISSN: Online ISSN: The online version of this article, along with updated information and services, is located on the World Wide Web at: Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: Subscriptions: Information about subscribing to Circulation Research is online at: